Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space

*Peter Norsk*

## **Abstract**

There are eight steps in the preparation, implementation and execution of a human spaceflight experiment: (1) writing a proposal, (2) being selected by a space agency, (3) finding funding, (4) flight feasibility assessment for flight, (5) implementation into a specific space platform (e.g. the Space Shuttle in the past and now the International Space Station), (6) experiment execution, (7) analysis of collected data and (8) publication. The unique features about spaceflight experiments are steps 4–6 because of the limitations of conducting experimental procedures in space. Furthermore, all of the associated equipment have to be developed and approved for spaceflight with all the safety aspects taken appropriately into consideration. In this chapter, two specific experiments from the Spacelab D2 mission in 1993 are used as illustration of these steps as well as describing the use of parabolic flights as a preparatory platform. It is important to have data collected of such a quality that they can be published in science journals with external peer review. It is also important that the data not only have operational spaceflight applications but also can advance knowledge for terrestrial science purposes.

**Keywords:** cardiac output, cardiovascular, central venous pressure, parabolic flight, weightlessness

## **1. Introduction**

On 22 March 1993, I was closely watching the big screen in the German Space Control Centre in Oberpfaffenhofen near Munich, Germany. The Space Shuttle with the Spacelab D2 mission was just about to lift off, and on board were seven astronauts and equipment for a host of experiments of which four were from Denmark. I was the responsible investigator for these four experiments. I was nervous. In the airplane from Copenhagen to Munich the day before, I had told myself that this would either be my stepping stone to a further career in space physiology and medicine or simply my big Waterloo. The Danish government had payed millions of Kroners in preparation of the studies, and failure was not an option. The problem, though, was that anything random, which was totally out of my and my colleagues' control, could happen at any time and jeopardise our experiments.

The four experiments had been under preparation for some 5 years and supported by Danish space research grants. Eight people had been full time involved in my laboratory: four engineers and four medical doctors. Equipment had been developed, tested and adapted for spaceflight, and many nervous moments had been overcome. It was exciting times and now—finally—everything was about to be launched into space to orbit the Earth at a speed of 28,000 km per hour in a free fall condition inducing chronic weightlessness for 10 days. I felt tense as the launch countdown approached zero.

A few seconds before launch, two of the three Space Shuttle engines ignited. The third one did not. Immediately we knew in the control room that something was wrong. One of the engines did not blast, and within a few seconds, all engines stopped. As water vapour evaporated away from the shuttle, we waited anxiously. What would happen now?

The ignition had failed, and within the next hour or so, the astronauts were let out, and the launch postponed until the end of April. In that interim period, we had to repeat some ground tests on the astronauts in collaboration with several other international teams from Europe and the United States. Finally, after some additional postponements of the launch, the Space Shuttle Columbia ignited on 26 April and went to space, where it completed a 10-day mission with great success. Almost 90 experiments were expertly conducted. Concerning the four Danish human physiology experiments, the data were successfully collected, which 2–3 years later led to three scientific papers [1–3] from our Danish space medicine and physiology laboratory, DAMEC Research Inc. (Danish Aerospace Medical Centre of Research Inc.), at the Copenhagen University Hospital.

The successful completion of our experiments on the Spacelab D2 mission in 1993 with the publications in 1995 and 1996 led to the later expert preparation, implementation and conduction of seven additional studies in space on the Russian Mir station, Space Shuttle Columbia and International Space Station. All studies had cardiovascular adaptation aspects, and parabolic flights were used—not only for preparation of the spaceflight experiments but also to obtain basic science knowledge of human physiological responses to very acute weightlessness of 20 s.

## **2. Experimental science background**

During changes in posture, blood pressure in humans is continuously and acutely regulated by pressure reflexes originating from receptors (baroreceptors) in the aorta close to the heart and in the two carotid (neck) arteries at the base of the skull. In addition, there are pressure sensors in the heart, from which reflexes also originate to participate in blood pressure control. This blood pressure regulation is for the central nervous system to make sure that the perfusion pressures to the brain and other organs are optimal despite the displacement of blood caused by the posture. As an example, the upright posture displaces blood downwards towards the lower body and the legs away from the head and heart. To counteract that so that the blood pressure at heart and head level does not fall too much, which would impede blood supply to the brain, the blood pressure reflexes sense the decrease in pressure and within a few heart beats initiate an increase in heart rate and constriction of the small arteries in the lower body. The opposite occurs, when the posture changes from upright to recumbent or supine.

We have for many years investigated, which blood pressure sensors are the most important for adjusting blood pressure to posture changes. In a whole host of investigations using a combination of various models as well as short-term weightlessness during parabolic flights, we have aimed at isolating the effects of some

**197**

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

receptors versus others and found that in order for blood pressure and heart rate to adapt to either the supine or upright posture, the low pressure reflexes originating from the heart and major central veins are pivotal. Without the inputs from these low pressure heart and venous reflexes, the new steady state cannot be achieved. These findings have changed our previous understanding of how the human cardio-

Since blood pressure is also determined by the amount of fluid in the body, we have used the human head-out water immersion model to investigate how the volume of fluid and amount of salt is controlled. By immersing humans in the seated posture to the level of the neck for hours, the fluid- and salt-excreting mechanisms through the kidneys are stimulated, because the headward shift of blood and fluid from the lower body to the heart through various mechanisms informs the central nervous system that the upper body vessels are being overloaded with blood and fluid so that this excess volume must be excreted. The mechanisms for this are still only partly understood, but the main opinion is that there is a connection between the heart and kidneys through what is termed the cardio-renal link so that when the heart chambers are stretched, it initiates a reflex response to the kidneys to excrete

Our research using the seated head-out water immersion model has shown that not only the cardio-renal link and associated hormones control the urinary excretion rates of sodium during shifting of blood and fluid to the upper body but also dilution of the blood with fluid from the tissues plays an important role. During shift of blood from the lower to the upper body caused by the surrounding water pressure, fluid is pushed into the circulation from the lower body tissues. This dilution can affect the kidneys directly as well as release of some kidney-regulating hormones. Weightlessness in space is a unique condition that cannot be replicated on the ground and where bodily functions can be studied without the intervening effects of gravity. In space, blood and fluids are chronically displaced towards the upper body segments (heart and head), and the daily fluctuations induced by posture changes do not occur. This gives us a unique opportunity to utilise weightlessness in space for exploring the cardiovascular and fluid volume-regulating systems in the

The experiments on the Spacelab D2 mission in 1993 aimed at understanding whether the blood and fluid shift into the heart during prolonged weightlessness would augment the urinary output of water and salt, just as we usually observe in ground-based simulation models. In one experiment we aimed at measuring how much the fluid pressure leading into the heart (central venous pressure, CVP) increases, which we at that time thought to be the stimulus for control of the urinary fluid and salt excretion. In another experiment we aimed at monitoring the urine production. It was the hypothesis that an increase in CVP induced by weight-

The CVP experiment was originally planned to be done on two of the Spacelab D2 astronauts, but for some technical reasons, it was only accomplished in one. The experimental plan was to shortly before launch insert a long catheter with a pressure transducer at the end into the vein directly leading into the right atrial chamber of the heart through a peripheral arm vein and connect it to a preamplifier

vascular system adapts to changes in postures and the effects of gravity.

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

more salt and fluid in the urine.

**3. Experiments: the spacelab D2 mission in 1993**

lessness would augment the excretion rate of fluid and salt.

human body.

**3.1 CVP experiment**

#### *Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space DOI: http://dx.doi.org/10.5772/intechopen.93466*

receptors versus others and found that in order for blood pressure and heart rate to adapt to either the supine or upright posture, the low pressure reflexes originating from the heart and major central veins are pivotal. Without the inputs from these low pressure heart and venous reflexes, the new steady state cannot be achieved. These findings have changed our previous understanding of how the human cardiovascular system adapts to changes in postures and the effects of gravity.

Since blood pressure is also determined by the amount of fluid in the body, we have used the human head-out water immersion model to investigate how the volume of fluid and amount of salt is controlled. By immersing humans in the seated posture to the level of the neck for hours, the fluid- and salt-excreting mechanisms through the kidneys are stimulated, because the headward shift of blood and fluid from the lower body to the heart through various mechanisms informs the central nervous system that the upper body vessels are being overloaded with blood and fluid so that this excess volume must be excreted. The mechanisms for this are still only partly understood, but the main opinion is that there is a connection between the heart and kidneys through what is termed the cardio-renal link so that when the heart chambers are stretched, it initiates a reflex response to the kidneys to excrete more salt and fluid in the urine.

Our research using the seated head-out water immersion model has shown that not only the cardio-renal link and associated hormones control the urinary excretion rates of sodium during shifting of blood and fluid to the upper body but also dilution of the blood with fluid from the tissues plays an important role. During shift of blood from the lower to the upper body caused by the surrounding water pressure, fluid is pushed into the circulation from the lower body tissues. This dilution can affect the kidneys directly as well as release of some kidney-regulating hormones.

Weightlessness in space is a unique condition that cannot be replicated on the ground and where bodily functions can be studied without the intervening effects of gravity. In space, blood and fluids are chronically displaced towards the upper body segments (heart and head), and the daily fluctuations induced by posture changes do not occur. This gives us a unique opportunity to utilise weightlessness in space for exploring the cardiovascular and fluid volume-regulating systems in the human body.

## **3. Experiments: the spacelab D2 mission in 1993**

The experiments on the Spacelab D2 mission in 1993 aimed at understanding whether the blood and fluid shift into the heart during prolonged weightlessness would augment the urinary output of water and salt, just as we usually observe in ground-based simulation models. In one experiment we aimed at measuring how much the fluid pressure leading into the heart (central venous pressure, CVP) increases, which we at that time thought to be the stimulus for control of the urinary fluid and salt excretion. In another experiment we aimed at monitoring the urine production. It was the hypothesis that an increase in CVP induced by weightlessness would augment the excretion rate of fluid and salt.

#### **3.1 CVP experiment**

The CVP experiment was originally planned to be done on two of the Spacelab D2 astronauts, but for some technical reasons, it was only accomplished in one. The experimental plan was to shortly before launch insert a long catheter with a pressure transducer at the end into the vein directly leading into the right atrial chamber of the heart through a peripheral arm vein and connect it to a preamplifier

*Preparation of Space Experiments*

countdown approached zero.

What would happen now?

Inc.), at the Copenhagen University Hospital.

**2. Experimental science background**

changes from upright to recumbent or supine.

The four experiments had been under preparation for some 5 years and supported by Danish space research grants. Eight people had been full time involved in my laboratory: four engineers and four medical doctors. Equipment had been developed, tested and adapted for spaceflight, and many nervous moments had been overcome. It was exciting times and now—finally—everything was about to be launched into space to orbit the Earth at a speed of 28,000 km per hour in a free fall condition inducing chronic weightlessness for 10 days. I felt tense as the launch

A few seconds before launch, two of the three Space Shuttle engines ignited. The third one did not. Immediately we knew in the control room that something was wrong. One of the engines did not blast, and within a few seconds, all engines stopped. As water vapour evaporated away from the shuttle, we waited anxiously.

The ignition had failed, and within the next hour or so, the astronauts were let out, and the launch postponed until the end of April. In that interim period, we had to repeat some ground tests on the astronauts in collaboration with several other international teams from Europe and the United States. Finally, after some additional postponements of the launch, the Space Shuttle Columbia ignited on 26 April and went to space, where it completed a 10-day mission with great success. Almost 90 experiments were expertly conducted. Concerning the four Danish human physiology experiments, the data were successfully collected, which 2–3 years later led to three scientific papers [1–3] from our Danish space medicine and physiology laboratory, DAMEC Research Inc. (Danish Aerospace Medical Centre of Research

The successful completion of our experiments on the Spacelab D2 mission in 1993 with the publications in 1995 and 1996 led to the later expert preparation, implementation and conduction of seven additional studies in space on the Russian Mir station, Space Shuttle Columbia and International Space Station. All studies had cardiovascular adaptation aspects, and parabolic flights were used—not only for preparation of the spaceflight experiments but also to obtain basic science knowledge of human physiological responses to very acute weightlessness of 20 s.

During changes in posture, blood pressure in humans is continuously and acutely regulated by pressure reflexes originating from receptors (baroreceptors) in the aorta close to the heart and in the two carotid (neck) arteries at the base of the skull. In addition, there are pressure sensors in the heart, from which reflexes also originate to participate in blood pressure control. This blood pressure regulation is for the central nervous system to make sure that the perfusion pressures to the brain and other organs are optimal despite the displacement of blood caused by the posture. As an example, the upright posture displaces blood downwards towards the lower body and the legs away from the head and heart. To counteract that so that the blood pressure at heart and head level does not fall too much, which would impede blood supply to the brain, the blood pressure reflexes sense the decrease in pressure and within a few heart beats initiate an increase in heart rate and constriction of the small arteries in the lower body. The opposite occurs, when the posture

We have for many years investigated, which blood pressure sensors are the most important for adjusting blood pressure to posture changes. In a whole host of investigations using a combination of various models as well as short-term weightlessness during parabolic flights, we have aimed at isolating the effects of some

**196**

#### *Preparation of Space Experiments*

and a recording system. The test astronaut would thus be inserted with the catheter and wear the CVP monitoring system until 3 hours into the mission following launch. Thereafter the catheter would be withdrawn. Before the launch of the Space Shuttle, control measurements in different body postures were performed.

The equipment used for measurements of central venous pressure during the Spacelab D2 mission in one astronaut is depicted in **Figure 1**. The central venous catheter (1) with a pressure transducer placed at one end and a connector box at the other was plugged into a preamplifier (2) that was connected to a recording unit (3). A calibration piston (4) could be connected to the reference opening of the catheter and thus induce predetermined pressure changes on the backside of the transducer membrane. In this way, it was tested to what degree the calibration characteristics of the pressure transducer might have changed over time after being inserted into the astronaut. Following the spaceflight, the catheter was brought back to the investigators and tested for change in drift of the tip transducer.

## **3.2 Urinary excretion experiment**

Before the spaceflight, four test astronauts would, over a 4.5-h period, empty their urine bladders in either supine or seated posture, on an hourly basis after being infused through a peripheral vein with isotonic saline in an amount of 2% of their body weight. About a week into the flight, the same would be done following a similar infusion while they thus would be free floating in the space vehicle. Blood was sampled on ground and during flight for determination of water-, salt- and blood pressure-regulating hormones. The volume of urine was measured after each void and samples taken for determination of salt (sodium and potassium) concentrations. In space, the volume of urine was determined by a urine monitoring system delivered by NASA, which was connected to the toilet. If the voids were felt by the subjects to be small, the bladders were emptied into bags and returned to

## **Figure 1.**

*Central venous pressure equipment, which flew on the Spacelab D2 mission in 1993 [3]. (See text for detailed explanation).*

**199**

**Figure 2.**

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

Earth through the trash system on the Shuttle. The saline infusion was conducted over some 20 min by manually inflating a cuff-pressure system, and the amount

After the Spacelab D2 mission in 1993, our research team in Denmark conducted additional experiments on various space platforms with one on the Russian space station, Mir, in 1995, where we monitored urine excretion rates over longer flight periods of up to 6 months by having three test astronauts collect urine into bags and the volumes measured by a scale system. The idea was to test whether urine production in weightlessness after intake of an oral water load would be enhanced—just like we tested the same hypothesis regarding urinary salt excretion on the previous

During later space missions, we have conducted several additional studies on the Space Shuttle and the International Space Station focusing on the cardiovascular adaptation to short (<30 days) and long (>30 days) duration flights. In particular, we have for this purpose conducted cardiac output measurements by a non-invasive rebreathing technique, which has been developed for spaceflight. Cardiac output is the amount of blood injected by the heart into the body per minute, and this variable is important for understanding the effects of weightlessness on not only cardiac function but also the vascular condition in general. The hypothesis was tested that the weightlessness-induced increase in central blood volume would increase cardiac output and at the same time through the cardiovascular reflexes dilate the arterial

Normally, accurate cardiac output estimations require insertion of catheters into the veins and arteries, which makes the measurement difficult on a routine basis in normal healthy humans. With the non-invasive foreign gas rebreathing technique developed for spaceflight, such estimations can be done anytime and anywhere with no harm to the test subject. As indicated in **Figure 2**, the tested person breathes

*The principle of foreign gas rebreathing for measurement of cardiac output [4]. (See text for explanation).*

**4. Experiments during subsequent missions (1995–2012)**

resistance vessels to counteract an increase in blood pressure.

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

Spacelab D2 mission.

varied on an average between 1.7 and 1.8 litres.

Earth through the trash system on the Shuttle. The saline infusion was conducted over some 20 min by manually inflating a cuff-pressure system, and the amount varied on an average between 1.7 and 1.8 litres.

## **4. Experiments during subsequent missions (1995–2012)**

After the Spacelab D2 mission in 1993, our research team in Denmark conducted additional experiments on various space platforms with one on the Russian space station, Mir, in 1995, where we monitored urine excretion rates over longer flight periods of up to 6 months by having three test astronauts collect urine into bags and the volumes measured by a scale system. The idea was to test whether urine production in weightlessness after intake of an oral water load would be enhanced—just like we tested the same hypothesis regarding urinary salt excretion on the previous Spacelab D2 mission.

During later space missions, we have conducted several additional studies on the Space Shuttle and the International Space Station focusing on the cardiovascular adaptation to short (<30 days) and long (>30 days) duration flights. In particular, we have for this purpose conducted cardiac output measurements by a non-invasive rebreathing technique, which has been developed for spaceflight. Cardiac output is the amount of blood injected by the heart into the body per minute, and this variable is important for understanding the effects of weightlessness on not only cardiac function but also the vascular condition in general. The hypothesis was tested that the weightlessness-induced increase in central blood volume would increase cardiac output and at the same time through the cardiovascular reflexes dilate the arterial resistance vessels to counteract an increase in blood pressure.

Normally, accurate cardiac output estimations require insertion of catheters into the veins and arteries, which makes the measurement difficult on a routine basis in normal healthy humans. With the non-invasive foreign gas rebreathing technique developed for spaceflight, such estimations can be done anytime and anywhere with no harm to the test subject. As indicated in **Figure 2**, the tested person breathes

**Figure 2.**

*The principle of foreign gas rebreathing for measurement of cardiac output [4]. (See text for explanation).*

*Preparation of Space Experiments*

**3.2 Urinary excretion experiment**

and a recording system. The test astronaut would thus be inserted with the catheter and wear the CVP monitoring system until 3 hours into the mission following launch. Thereafter the catheter would be withdrawn. Before the launch of the Space

The equipment used for measurements of central venous pressure during the Spacelab D2 mission in one astronaut is depicted in **Figure 1**. The central venous catheter (1) with a pressure transducer placed at one end and a connector box at the other was plugged into a preamplifier (2) that was connected to a recording unit (3). A calibration piston (4) could be connected to the reference opening of the catheter and thus induce predetermined pressure changes on the backside of the transducer membrane. In this way, it was tested to what degree the calibration characteristics of the pressure transducer might have changed over time after being inserted into the astronaut. Following the spaceflight, the catheter was brought back to the investigators and tested for change in drift of the tip transducer.

Before the spaceflight, four test astronauts would, over a 4.5-h period, empty their urine bladders in either supine or seated posture, on an hourly basis after being infused through a peripheral vein with isotonic saline in an amount of 2% of their body weight. About a week into the flight, the same would be done following a similar infusion while they thus would be free floating in the space vehicle. Blood was sampled on ground and during flight for determination of water-, salt- and blood pressure-regulating hormones. The volume of urine was measured after each void and samples taken for determination of salt (sodium and potassium) concentrations. In space, the volume of urine was determined by a urine monitoring system delivered by NASA, which was connected to the toilet. If the voids were felt by the subjects to be small, the bladders were emptied into bags and returned to

*Central venous pressure equipment, which flew on the Spacelab D2 mission in 1993 [3]. (See text for detailed* 

Shuttle, control measurements in different body postures were performed.

**198**

**Figure 1.**

*explanation).*

back and forth into a rebreathing bag with a gas mixture, in which a tracer gas (e.g. N2O) is taken up by the blood flowing through the lungs. The disappearance rate of the tracer gas from the lungs to blood is detected by an infrared photoacoustic gas analyser connected to the mouthpiece of the rebreathing person. By knowing the solubility of the gas in the blood, the amount of blood flowing through the lungs per unit of time can be calculated. This amount is equal to cardiac output. The measurements take less than 30 s and are pivotal for understanding how cardiac output adapts to various conditions such as weightlessness in space [4].

This methodology is currently being used on the International Space Station for various research projects and has been developed from a mass spectrometry detection technique for measuring gas concentrations to using infrared photoacoustic gas detection. This has made it possible to have a much less voluminous and more user-friendly equipment on board the space station. Also, this technique has been tested against golden standard invasive clinical techniques, where excellent correlations have been found [5].

## **5. Microgravity experiment implementation and execution**

The cardiovascular experiments that we have conducted in space through the past three decades entail the following steps: (1) proposal, (2) selection, (3) funding, (4) feasibility assessment, (5) implementation, (6) execution, (7) data analysis and (8) publication. The unique features about spaceflight experiments are steps 4–6, because of the limitations of conducting experimental procedures in space. Furthermore, all of the associated equipment have to be developed and approved for spaceflight with all the safety aspects taken appropriately into consideration.

### **5.1 Proposal**

The first step for performing experiments in space is to develop a proposal and respond to a space agency solicitation from, e.g. the European Space Agency (ESA) or National Aeronautics and Space Administration (NASA). ESA's topics are usually broader than NASA's, because the latter are mostly focused on operational aspects. In both cases the proposal formats are rather similar and the subsequent selection process very much like the way it is done at the national levels with peer review panels and scientific merit scorings. In order for a proposal to be successful, the following criteria must generally be fulfilled:


One thing to keep in mind when writing a proposal is to formulate it so that non-experts in the field can also get something meaningful out of it, because at the

**201**

the space agencies.

**5.4 Feasibility**

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

time of selection and thereafter, decision-makers who may not be scientists might make a judgement as to the appropriateness of spending resources in space for this

When a proposal is submitted in response to a research announcement, the first step is that it will be evaluated by scientists appointed by the space agency or a group of space agencies. The scientists—or peer reviewers—are usually experts within the field of the topic of the solicitation, who are not involved in collaborations with the proposers. The peer review panels are usually led by an agency representative, and the panel will score each proposal on a scale between zero and 100. Scores between 90 and 100 are categorised as "Excellent" or "Outstanding", 80 and 89 as "Very Good", 70 and 79 as "Good", 50 and 69 as "Fair" and below 50 as "Poor". The score threshold for selection may vary between space agencies, but

The next step for the space agency representatives is to—based on the peer review scores—perform final selections. In this case, not only the scientific merit scores play a role but also the relevancy of the proposal for the agency. Usually a subset of the highest scientifically scoring proposals are selected, but sometimes even proposals with the highest scores may not be finally selected because of less relevancy for the operational purposes of the agency. In this regard, there are different policies between the space agencies. For NASA, deep space explorations are the main drivers, while ESA usually focuses almost entirely on the scientific merit and the proposal's ability to produce new fundamental knowledge to the scientific

In order for a selected proposal to be executed in space, funding has to be obtained. This can either be done by grants from local and national authorities or from the space agency itself. The problem in particular for European researchers is that for ESA to consider selection of a proposal, it is advantageous to have obtained national funding or a declaration of intention of funding already before submission. In many cases, national authorities will only indicate intention of funding, should the proposal be selected, but they usually will not guarantee it. This can sometimes create a hen and an egg problem: The space agency will—before it actually finally selects a proposal—require guaranteed funding from a national authority, whereas the national authority requires that the agency selects the proposal. The proposers usually obtain intention for funding in letters from the national funding authorities, and usually the space agency will accept that. In our case, when the research team was supported for selected experiments, we referred to an existing grant that could

Funding of a grant for experiments in space usually only covers the expenses incurred by the experimental research team. The space infrastructure, such as access to a space vehicle and its astronauts (e.g. the Space Shuttle), is delivered by

When selection and funding are obtained, the space agency will conduct a feasibility study to evaluate whether the experiment can be implemented in space and whether there are technical or other obstacles. If these cannot be overcome,

usually no proposals are selected with a score lower than 70.

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

particular experiment.

**5.2 Selection**

community.

**5.3 Funding**

overlap with selection of a new proposal.

time of selection and thereafter, decision-makers who may not be scientists might make a judgement as to the appropriateness of spending resources in space for this particular experiment.

## **5.2 Selection**

*Preparation of Space Experiments*

tions have been found [5].

**5.1 Proposal**

following criteria must generally be fulfilled:

space research is an advantage but not a requirement.

back and forth into a rebreathing bag with a gas mixture, in which a tracer gas (e.g. N2O) is taken up by the blood flowing through the lungs. The disappearance rate of the tracer gas from the lungs to blood is detected by an infrared photoacoustic gas analyser connected to the mouthpiece of the rebreathing person. By knowing the solubility of the gas in the blood, the amount of blood flowing through the lungs per unit of time can be calculated. This amount is equal to cardiac output. The measurements take less than 30 s and are pivotal for understanding how cardiac output

This methodology is currently being used on the International Space Station for various research projects and has been developed from a mass spectrometry detection technique for measuring gas concentrations to using infrared photoacoustic gas detection. This has made it possible to have a much less voluminous and more user-friendly equipment on board the space station. Also, this technique has been tested against golden standard invasive clinical techniques, where excellent correla-

The cardiovascular experiments that we have conducted in space through the past three decades entail the following steps: (1) proposal, (2) selection, (3) funding, (4) feasibility assessment, (5) implementation, (6) execution, (7) data analysis and (8) publication. The unique features about spaceflight experiments are steps 4–6, because of the limitations of conducting experimental procedures in space. Furthermore, all of the associated equipment have to be developed and approved for spaceflight with all the safety aspects taken appropriately into consideration.

The first step for performing experiments in space is to develop a proposal and respond to a space agency solicitation from, e.g. the European Space Agency (ESA) or National Aeronautics and Space Administration (NASA). ESA's topics are usually broader than NASA's, because the latter are mostly focused on operational aspects. In both cases the proposal formats are rather similar and the subsequent selection process very much like the way it is done at the national levels with peer review panels and scientific merit scorings. In order for a proposal to be successful, the

1.Qualified research team with experimental experience from a university or a recognised company. Most proposals are from universities, and experience in

2.Clearly written proposal, which fulfils the requirements stated in the call.

3.Adherence to all of the rules and instructions—otherwise the proposals will be rejected upon receipt, and this includes adhering to the stated deadlines for

4.Relevancy for utilising spaceflight meaning that what is proposed to be measured should be responsive to the flight environment (e.g. weightlessness).

One thing to keep in mind when writing a proposal is to formulate it so that non-experts in the field can also get something meaningful out of it, because at the

adapts to various conditions such as weightlessness in space [4].

**5. Microgravity experiment implementation and execution**

**200**

submission.

When a proposal is submitted in response to a research announcement, the first step is that it will be evaluated by scientists appointed by the space agency or a group of space agencies. The scientists—or peer reviewers—are usually experts within the field of the topic of the solicitation, who are not involved in collaborations with the proposers. The peer review panels are usually led by an agency representative, and the panel will score each proposal on a scale between zero and 100. Scores between 90 and 100 are categorised as "Excellent" or "Outstanding", 80 and 89 as "Very Good", 70 and 79 as "Good", 50 and 69 as "Fair" and below 50 as "Poor". The score threshold for selection may vary between space agencies, but usually no proposals are selected with a score lower than 70.

The next step for the space agency representatives is to—based on the peer review scores—perform final selections. In this case, not only the scientific merit scores play a role but also the relevancy of the proposal for the agency. Usually a subset of the highest scientifically scoring proposals are selected, but sometimes even proposals with the highest scores may not be finally selected because of less relevancy for the operational purposes of the agency. In this regard, there are different policies between the space agencies. For NASA, deep space explorations are the main drivers, while ESA usually focuses almost entirely on the scientific merit and the proposal's ability to produce new fundamental knowledge to the scientific community.

## **5.3 Funding**

In order for a selected proposal to be executed in space, funding has to be obtained. This can either be done by grants from local and national authorities or from the space agency itself. The problem in particular for European researchers is that for ESA to consider selection of a proposal, it is advantageous to have obtained national funding or a declaration of intention of funding already before submission. In many cases, national authorities will only indicate intention of funding, should the proposal be selected, but they usually will not guarantee it. This can sometimes create a hen and an egg problem: The space agency will—before it actually finally selects a proposal—require guaranteed funding from a national authority, whereas the national authority requires that the agency selects the proposal. The proposers usually obtain intention for funding in letters from the national funding authorities, and usually the space agency will accept that. In our case, when the research team was supported for selected experiments, we referred to an existing grant that could overlap with selection of a new proposal.

Funding of a grant for experiments in space usually only covers the expenses incurred by the experimental research team. The space infrastructure, such as access to a space vehicle and its astronauts (e.g. the Space Shuttle), is delivered by the space agencies.

## **5.4 Feasibility**

When selection and funding are obtained, the space agency will conduct a feasibility study to evaluate whether the experiment can be implemented in space and whether there are technical or other obstacles. If these cannot be overcome,

the proposal will be de-selected. Usually the experiment is modified, if obstacles are detected. An obstacle for implementation may not just be technical such as lack of availability of a technique or equipment but may also be lack of astronaut crew time for execution of the experiment. In that case, the experiment is usually modified in close collaboration with the research team. If the experiment changes considerably, the space agency may require an additional scientific peer review to evaluate whether the scientific quality is still high enough, but this is not the usual process.

## **5.5 Implementation**

When the space agency feasibility assessment has been successfully completed, meaning that the experiment can be conducted in space, the implementation process begins. All of our previous research team's selected experiments (10 in total) have been somehow modified during the feasibility assessment and implementation processes. The renal experiment on the Spacelab D2 mission in 1993 was changed by decreasing the number of inflight sessions from two to one, because of limited crew time. The purpose was as previously described to evaluate the effects of applying an intravenous saline fluid load to the test astronauts on renal excretion rates of fluid and sodium. We had originally planned a session with infusion and a session without, but only the infusion session could be implemented in space. Otherwise the experiment was kept intact except that it was actually improved by changing our proposed saline loading from an oral saltwater load to intravenous infusion. This was done because an experimental complement was created for the space mission, whereby several experiments were to be executed in an integrated fashion, and a US experiment had suggested to use saline loading by infusion. Thus, this intravenous infusion of isotonic saline was planned to be done for the first time ever in space.

Although the urinary experiment was not difficult to implement from a technical point of view, it was totally different regarding the CVP experiment. After selection of this experiment, many managers within ESA and NASA thought it would be impossible to be allowed to conduct such an invasive study. We succeeded anyway, which was because of one important thing: the backing of the appointed payload commander. Without this support for the experiment, it would never have been executed. The reason for the astronaut support was because of thrust in our research group's ability to conduct the study, which we obtained by always being well prepared for pre-flight briefings of the astronauts as well as for the pre-flight control studies. We always made sure that as much of the data that had been collected were properly analysed between the different ground tests and that the data were presented to the astronauts during the subsequent tests so that they together with the investigators could follow the progress of the study throughout the preflight period.

## **5.6 Execution**

During the Spacelab D2 mission, I was standing in the mission control centre in Oberpfaffenhofen near Munich in Germany holding my breath and watching the big screen. All investigators followed the executions of their experiments from the mission control centre, and just before initiation of our renal experiment, which as described earlier was integrated into one complement, a valve was stuck in some of the equipment. If the valve problem was not solved, we would all risk that none of the experiments in the complement would be conducted. It was a tense moment, when the payload commander after directions from mission control finally got the valve to work and initiated the flow of measurements. What a relief!

**203**

from the mission.

developed and built the equipment.

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

During execution of our urinary excretion experiment a mistake did happen, whereby urine bags, which were to be collected after flight directly from the trash can in the Space Shuttle, were not correctly labelled. It meant that we could not readily identify, from which crew members the urine in the bags derived. The way the problem of identification was solved was by measuring the concentrations of five different substances in each urine bag and comparing them to samples that had been collected inflight from each bag before trash storage. Each of the samples had been correctly labelled. Since each crew member had a unique pattern of concentrations of the selected substances, the matching and identification of the bags with

Spaceflight experimentation often requires creative solutions to unexpected

The CVP experiment went well in one astronaut. Originally, we had planned for two astronauts to be instrumented, but unfortunately the catheter broke in one during an extended prelaunch period, where the catheters had been successfully inserted into two astronauts, but where the prelaunch period was extended for 2 days over a weekend because of a failure in one of the shuttle's navigation systems that had to be changed. During some leisure activities, it broke and was withdrawn

During execution of the experiment on ground before the flight, which is called

the baseline data collection to which all inflight data were to be compared, the biggest obstacle occurred during the execution process of one of the pre-flight test sessions on the ground. The obstacle demonstrated that it is not only a challenge to implement and execute an experiment during the flight phase but that ground tests can be limiting factors too. What happened is that the gas analyser used for rebreathing experiments to determine cardiac output and respiratory variables (**Figure 3**) for some reason did not work. Even though these measurements were not directly involved with our urinary experiment, it sent our experiment to jeopardy, because it was totally integrated into an experimental complement to be executed in concert. The breakdown happened at the Aerospace Medical Institute at the German space centre, DLR, and since the astronauts' test time was extremely limited with many other obligations, it was made clear to the experimental team that the astronauts would withdraw from the experiments, if the equipment was

not working the next morning. We knew then that we were in trouble!

mental efforts would go down the drain. What could we do?

We were otherwise all ready to conduct the baseline data collections the next day, and if the gas analyser could not be fixed, it would mean that all of our experi-

Ingenuity, imagination and thinking out of the box are usually essential in solving unexpected problems associated with spaceflight. In fact, this is what characterises this discipline. To my disappointment most of the officials and investigators gave up immediately. It was late afternoon, and the experiments were to be commenced early next morning at 07.00 am. The payload commander had left with a statement that he and his astronaut team would show up on time the next morning, and if the equipment did not work, the experimental complement would be deleted

I and one of our ESA representatives soon conferred with each other, and we promised that we would demonstrate to the payload commander that this problem could be fixed in time. The only question was how? At the time we did not know that the problem was a burned capacitor, so we planned to have a technician immediately flown down from the company, Innovision A/S, in Denmark, which had

What we did was risky, unusual and not according to the normal rules and regulations, but we were running out of time. We had to rent a private airplane

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

the samples were possible.

problems.

before launch.

#### *Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space DOI: http://dx.doi.org/10.5772/intechopen.93466*

During execution of our urinary excretion experiment a mistake did happen, whereby urine bags, which were to be collected after flight directly from the trash can in the Space Shuttle, were not correctly labelled. It meant that we could not readily identify, from which crew members the urine in the bags derived. The way the problem of identification was solved was by measuring the concentrations of five different substances in each urine bag and comparing them to samples that had been collected inflight from each bag before trash storage. Each of the samples had been correctly labelled. Since each crew member had a unique pattern of concentrations of the selected substances, the matching and identification of the bags with the samples were possible.

Spaceflight experimentation often requires creative solutions to unexpected problems.

The CVP experiment went well in one astronaut. Originally, we had planned for two astronauts to be instrumented, but unfortunately the catheter broke in one during an extended prelaunch period, where the catheters had been successfully inserted into two astronauts, but where the prelaunch period was extended for 2 days over a weekend because of a failure in one of the shuttle's navigation systems that had to be changed. During some leisure activities, it broke and was withdrawn before launch.

During execution of the experiment on ground before the flight, which is called the baseline data collection to which all inflight data were to be compared, the biggest obstacle occurred during the execution process of one of the pre-flight test sessions on the ground. The obstacle demonstrated that it is not only a challenge to implement and execute an experiment during the flight phase but that ground tests can be limiting factors too. What happened is that the gas analyser used for rebreathing experiments to determine cardiac output and respiratory variables (**Figure 3**) for some reason did not work. Even though these measurements were not directly involved with our urinary experiment, it sent our experiment to jeopardy, because it was totally integrated into an experimental complement to be executed in concert. The breakdown happened at the Aerospace Medical Institute at the German space centre, DLR, and since the astronauts' test time was extremely limited with many other obligations, it was made clear to the experimental team that the astronauts would withdraw from the experiments, if the equipment was not working the next morning. We knew then that we were in trouble!

We were otherwise all ready to conduct the baseline data collections the next day, and if the gas analyser could not be fixed, it would mean that all of our experimental efforts would go down the drain. What could we do?

Ingenuity, imagination and thinking out of the box are usually essential in solving unexpected problems associated with spaceflight. In fact, this is what characterises this discipline. To my disappointment most of the officials and investigators gave up immediately. It was late afternoon, and the experiments were to be commenced early next morning at 07.00 am. The payload commander had left with a statement that he and his astronaut team would show up on time the next morning, and if the equipment did not work, the experimental complement would be deleted from the mission.

I and one of our ESA representatives soon conferred with each other, and we promised that we would demonstrate to the payload commander that this problem could be fixed in time. The only question was how? At the time we did not know that the problem was a burned capacitor, so we planned to have a technician immediately flown down from the company, Innovision A/S, in Denmark, which had developed and built the equipment.

What we did was risky, unusual and not according to the normal rules and regulations, but we were running out of time. We had to rent a private airplane

*Preparation of Space Experiments*

process.

**5.5 Implementation**

the proposal will be de-selected. Usually the experiment is modified, if obstacles are detected. An obstacle for implementation may not just be technical such as lack of availability of a technique or equipment but may also be lack of astronaut crew time for execution of the experiment. In that case, the experiment is usually modified in close collaboration with the research team. If the experiment changes considerably, the space agency may require an additional scientific peer review to evaluate whether the scientific quality is still high enough, but this is not the usual

When the space agency feasibility assessment has been successfully completed, meaning that the experiment can be conducted in space, the implementation process begins. All of our previous research team's selected experiments (10 in total) have been somehow modified during the feasibility assessment and implementation processes. The renal experiment on the Spacelab D2 mission in 1993 was changed by decreasing the number of inflight sessions from two to one, because of limited crew time. The purpose was as previously described to evaluate the effects of applying an intravenous saline fluid load to the test astronauts on renal excretion rates of fluid and sodium. We had originally planned a session with infusion and a session without, but only the infusion session could be implemented in space. Otherwise the experiment was kept intact except that it was actually improved by changing our proposed saline loading from an oral saltwater load to intravenous infusion. This was done because an experimental complement was created for the space mission, whereby several experiments were to be executed in an integrated fashion, and a US experiment had suggested to use saline loading by infusion. Thus, this intravenous infusion of isotonic saline was planned to be done for the first time ever in space. Although the urinary experiment was not difficult to implement from a techni-

cal point of view, it was totally different regarding the CVP experiment. After selection of this experiment, many managers within ESA and NASA thought it would be impossible to be allowed to conduct such an invasive study. We succeeded anyway, which was because of one important thing: the backing of the appointed payload commander. Without this support for the experiment, it would never have been executed. The reason for the astronaut support was because of thrust in our research group's ability to conduct the study, which we obtained by always being well prepared for pre-flight briefings of the astronauts as well as for the pre-flight control studies. We always made sure that as much of the data that had been collected were properly analysed between the different ground tests and that the data were presented to the astronauts during the subsequent tests so that they together with the investigators could follow the progress of the study throughout the pre-

During the Spacelab D2 mission, I was standing in the mission control centre in Oberpfaffenhofen near Munich in Germany holding my breath and watching the big screen. All investigators followed the executions of their experiments from the mission control centre, and just before initiation of our renal experiment, which as described earlier was integrated into one complement, a valve was stuck in some of the equipment. If the valve problem was not solved, we would all risk that none of the experiments in the complement would be conducted. It was a tense moment, when the payload commander after directions from mission control finally got the

valve to work and initiated the flow of measurements. What a relief!

**202**

flight period.

**5.6 Execution**

#### **Figure 3.**

*This rack was called Anthrorack and developed for the spacelab D2 mission. It consisted of several pieces of equipment of which one was a mass spectrometer gas analyser used for respiratory analysis and cardiac output determinations by rebreathing (Figure 2). During the ground-based data collections, a capacitor was burned and had to be replaced at a very critical time before the mission.*

within a few hours, because there were no commercial flights at that time. We had to establish an ESA guarantee for payment to the airline company, and we—above all—had to get in contact with the technician in Denmark. It soon turned out that he was available and the money for renting the airplane could be secured (after tough negotiations with ESA) and everything seemed possible.

The technician was late at night transported by taxi to a nearby local airport some 200 km away, from where he lived, entered the plane and came to Cologne around 4 am in the morning. We picked him up at the airport in Cologne and brought him to the aerospace facility, and by a miracle, he quickly identified the problem to be a burned capacitor and substituted it by another from a similar equipment.

At exactly 07.00 am before the baseline data collection was to begin, we were ready. The astronauts entered with the expectation that the tests would be cancelled. We could inform them otherwise, and with a rare expression of facial recognition, the payload commander and his fellow astronauts professionally moved ahead to be ready for the tests.

Everything went smoothly!

This as well as the inflight incident with the stuck valve were pivotal obstacles for the outcome of many of the physiological experiments during the Spacelab D2 mission. Had they not been overcome, I would probably not have been able to continue my space physiology career for the next 30 years.

**205**

to Mars.

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

The investigators must also be proactive and tenacious in obtaining the collected data in space that usually are stored on inflight computers. One way to make sure that the data are correctly handled is to push the mission controllers to download as much as possible from space to ground as soon as the data are collected, because no one knows what could happen afterwards to the storage. Usually, it is not a problem should downloading not be possible, but in this case, it can also delay the post-flight analysis of the data because of bureaucratic impedi-

During the very sad and unfortunate accident of the STS-107 mission on February 1, 2003, where the Space Shuttle, Columbia, and crew were lost during re-entry in the atmosphere, I was in charge of an experiment. We conducted inflight cardiac output rebreathing experiments as well as blood pressure monitoring. The data were downloaded to the mission control centre during the mission, which made it possible for us to publish the data so that the experiments—despite the very

During the Euromir 95 mission, which was a long-duration mission on the Russian space station Mir, where the ESA astronaut, Thomas Reiter, stayed for 179 days in space, I was in charge of a urinary collection experiment following an oral water load. I obtained the inflight data directly from Thomas Reiter himself shortly after his flight, which is unusual, but we did so to bypass the bureaucracy.

The most important part of all investigations including those in conjunction with human spaceflights is to publish the results in as widely distributed scientific journals as possible. The whole purpose of obtaining the data is to gain new knowledge, and by publishing in science journals with external peer review, there is a certain guarantee for data quality and interpretation. It usually takes 2 years after the end of a spaceflight mission to have the data published, but many times, it takes longer. The investigators, however, owe it to everybody involved as well as society in

From the Spacelab D2 mission in 1993, our research team succeeded in publishing three papers within 3 years of the mission [1–3]. During later missions on the Russian space station Mir, the Space Shuttle Columbia (STS-107) and the International Space Station, we conducted five additional experiments focusing on how the human cardiovascular system adapts to short- and long-duration flights [6–10]. This is important for understanding the long-duration health effects of future deep space missions that may last up to 3 years on a mission

It is pivotal to make sure that the data collected are correct. One basic rule is for the principal investigator to always be present or to have proper representation at each of the pre- and post-flight experimental sessions and to be monitoring how the data collections are done during flight—preferably from a mission control centre. If that is not made sure of by the investigators of a study, one cannot be sure that the circumstances surrounding the collections are fully understood and that handling of blood samples is done correctly and according to specifications. Furthermore, the investigators have to make sure to be readily available during executions of their experiment should inquiries from space agency representatives need acute responses and interventions. Otherwise, it is unlikely that the data can

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

**5.7 Data analysis**

be trusted.

ments to obtain it.

**5.8 Publication**

sad circumstances—were not done in vain.

Otherwise it could have taken weeks to obtain it.

general to produce a scientific publication as fast as possible.

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space DOI: http://dx.doi.org/10.5772/intechopen.93466*

## **5.7 Data analysis**

*Preparation of Space Experiments*

**204**

**Figure 3.**

equipment.

ahead to be ready for the tests. Everything went smoothly!

*This rack was called Anthrorack and developed for the spacelab D2 mission. It consisted of several pieces of equipment of which one was a mass spectrometer gas analyser used for respiratory analysis and cardiac output determinations by rebreathing (Figure 2). During the ground-based data collections, a capacitor was burned* 

within a few hours, because there were no commercial flights at that time. We had to establish an ESA guarantee for payment to the airline company, and we—above all—had to get in contact with the technician in Denmark. It soon turned out that he was available and the money for renting the airplane could be secured (after tough

The technician was late at night transported by taxi to a nearby local airport some 200 km away, from where he lived, entered the plane and came to Cologne around 4 am in the morning. We picked him up at the airport in Cologne and brought him to the aerospace facility, and by a miracle, he quickly identified the problem to be a burned capacitor and substituted it by another from a similar

At exactly 07.00 am before the baseline data collection was to begin, we were ready. The astronauts entered with the expectation that the tests would be cancelled. We could inform them otherwise, and with a rare expression of facial recognition, the payload commander and his fellow astronauts professionally moved

This as well as the inflight incident with the stuck valve were pivotal obstacles for the outcome of many of the physiological experiments during the Spacelab D2 mission. Had they not been overcome, I would probably not have been able to

*and had to be replaced at a very critical time before the mission.*

negotiations with ESA) and everything seemed possible.

continue my space physiology career for the next 30 years.

It is pivotal to make sure that the data collected are correct. One basic rule is for the principal investigator to always be present or to have proper representation at each of the pre- and post-flight experimental sessions and to be monitoring how the data collections are done during flight—preferably from a mission control centre. If that is not made sure of by the investigators of a study, one cannot be sure that the circumstances surrounding the collections are fully understood and that handling of blood samples is done correctly and according to specifications. Furthermore, the investigators have to make sure to be readily available during executions of their experiment should inquiries from space agency representatives need acute responses and interventions. Otherwise, it is unlikely that the data can be trusted.

The investigators must also be proactive and tenacious in obtaining the collected data in space that usually are stored on inflight computers. One way to make sure that the data are correctly handled is to push the mission controllers to download as much as possible from space to ground as soon as the data are collected, because no one knows what could happen afterwards to the storage. Usually, it is not a problem should downloading not be possible, but in this case, it can also delay the post-flight analysis of the data because of bureaucratic impediments to obtain it.

During the very sad and unfortunate accident of the STS-107 mission on February 1, 2003, where the Space Shuttle, Columbia, and crew were lost during re-entry in the atmosphere, I was in charge of an experiment. We conducted inflight cardiac output rebreathing experiments as well as blood pressure monitoring. The data were downloaded to the mission control centre during the mission, which made it possible for us to publish the data so that the experiments—despite the very sad circumstances—were not done in vain.

During the Euromir 95 mission, which was a long-duration mission on the Russian space station Mir, where the ESA astronaut, Thomas Reiter, stayed for 179 days in space, I was in charge of a urinary collection experiment following an oral water load. I obtained the inflight data directly from Thomas Reiter himself shortly after his flight, which is unusual, but we did so to bypass the bureaucracy. Otherwise it could have taken weeks to obtain it.

#### **5.8 Publication**

The most important part of all investigations including those in conjunction with human spaceflights is to publish the results in as widely distributed scientific journals as possible. The whole purpose of obtaining the data is to gain new knowledge, and by publishing in science journals with external peer review, there is a certain guarantee for data quality and interpretation. It usually takes 2 years after the end of a spaceflight mission to have the data published, but many times, it takes longer. The investigators, however, owe it to everybody involved as well as society in general to produce a scientific publication as fast as possible.

From the Spacelab D2 mission in 1993, our research team succeeded in publishing three papers within 3 years of the mission [1–3]. During later missions on the Russian space station Mir, the Space Shuttle Columbia (STS-107) and the International Space Station, we conducted five additional experiments focusing on how the human cardiovascular system adapts to short- and long-duration flights [6–10]. This is important for understanding the long-duration health effects of future deep space missions that may last up to 3 years on a mission to Mars.

## **6. Parabolic flights**

In preparation of the CVP experiment for the Spacelab D2 mission, we in 1991 participated in a series of ESA-funded parabolic flights at an air base in Bretignysur-Orge, near Paris in France. The purpose of participating in these flights was not only to test the technical feasibility of the equipment in weightlessness but also to obtain short-term data during this condition and compare them to longer effects of spaceflight (see **Figure 4**). At that time, the parabolic flights were conducted by a Caravelle, which flew in a Keplerian trajectory, thereby creating a free fall condition (0 g) symmetrically around the top of the trajectory for 20 s. Some 20 s before and after the 0 g period, the plane underwent a period of increased g's from 1 up to 2. Thus, it is a very short period of weightlessness that is created in this way, but it is the only way to induce real weightlessness in humans without going into actual space.

The CVP equipment was also tested during longer weightless periods (some 60 s) in a fighter airplane (Draken) in Denmark in one of the investigators. This test was supported by the Royal Danish Air Force. All of these tests were conducted in seven subjects (Caravelle) and in an additional one subject (Draken) and demonstrated that the equipment worked during short-term variations in g's between 0 and 4. In addition we obtained data on effects of short-term changes in g-loads on CVP including effects of weightlessness for comparisons with spaceflight.

For further interpretation of the data, we later after completion of the D2 mission performed another series of CVP experiments during 20 s of weightlessness during parabolic flights [11]. In that context, we added measurements of oesophageal pressures through an air tube that was swallowed by the test subject

#### **Figure 4.**

*Dr. Regitze Videbaek measuring the size of the heart chambers in a subject during an ESA parabolic flight campaign. The airplane ascents into a parabolic (Keplerian) trajectory to create weightlessness for 20 s. The subject is also instrumented with invasive monitoring equipment for estimating central venous pressure (CVP), which was also used for the D2 spacelab mission in 1993 on board the space shuttle Columbia [3].*

**207**

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

through the nose for obtaining intrathoracic pressures. Intrathoracic pressures are the pressures surrounding the heart. Those pressures were not measured during the Spacelab D2 mission, so the parabolic flight data helped us interpret the CVP data

The process of getting access to parabolic flights is not very different from getting access to spaceflight. Investigators must usually respond to solicitations put forward by a space agency and go through the scientific selection and feasibility assessment processes. The space agency will supply the investigators with the infrastructure such as the flights, but investigators must find their own funding, which usually also applies for adjustments of the equipment to fit into the airplane. In some cases, investigators will have more direct access to the parabolic flight venue, if their experiments concern technical feasibility assessments for a spaceflight. Obtaining experimental baseline data from these flights for comparisons with space

From the Spacelab D2 mission, our CVP and urinary experiments showed us a new mechanism as to how blood and fluid are shifted from the lower to the upper portions of the body in weightlessness and that the excretion rate of a saline load is not faster than on Earth. Both results were surprising and revealed new insight. Likewise, it was a surprise that the agitating (sympathetic) part of the autonomous nervous system was stimulated during weightlessness and that it was not—as

rest or acute seated head-out water immersion, the opposite is usually seen. Thus, there is a difference in effects of weightlessness in space and the simulation models

Despite the upward blood shift to the heart and head, CVP was measured to decrease in space compared to being horizontal supine on the ground (see **Figure 5**). We had expected it to be increased. The data we obtained were only from one astronaut, but a US-led team during two other missions also measured CVP directly with invasive catheters and found decreases. We thereafter performed a parabolic flight study and measured CVP with same technology as during the D2 mission and found similar acute decreases during the 20 s of weightlessness [11]. However, we also observed that the heart was expanded despite the decreased CVP, because simultaneously the oesophageal pressure also decreased and even more so. From ultrasound images taken of the heart during the parabolic weightless period, we observed an expansion of the cardiac chambers, so the ostensible discrepancy between the decrease in CVP and the expanded heart could be reconciled by the expansion of the thorax that further stretches the heart and gives an erroneous

From our later inflight experiments [6–10] following the Spacelab D2 mission,

• Cardiac output and stroke volume increase by some 35–40% during months of flight in space, which is caused by the weightlessness-induced upward

head-down bed

data can also be allowed at the discretion of the relevant space agency.

expected—supressed. In ground-based simulation studies using 60

impression of the change in its feeding pressure (CVP, **Figure 6**).

the main conclusions can be briefly summarised as follows:

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

**7. Results and conclusions**

**7.2 Subsequent space missions**

fluid shift.

**7.1 Spacelab D2 mission**

on the ground.

from space.

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space DOI: http://dx.doi.org/10.5772/intechopen.93466*

through the nose for obtaining intrathoracic pressures. Intrathoracic pressures are the pressures surrounding the heart. Those pressures were not measured during the Spacelab D2 mission, so the parabolic flight data helped us interpret the CVP data from space.

The process of getting access to parabolic flights is not very different from getting access to spaceflight. Investigators must usually respond to solicitations put forward by a space agency and go through the scientific selection and feasibility assessment processes. The space agency will supply the investigators with the infrastructure such as the flights, but investigators must find their own funding, which usually also applies for adjustments of the equipment to fit into the airplane. In some cases, investigators will have more direct access to the parabolic flight venue, if their experiments concern technical feasibility assessments for a spaceflight. Obtaining experimental baseline data from these flights for comparisons with space data can also be allowed at the discretion of the relevant space agency.

## **7. Results and conclusions**

## **7.1 Spacelab D2 mission**

*Preparation of Space Experiments*

In preparation of the CVP experiment for the Spacelab D2 mission, we in 1991 participated in a series of ESA-funded parabolic flights at an air base in Bretignysur-Orge, near Paris in France. The purpose of participating in these flights was not only to test the technical feasibility of the equipment in weightlessness but also to obtain short-term data during this condition and compare them to longer effects of spaceflight (see **Figure 4**). At that time, the parabolic flights were conducted by a Caravelle, which flew in a Keplerian trajectory, thereby creating a free fall condition (0 g) symmetrically around the top of the trajectory for 20 s. Some 20 s before and after the 0 g period, the plane underwent a period of increased g's from 1 up to 2. Thus, it is a very short period of weightlessness that is created in this way, but it is the only way to induce real weightlessness in humans without going into

The CVP equipment was also tested during longer weightless periods (some 60 s) in a fighter airplane (Draken) in Denmark in one of the investigators. This test was supported by the Royal Danish Air Force. All of these tests were conducted in seven subjects (Caravelle) and in an additional one subject (Draken) and demonstrated that the equipment worked during short-term variations in g's between 0 and 4. In addition we obtained data on effects of short-term changes in g-loads on

CVP including effects of weightlessness for comparisons with spaceflight. For further interpretation of the data, we later after completion of the D2 mission performed another series of CVP experiments during 20 s of weightlessness during parabolic flights [11]. In that context, we added measurements of oesophageal pressures through an air tube that was swallowed by the test subject

*Dr. Regitze Videbaek measuring the size of the heart chambers in a subject during an ESA parabolic flight campaign. The airplane ascents into a parabolic (Keplerian) trajectory to create weightlessness for 20 s. The subject is also instrumented with invasive monitoring equipment for estimating central venous pressure (CVP),* 

*which was also used for the D2 spacelab mission in 1993 on board the space shuttle Columbia [3].*

**6. Parabolic flights**

actual space.

**206**

**Figure 4.**

From the Spacelab D2 mission, our CVP and urinary experiments showed us a new mechanism as to how blood and fluid are shifted from the lower to the upper portions of the body in weightlessness and that the excretion rate of a saline load is not faster than on Earth. Both results were surprising and revealed new insight. Likewise, it was a surprise that the agitating (sympathetic) part of the autonomous nervous system was stimulated during weightlessness and that it was not—as expected—supressed. In ground-based simulation studies using 60 head-down bed rest or acute seated head-out water immersion, the opposite is usually seen. Thus, there is a difference in effects of weightlessness in space and the simulation models on the ground.

Despite the upward blood shift to the heart and head, CVP was measured to decrease in space compared to being horizontal supine on the ground (see **Figure 5**). We had expected it to be increased. The data we obtained were only from one astronaut, but a US-led team during two other missions also measured CVP directly with invasive catheters and found decreases. We thereafter performed a parabolic flight study and measured CVP with same technology as during the D2 mission and found similar acute decreases during the 20 s of weightlessness [11]. However, we also observed that the heart was expanded despite the decreased CVP, because simultaneously the oesophageal pressure also decreased and even more so. From ultrasound images taken of the heart during the parabolic weightless period, we observed an expansion of the cardiac chambers, so the ostensible discrepancy between the decrease in CVP and the expanded heart could be reconciled by the expansion of the thorax that further stretches the heart and gives an erroneous impression of the change in its feeding pressure (CVP, **Figure 6**).

#### **7.2 Subsequent space missions**

From our later inflight experiments [6–10] following the Spacelab D2 mission, the main conclusions can be briefly summarised as follows:

• Cardiac output and stroke volume increase by some 35–40% during months of flight in space, which is caused by the weightlessness-induced upward fluid shift.

#### **Figure 5.**

*Central venous pressure (in mm Hg) as a function of time in one astronaut before launch of the space shuttle in the suit room with the space suit on and in the shuttle on the launch pad in the supine leg-up position. (A) Closing of the helmet visor. (B) ignition. (C) Release of the solid rocket boosters from the ascending shuttle. (D) Entering weightlessness. The g-load (G) is indicated at the bottom [3].*

#### **Figure 6.**

*The parabolic flight experiment which helped us interpret the Spacelab D2 mission data. Central venous pressure (CVP) was measured directly with long catheters with transducers at their tip placed near to the heart chambers in supine subjects during the parabolic manoeuvre. Simultaneously, the intrathoracic pressures (IPP) were also measured through long air-filled tubes with balloons at the end in the oesophagus. By subtracting IPP from CVP, the transmural heart distension pressure (tCVP) can be estimated. As can be seen, the tCVP increased in weightlessness (0 G) by 4.3 mm Hg (Delta) even though CVP fell by 1.3. Thus, parabolic flight data could help interpret those obtained during spaceflight [3, 11].*


**209**

**Figure 7.**

lation (**Figures 6** and **7**).

**Acknowledgements**

Health (TRISH).

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

Thus, to our experience, experiments in space have revealed some new insight and mechanisms into human physiology, which could be of importance in interpreting the health consequences of long-duration flights in the future. By comparing the effects of long-duration (3–6 months) spaceflight on the International Space Station [12] with those of short-term shuttle flights [8], there are at least two important and surprising observations: (1) The shift of blood and fluid from the lower body segments into the heart, which increases cardiac output, is even bigger, and (2) blood pressure is more decreased by a more pronounced peripheral vasodi-

*During missions to the international Space Station in the period of 2006 to 2012, we conducted measurements of cardiac output by a non-invasive rebreathing method in eight astronauts and found a clear-cut increase in some 35% between the 3rd and 6th month in space [10]. At the same time, blood pressure is decreased, which indicates that the total vascular resistance is decreased by almost 40%. In contradiction to this, noradrenaline levels are not suppressed but maintained unchanged from ground-based upright levels. Thus, the mechanism of* 

*chronic peripheral vasodilatation in space is still unknown [10, 12].*

This paper was supported by the NNX16A069A, NASA Cooperative Agreement to Baylor College of Medicine for the Translational Research Institute for Space

The help and dedication of Mr. Poul Knudsen, technician at Innovision, is deeply

appreciated for the repair of the faulty component described in Section 5.6.

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

• The dilatation of the arteries and the high sympathetic nervous activity is in contradiction to each other, and the mechanism is not yet known (**Figure 7**).

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space DOI: http://dx.doi.org/10.5772/intechopen.93466*

#### **Figure 7.**

*Preparation of Space Experiments*

**208**

**Figure 6.**

**Figure 5.**

(**Figure 7**).

• The arterial resistance vessels dilate and decrease the circulatory resistance by

*The parabolic flight experiment which helped us interpret the Spacelab D2 mission data. Central venous pressure (CVP) was measured directly with long catheters with transducers at their tip placed near to the heart chambers in supine subjects during the parabolic manoeuvre. Simultaneously, the intrathoracic pressures (IPP) were also measured through long air-filled tubes with balloons at the end in the oesophagus. By subtracting IPP from CVP, the transmural heart distension pressure (tCVP) can be estimated. As can be seen, the tCVP increased in weightlessness (0 G) by 4.3 mm Hg (Delta) even though CVP fell by 1.3. Thus, parabolic flight* 

*Central venous pressure (in mm Hg) as a function of time in one astronaut before launch of the space shuttle in the suit room with the space suit on and in the shuttle on the launch pad in the supine leg-up position. (A) Closing of the helmet visor. (B) ignition. (C) Release of the solid rocket boosters from the ascending shuttle.* 

• The sympathetic nervous activity is not decreased in space and is at the level of being upright on ground, which is supported by the attenuated urinary excre-

• The dilatation of the arteries and the high sympathetic nervous activity is in contradiction to each other, and the mechanism is not yet known

some 40% and blood pressure by 10 mm Hg.

*(D) Entering weightlessness. The g-load (G) is indicated at the bottom [3].*

*data could help interpret those obtained during spaceflight [3, 11].*

tion rates of fluid and sodium.

*During missions to the international Space Station in the period of 2006 to 2012, we conducted measurements of cardiac output by a non-invasive rebreathing method in eight astronauts and found a clear-cut increase in some 35% between the 3rd and 6th month in space [10]. At the same time, blood pressure is decreased, which indicates that the total vascular resistance is decreased by almost 40%. In contradiction to this, noradrenaline levels are not suppressed but maintained unchanged from ground-based upright levels. Thus, the mechanism of chronic peripheral vasodilatation in space is still unknown [10, 12].*

Thus, to our experience, experiments in space have revealed some new insight and mechanisms into human physiology, which could be of importance in interpreting the health consequences of long-duration flights in the future. By comparing the effects of long-duration (3–6 months) spaceflight on the International Space Station [12] with those of short-term shuttle flights [8], there are at least two important and surprising observations: (1) The shift of blood and fluid from the lower body segments into the heart, which increases cardiac output, is even bigger, and (2) blood pressure is more decreased by a more pronounced peripheral vasodilation (**Figures 6** and **7**).

## **Acknowledgements**

This paper was supported by the NNX16A069A, NASA Cooperative Agreement to Baylor College of Medicine for the Translational Research Institute for Space Health (TRISH).

The help and dedication of Mr. Poul Knudsen, technician at Innovision, is deeply appreciated for the repair of the faulty component described in Section 5.6.

*Preparation of Space Experiments*

## **Author details**

Peter Norsk

Center for Space Medicine and Department Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, USA

\*Address all correspondence to: norsk@bcm.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**211**

2007;**103**:959-962

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space*

[10] Norsk P, Asmar A, Damgaard M, Christensen NJ. Fluid shifts, vasodilation and ambulatory blood pressure reduction during long duration spaceflight. The Journal of Physiology. 2015;**593**:573-584

[11] Videbaek R, Norsk P. Atrial distension in humans during microgravity induced by parabolic flights. Journal of Applied Physiology.

[12] Norsk P. Adaptation of the

for deep space missions. Acta Physiologica (Oxford, England). 2020;**228**(3):e13434. DOI: 10.1111/

cardiovascular system to weightlessness: Surprises, paradoxes and implications

1997;**83**:1862-1866

apha.13434

*DOI: http://dx.doi.org/10.5772/intechopen.93466*

[1] Norsk P, Drummer C, Rocker L, et al. Renal and endocrine responses in humans to isotonic saline infusion during microgravity. Journal of Applied

[2] Gabrielsen A, Norsk P, Videbaek R, Henriksen O. Effect of microgravity on forearm subcutaneous vascular resistance in humans. Journal of Applied

[3] Foldager N, Andersen TAE, Jessen FB, et al. Central venous pressure in humans during microgravity. Journal of Applied

[4] Clemensen P, Christensen P, Norsk P, Groenlund J. A modified photo- and magnetoacoustic multigas analyzer applied in gas exchange measurements.

[5] Gabrielsen A, Videbaek R, Schou M, Damgaard M, Kastrup J, Norsk P. Noninvasive measurement of cardiac output in heart failure patients using a new foreign gas rebreathing technique. Clinical Science. 2002;**102**:247-252

[6] Norsk P, Christensen NJ, Bie P, Gabrielsen A, Heer M, Drummer C. Unexpected renal responses in space. The Lancet. 2000;**356**:1577-1578

[7] Christensen NJ, Heer M, Ivanova K, Norsk P. Sympathetic nervous activity decreases during head-down bed rest but not during microgravity. Journal of Applied Physiology. 2005;**99**:1552-1557

[8] Norsk P, Damgaard M, Petersen L,

et al. Vasorelaxation in space. Hypertension. 2006;**47**:69-73

[9] Gabrielsen A, Norsk P. Effect of spaceflight on the subcutaneous venoarteriolar reflex in the human lower leg. Journal of Applied Physiology.

Physiology. 1995;**78**:2253-2259

Physiology. 1995;**79**:434-438

Physiology. 1996;**81**:408-412

Journal of Applied Physiology.

1994;**76**:2832-2839

**References**

*Preparation, Implementation and Execution of Human Cardiovascular Experiments in Space DOI: http://dx.doi.org/10.5772/intechopen.93466*

## **References**

*Preparation of Space Experiments*

**210**

**Author details**

Center for Space Medicine and Department Molecular Physiology and Biophysics,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Baylor College of Medicine, Houston, Texas, USA

\*Address all correspondence to: norsk@bcm.edu

provided the original work is properly cited.

Peter Norsk

[1] Norsk P, Drummer C, Rocker L, et al. Renal and endocrine responses in humans to isotonic saline infusion during microgravity. Journal of Applied Physiology. 1995;**78**:2253-2259

[2] Gabrielsen A, Norsk P, Videbaek R, Henriksen O. Effect of microgravity on forearm subcutaneous vascular resistance in humans. Journal of Applied Physiology. 1995;**79**:434-438

[3] Foldager N, Andersen TAE, Jessen FB, et al. Central venous pressure in humans during microgravity. Journal of Applied Physiology. 1996;**81**:408-412

[4] Clemensen P, Christensen P, Norsk P, Groenlund J. A modified photo- and magnetoacoustic multigas analyzer applied in gas exchange measurements. Journal of Applied Physiology. 1994;**76**:2832-2839

[5] Gabrielsen A, Videbaek R, Schou M, Damgaard M, Kastrup J, Norsk P. Noninvasive measurement of cardiac output in heart failure patients using a new foreign gas rebreathing technique. Clinical Science. 2002;**102**:247-252

[6] Norsk P, Christensen NJ, Bie P, Gabrielsen A, Heer M, Drummer C. Unexpected renal responses in space. The Lancet. 2000;**356**:1577-1578

[7] Christensen NJ, Heer M, Ivanova K, Norsk P. Sympathetic nervous activity decreases during head-down bed rest but not during microgravity. Journal of Applied Physiology. 2005;**99**:1552-1557

[8] Norsk P, Damgaard M, Petersen L, et al. Vasorelaxation in space. Hypertension. 2006;**47**:69-73

[9] Gabrielsen A, Norsk P. Effect of spaceflight on the subcutaneous venoarteriolar reflex in the human lower leg. Journal of Applied Physiology. 2007;**103**:959-962

[10] Norsk P, Asmar A, Damgaard M, Christensen NJ. Fluid shifts, vasodilation and ambulatory blood pressure reduction during long duration spaceflight. The Journal of Physiology. 2015;**593**:573-584

[11] Videbaek R, Norsk P. Atrial distension in humans during microgravity induced by parabolic flights. Journal of Applied Physiology. 1997;**83**:1862-1866

[12] Norsk P. Adaptation of the cardiovascular system to weightlessness: Surprises, paradoxes and implications for deep space missions. Acta Physiologica (Oxford, England). 2020;**228**(3):e13434. DOI: 10.1111/ apha.13434

**Chapter 11**

**Abstract**

**1. Introduction**

**213**

Spaceflight

*Tricia L. Larose*

Tumors in Space: Preparation for

Tumors in Space is a cutting-edge cancer research experiment at the intersection of stem-cell biology and space technology selected by the United Nations Office for Outer Space Affairs and the China Manned Space Agency for a 31-day space mission on board the China Space Station. Anchored in Norway, Tumors in Space includes an international team of exceptional scientists at several European partner organizations including the University of Oslo and the Norwegian University of Science and Technology in Norway, the International Space University in France, the Belgian Nuclear Research Center in Belgium, and Vrije University Amsterdam as well as the Hubrecht Institute in The Netherlands. This chapter first presents our two novel hypotheses including the current state of scientific evidence upon which our hypotheses are based. Following, the seven main steps of our spaceflight preparation are discussed within the context of our 2025 launch date from China. Finally, some thoughts on impact, including support for the United Nation's Sustainable Development Goals and commitment to science communication in the public domain, are given. Tumors is Space is under a programme of, and funded by the European Space Agency with the support of the Norwegian Space Agency.

**Keywords:** China Space Station, cancer, organoids, microgravity, cosmic radiation

Spaceflight is a high-risk/high-gain endeavor (**Figure 1**) that is resource intensive. For these reasons, spaceflight experiments are rare, and selection is highly competitive at the international level. Selection criteria for spaceflight experiments include scientific excellence, innovation, feasibility, impact, competency of the scientific team to achieve the proposed research goals, and timely delivery. Moreover, a selected spaceflight experiment must be novel in that it must address a critical issue for which knowledge and understanding are lacking; it must be ground-breaking in nature, both literally and figuratively. In the case of medical research in space, a selected spaceflight experiment must create value for astro-/ cosmo-/taikonaut health applied to short- and long-duration space missions and solar system exploration. At the same time, space medicine must also bring value to public health on Earth. In this vein,*Tumors in Space* is a ground-breaking cancer research experiment at the intersection of space technology and stem-cell biology recently selected for a 31-day space mission on board the China Space Station (CSS). Not only will *Tumors in Space* address cosmic radiation as a potential cause of cancer for humans in space, it will also address the side effects of radiation therapy

## **Chapter 11**

## Tumors in Space: Preparation for Spaceflight

*Tricia L. Larose*

## **Abstract**

Tumors in Space is a cutting-edge cancer research experiment at the intersection of stem-cell biology and space technology selected by the United Nations Office for Outer Space Affairs and the China Manned Space Agency for a 31-day space mission on board the China Space Station. Anchored in Norway, Tumors in Space includes an international team of exceptional scientists at several European partner organizations including the University of Oslo and the Norwegian University of Science and Technology in Norway, the International Space University in France, the Belgian Nuclear Research Center in Belgium, and Vrije University Amsterdam as well as the Hubrecht Institute in The Netherlands. This chapter first presents our two novel hypotheses including the current state of scientific evidence upon which our hypotheses are based. Following, the seven main steps of our spaceflight preparation are discussed within the context of our 2025 launch date from China. Finally, some thoughts on impact, including support for the United Nation's Sustainable Development Goals and commitment to science communication in the public domain, are given. Tumors is Space is under a programme of, and funded by the European Space Agency with the support of the Norwegian Space Agency.

**Keywords:** China Space Station, cancer, organoids, microgravity, cosmic radiation

## **1. Introduction**

Spaceflight is a high-risk/high-gain endeavor (**Figure 1**) that is resource intensive. For these reasons, spaceflight experiments are rare, and selection is highly competitive at the international level. Selection criteria for spaceflight experiments include scientific excellence, innovation, feasibility, impact, competency of the scientific team to achieve the proposed research goals, and timely delivery. Moreover, a selected spaceflight experiment must be novel in that it must address a critical issue for which knowledge and understanding are lacking; it must be ground-breaking in nature, both literally and figuratively. In the case of medical research in space, a selected spaceflight experiment must create value for astro-/ cosmo-/taikonaut health applied to short- and long-duration space missions and solar system exploration. At the same time, space medicine must also bring value to public health on Earth. In this vein,*Tumors in Space* is a ground-breaking cancer research experiment at the intersection of space technology and stem-cell biology recently selected for a 31-day space mission on board the China Space Station (CSS).

Not only will *Tumors in Space* address cosmic radiation as a potential cause of cancer for humans in space, it will also address the side effects of radiation therapy

Interspersed throughout this section are some details on our scientific approach,

The cancer burden on Earth is substantial. Currently, cancer is the second leading cause of death worldwide [1]. As of 2018, an estimated 18.1 million people had been diagnosed with cancer, and nearly 10 million people had died from the disease [2]. Moreover, new cancer cases are expected to increase by 70% over the next two decades [1]. As of 2009, the global cost of treating cancer patients was estimated to be \$285.8 billion USD per year, and the indirect costs associated with premature death and loss of productivity was estimated at \$1.16 trillion USD per

The scientific community has made increasing progress toward understanding lifestyle, environmental, and genetic causes of cancer [3], yet approximately 50% of

In space, radiation risk is the most dangerous issue for crew health and safety.

) ions from three natural sources of radiation that could lead to crew sickness or death [5]. During space exploration missions, crew are exposed to HZE ions from trapped particle belts, solar particle events, and background galactic cosmic rays [5]. When primary HZE ions interact with spacecraft materials, they can generate harmful secondary radiation that can penetrate deeply into the human body. Cancer risk from human spaceflight and exploration is assumed to be high, but the shortand long-term effects of harmful HZE ion radiation during deep space missions are still undetermined and have large uncertainties [5]. As stated by the European Space Agency (ESA), most of this uncertainty is due to poor knowledge on the effects of galactic cosmic rays [6]. *Tumors in Space* will address some of the uncertainty associated with cosmic radiation as a potential cause of cancer among crew. At the same time, advances in space-based cancer research have the potential to improve charged particle therapy in oncology for the benefit of cancer patients on

The spaceflight environment is characterized by high atomic number energy

All cancers are caused by somatic mutations, meaning an accumulation of genetic alterations that damage our deoxyribonucleic acid (DNA) at the cellular level [7]. Cell damage from exogenous exposures (e.g., HZE ion radiation), as well as endogenous exposures (e.g., circulating hormones), and modifications to DNA can lead to cancer. DNA damage in cancerous cells can be observed as distinct patterns known as mutational signatures. Just like fingerprints are unique to every individual, mutational signatures are unique to specific cancer-causing exposures. Each mutational signature is the outcome of a series of biological processes that include DNA modification or damage, DNA repair or absence of repair, and DNA replication. Using cancer genome sequencing [8], these mutational signatures can be observed, catalogued, and compared to identify unique causes of different cancer types. This was first demonstrated when mutational signatures specific to UV<sup>2</sup>

<sup>1</sup> The abbreviation HZE comes from high (H) atomic number (Z) and energy (E).

<sup>2</sup> UV stands for ultraviolet rays from sunlight that induces a unique mutation found specifically in skin

all cancer cases have no known cause. There is no cure for cancer. We must therefore adopt a broad, interdisciplinary perspective and exploit all scientific and technical capabilities to better understand this disease, including the use of orbital platforms [4]. *Tumors in Space* will exploit the natural spaceflight environment on the China Space Station to address cosmic radiation as a potential unknown cause of cancer among crew and to harness microgravity as a potential source for cancer

more of which will be detailed later in this chapter.

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

year [2]. Over time, these costs will continue to rise.

therapy in space and on Earth.

(HZE<sup>1</sup>

Earth.

cancer.

**215**

#### **Figure 1.** *Spaceflight is high risk and high gain.*

for cancer patients on Earth. In addition to radiation biology experiments,*Tumors in Space* will exploit the microgravity environment on the CSS to investigate whether lack of gravity can slow or stop the growth of cancer. Like all selected spaceflight experiments,*Tumors in Space* will undergo several years of preparation before spaceflight launch. This preparation period is broken down into seven main steps over 6 years, culminating in our spaceflight experiment scheduled for a 2025 launch date out of China. This chapter will discuss the different steps of our spaceflight preparation as depicted in **Figure 2**.

#### **Figure 2.**

Tumors in Space *project timeline over a 6-year period from 2020 to 2025 and according to seven steps of spaceflight preparation including launch to China Space Station and safe return to Earth in 2025.*

In addition to these seven steps, other important aspects of *Tumors in Space* will be presented, namely, the scientific background upon which the hypotheses are built, team structure and expertise, the importance of impact including response to the United Nations Sustainable Development Goals, and science communication in the public domain. Let us first begin with a presentation of the problem statement and the supporting evidence upon which the *Tumors in Space* hypotheses are built.

## **2. The problem statement and supporting evidence**

As stated in Section 1, medical experiments in space must address crew health and safety while simultaneously bringing value to medicine on Earth. What follows is a short paragraph on the burden of cancer on Earth, followed by a second paragraph on space radiation risk for crewed missions. To end this section, evidence on the potential use of microgravity for cancer therapeutics will be given.

## *Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

Interspersed throughout this section are some details on our scientific approach, more of which will be detailed later in this chapter.

The cancer burden on Earth is substantial. Currently, cancer is the second leading cause of death worldwide [1]. As of 2018, an estimated 18.1 million people had been diagnosed with cancer, and nearly 10 million people had died from the disease [2]. Moreover, new cancer cases are expected to increase by 70% over the next two decades [1]. As of 2009, the global cost of treating cancer patients was estimated to be \$285.8 billion USD per year, and the indirect costs associated with premature death and loss of productivity was estimated at \$1.16 trillion USD per year [2]. Over time, these costs will continue to rise.

The scientific community has made increasing progress toward understanding lifestyle, environmental, and genetic causes of cancer [3], yet approximately 50% of all cancer cases have no known cause. There is no cure for cancer. We must therefore adopt a broad, interdisciplinary perspective and exploit all scientific and technical capabilities to better understand this disease, including the use of orbital platforms [4]. *Tumors in Space* will exploit the natural spaceflight environment on the China Space Station to address cosmic radiation as a potential unknown cause of cancer among crew and to harness microgravity as a potential source for cancer therapy in space and on Earth.

In space, radiation risk is the most dangerous issue for crew health and safety. The spaceflight environment is characterized by high atomic number energy (HZE<sup>1</sup> ) ions from three natural sources of radiation that could lead to crew sickness or death [5]. During space exploration missions, crew are exposed to HZE ions from trapped particle belts, solar particle events, and background galactic cosmic rays [5]. When primary HZE ions interact with spacecraft materials, they can generate harmful secondary radiation that can penetrate deeply into the human body. Cancer risk from human spaceflight and exploration is assumed to be high, but the shortand long-term effects of harmful HZE ion radiation during deep space missions are still undetermined and have large uncertainties [5]. As stated by the European Space Agency (ESA), most of this uncertainty is due to poor knowledge on the effects of galactic cosmic rays [6]. *Tumors in Space* will address some of the uncertainty associated with cosmic radiation as a potential cause of cancer among crew. At the same time, advances in space-based cancer research have the potential to improve charged particle therapy in oncology for the benefit of cancer patients on Earth.

All cancers are caused by somatic mutations, meaning an accumulation of genetic alterations that damage our deoxyribonucleic acid (DNA) at the cellular level [7]. Cell damage from exogenous exposures (e.g., HZE ion radiation), as well as endogenous exposures (e.g., circulating hormones), and modifications to DNA can lead to cancer. DNA damage in cancerous cells can be observed as distinct patterns known as mutational signatures. Just like fingerprints are unique to every individual, mutational signatures are unique to specific cancer-causing exposures. Each mutational signature is the outcome of a series of biological processes that include DNA modification or damage, DNA repair or absence of repair, and DNA replication. Using cancer genome sequencing [8], these mutational signatures can be observed, catalogued, and compared to identify unique causes of different cancer types. This was first demonstrated when mutational signatures specific to UV<sup>2</sup>

for cancer patients on Earth. In addition to radiation biology experiments,*Tumors in Space* will exploit the microgravity environment on the CSS to investigate whether lack of gravity can slow or stop the growth of cancer. Like all selected spaceflight experiments,*Tumors in Space* will undergo several years of preparation before spaceflight launch. This preparation period is broken down into seven main steps over 6 years, culminating in our spaceflight experiment scheduled for a 2025 launch date out of China. This chapter will discuss the different steps of our spaceflight

In addition to these seven steps, other important aspects of *Tumors in Space* will be presented, namely, the scientific background upon which the hypotheses are built, team structure and expertise, the importance of impact including response to the United Nations Sustainable Development Goals, and science communication in the public domain. Let us first begin with a presentation of the problem statement and the supporting evidence upon which the *Tumors in Space*

Tumors in Space *project timeline over a 6-year period from 2020 to 2025 and according to seven steps of spaceflight preparation including launch to China Space Station and safe return to Earth in 2025.*

As stated in Section 1, medical experiments in space must address crew health and safety while simultaneously bringing value to medicine on Earth. What follows

paragraph on space radiation risk for crewed missions. To end this section, evidence

is a short paragraph on the burden of cancer on Earth, followed by a second

on the potential use of microgravity for cancer therapeutics will be given.

**2. The problem statement and supporting evidence**

preparation as depicted in **Figure 2**.

*Spaceflight is high risk and high gain.*

*Preparation of Space Experiments*

**Figure 1.**

**Figure 2.**

**214**

hypotheses are built.

<sup>1</sup> The abbreviation HZE comes from high (H) atomic number (Z) and energy (E).

<sup>2</sup> UV stands for ultraviolet rays from sunlight that induces a unique mutation found specifically in skin cancer.

exposure from the sun were observed in the p53 tumor suppressor gene<sup>3</sup> [9]. Most cancer genomes are now characterized by multiple mutational signatures across multiple mutational processes that generate more than one somatic mutation in each cancer cell [10]. This evidence leads us to our first *Tumors in Space* hypothesis.

expect to find a unique mutational signature of cosmic radiation. We also expect that cancer organoid growth will slow or stop after exposure to microgravity.

As mentioned previously, excellent science in line with the current state of evidence does not fulfill the stringent selection criteria for spaceflight experiments. Space medicine research must be novel, go beyond current scientific paradigms, and have application to crew health in space and public health on Earth. In other words, selected space medicine experiments must go beyond state of the art. Not only does *Tumors in Space* more than double the mission length of all previous cancer experiments in space, we also use the most advanced and physiologically relevant bio-samples in our experiments—human organoids. Until now the term "organoids" has been introduced without explanation. In fact, organoid technology is so new that many scientists are unfamiliar with the term. So, what exactly is an organoid, and why is an organoid so much more advanced than a cell line that

Human organoids are multicellular, stem-cell-derived, 3D constructs that selforganize to mimic the structure and function of the source tissue [15]. More simply, a human organoid is a 3D clump of human cells and extracellular matrix, taken as a biopsy from living human tissue, but organoids "live" in a lab. What is amazing about organoids is that they can be expanded, cloned and cared for in a way that allows them to replicate and grow while retaining the structure and function of the original source tissue or organ. For example, a colon cancer organoid derived from a tumor biopsy taken (with permission) from a colon cancer patient will not only grow into a 3D structure under laboratory conditions; it will also behave like we

**Figure 4** shows an artist rendition of the growth of a colon organoid. A small sample, or biopsy, is taken from the colon tissue of a patient. Both the original colon tissue and the biopsy are 3D in nature. Once the biopsy is removed from the patient, it can be nurtured in the lab so that cell division continues, resulting in larger and

would expect colon cancer to behave. Same goes for healthy tissue.

*Artist rendition of colon organoid growth (credit: Hans Clevers, the HUB).*

**3. Going beyond state of the art**

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

is typically used in medical research?

**Figure 4.**

**217**

Hypothesis 1: We will identify a novel mutational signature of cosmic radiation in healthy and cancer human organoids after 31-days exposure to cosmic radiation in space. The mutational signature of cosmic radiation can then be used as a biological marker for early detection of cancer and cancer susceptibility among spaceflight crew.

Beyond cosmic radiation, the space environment provides unique microgravity conditions that Earth-bound laboratories can simulate but never replicate. Microgravity can be exploited to study biological mechanisms and pathways to improve our understanding of many diseases, including cancer. Previous spaceflight experiments have shown that exposure to the space environment alters immune cell growth and function [11], causes cytoskeleton and cell shape changes [12], and alters cellular gene expression including genes involved in replication and suppression of tumors [13, 14]. This brings us to our second *Tumors in Space* hypothesis.

Hypothesis 2: Exposure to microgravity during a 31-day space mission will slow or stop the growth of cancer.

We will conduct a tumor colony survival analysis which is as simple as counting the number and/or expansion of our organoids after long-term exposure to microgravity and then compare those results to our ground-based controls. It is crucial to block out cosmic radiation that may interfere with this microgravity experimental arm. For that reason, we are building radiation shielding to ensure the cancer organoids exposed to microgravity are not hit by cosmic radiation.

So, the China Manned Space Agency (CMSA) in collaboration with the United Nations Office for Outer Space Affairs has selected *Tumors in Space* for a 31-day space mission on board the China Space Station (**Figure 3**). *Tumors in Space* utilizes space technology and three-dimensional (3D) organoid technology (see further) to study early mutational events in human DNA due to spaceflight exposure. We

**Figure 3.** *Artist rendition of the China Space Station (credit: China Manned Space Agency).*

<sup>3</sup> The p53 gene provides instructions for building a protein that acts as a tumor suppressor. Wellcharacterized mutations in this gene have been attributed to UV exposure and are found in many types of skin cancer.

expect to find a unique mutational signature of cosmic radiation. We also expect that cancer organoid growth will slow or stop after exposure to microgravity.

## **3. Going beyond state of the art**

exposure from the sun were observed in the p53 tumor suppressor gene<sup>3</sup> [9]. Most cancer genomes are now characterized by multiple mutational signatures across multiple mutational processes that generate more than one somatic mutation in each cancer cell [10]. This evidence leads us to our first *Tumors in Space* hypothesis. Hypothesis 1: We will identify a novel mutational signature of cosmic radiation in healthy and cancer human organoids after 31-days exposure to cosmic radiation in space. The mutational signature of cosmic radiation can then be used as a biological marker for early detection of cancer and cancer susceptibility among

Beyond cosmic radiation, the space environment provides unique microgravity conditions that Earth-bound laboratories can simulate but never replicate. Microgravity can be exploited to study biological mechanisms and pathways to improve our understanding of many diseases, including cancer. Previous spaceflight experiments have shown that exposure to the space environment alters immune cell growth and function [11], causes cytoskeleton and cell shape changes [12], and alters cellular gene expression including genes involved in replication and suppression of tumors [13, 14]. This brings us to our second *Tumors in Space* hypothesis. Hypothesis 2: Exposure to microgravity during a 31-day space mission will slow

We will conduct a tumor colony survival analysis which is as simple as counting the number and/or expansion of our organoids after long-term exposure to microgravity and then compare those results to our ground-based controls. It is crucial to block out cosmic radiation that may interfere with this microgravity experimental arm. For that reason, we are building radiation shielding to ensure the cancer

So, the China Manned Space Agency (CMSA) in collaboration with the United Nations Office for Outer Space Affairs has selected *Tumors in Space* for a 31-day space mission on board the China Space Station (**Figure 3**). *Tumors in Space* utilizes space technology and three-dimensional (3D) organoid technology (see further) to study early mutational events in human DNA due to spaceflight exposure. We

organoids exposed to microgravity are not hit by cosmic radiation.

*Artist rendition of the China Space Station (credit: China Manned Space Agency).*

<sup>3</sup> The p53 gene provides instructions for building a protein that acts as a tumor suppressor. Wellcharacterized mutations in this gene have been attributed to UV exposure and are found in many types

spaceflight crew.

**Figure 3.**

of skin cancer.

**216**

or stop the growth of cancer.

*Preparation of Space Experiments*

As mentioned previously, excellent science in line with the current state of evidence does not fulfill the stringent selection criteria for spaceflight experiments. Space medicine research must be novel, go beyond current scientific paradigms, and have application to crew health in space and public health on Earth. In other words, selected space medicine experiments must go beyond state of the art.

Not only does *Tumors in Space* more than double the mission length of all previous cancer experiments in space, we also use the most advanced and physiologically relevant bio-samples in our experiments—human organoids. Until now the term "organoids" has been introduced without explanation. In fact, organoid technology is so new that many scientists are unfamiliar with the term. So, what exactly is an organoid, and why is an organoid so much more advanced than a cell line that is typically used in medical research?

Human organoids are multicellular, stem-cell-derived, 3D constructs that selforganize to mimic the structure and function of the source tissue [15]. More simply, a human organoid is a 3D clump of human cells and extracellular matrix, taken as a biopsy from living human tissue, but organoids "live" in a lab. What is amazing about organoids is that they can be expanded, cloned and cared for in a way that allows them to replicate and grow while retaining the structure and function of the original source tissue or organ. For example, a colon cancer organoid derived from a tumor biopsy taken (with permission) from a colon cancer patient will not only grow into a 3D structure under laboratory conditions; it will also behave like we would expect colon cancer to behave. Same goes for healthy tissue.

**Figure 4** shows an artist rendition of the growth of a colon organoid. A small sample, or biopsy, is taken from the colon tissue of a patient. Both the original colon tissue and the biopsy are 3D in nature. Once the biopsy is removed from the patient, it can be nurtured in the lab so that cell division continues, resulting in larger and

**Figure 4.** *Artist rendition of colon organoid growth (credit: Hans Clevers, the HUB).*

## *Preparation of Space Experiments*

more complex tissue samples that mimic the structure and function of the source tissue (in this case, the human colon). Organoids are much smaller than the organ they mimic but can be up to 5 mm in size. These organoids are the most advanced and physiologically relevant bio-samples available for medical research today. This means that we can conduct cutting-edge cancer research using human organoids rather than cell lines.

Our additional collaborators are based in Europe and are part of the European Union. The Netherlands is a collaborating country where two critical organizations are located. Firstly, our patient-derived organoids come from Hubrecht Organoid Technology (the HUB) in Utrecht, and some of our ground-based experiments will take place at the European Space Research and Technology Center of ESA in Noordwijk. Our radiation biology experts are located in Belgium at the Belgian Nuclear Research Center where our ground radiation experiments in preparation for spaceflight will be conducted. Not only do they house safe infrastructure for radiation biology experiments, our Belgian colleagues have years of space experience and substantial experience in the safe design of nuclear reactors. For the latter reason, our Belgian colleagues will also participate in the design and development of our spaceflight hardware in close collaboration with Norway and China. Finally, France is the location of the International Space University, to which the principal investigator and other *Tumors in Space* team members are affiliated. Our aircraft

This brief summary shows how important international collaboration is. With-

The step-by-step preparation of our spaceflight experiment is now described. Each one of these steps could be a chapter in itself. Certainly, some details must be omitted. The success of this entire project relies heavily on the success of each step,

**5.1 Ethics, regulatory affairs, and information management: step 1**

This step ensures proper procedures for the selection, release, and use of intraindividual healthy and cancer organoids according to biobanking protocols and informed consent procedures. Procedures and consents are subject not only to the approval of the HUB biobank in the Netherlands but also from the host institute in Norway, as well as our collaborating institutes where our organoid research will be conducted (the European Space Agency in the Netherlands, the Belgian Nuclear Research Center in Belgium, parabolic flight laboratory in France, and the China Manned Space Agency in China for our spaceflight). Each institute has its own procedure, and each country or set of countries have their own legislations. Shipping bio-samples involves material transfer agreements and customs paperwork, and since human organoids contain human DNA, special ethical requirements

First and foremost, all patients provide written, informed consent to the HUB for future use of their bio-samples in medical research according to very specific guidelines. Research ethics approval for each experiment is required from the HUB and from the host institute in Norway. No experimentation can begin without these ethics approvals that assess our intended use of the samples across all of our collaborating sites in Europe and China. Since the HUB biobank is located in Europe, many of the intricacies with regard to ethics and legislation across our European collaborating sites are already in line. However, human DNA can be imported, but not exported from China. This means that we can ship our

organoid samples into China in preparation for our spaceflight launch, but after our

out all of these partners, realizing the most advanced cancer experiment ever conducted in space would not be possible. It is because of the unique strengths and expertise of all collaborating partners that the *Tumors in Space* project has become a

parabolic flight campaigns will also be flown from France.

**5. The** *Tumors in Space* **experiment in seven steps**

both independently and collectively.

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

reality.

apply.

**219**

Although monolayer cancer cell lines are easily accessible, well understood, and used in all previous cancer experiments in space, they lack spatial cellular organization and physiological relevance [16]. Cell lines cannot mimic conditions found in real humans, whereas organoids can. Using human organoids ensures that our results are applicable to patient diagnosis and treatment. This is crucial since we need to get as close as possible to studying the real human body in space. Organoid technology bridges the gap between cell lines and humans, or in vitro and in vivo. In 2017, organoids were named Method of the Year by *Nature Methods* for their unparalleled potential to advance our understanding of human biology, especially cancer [17]. We are the first team to send healthy and cancer human organoids into space.

## **4. International collaboration**

Before we dig into the seven steps of preparation for our spaceflight experiment, a quick note on international collaboration is warranted. Success of such a complex space medicine experiment requires world experts across many different fields. **Figure 5** is color coded to show collaborating countries, not only according to levels of responsibility with regard to the project but also according to geopolitical boundaries. You can see from **Figure 5** that Norway (in light gray) and China (in dark green) stand apart from the other collaborating countries like the Netherlands, Belgium, and France (in dark blue).

Norway, the center of the figure, is the coordinating country because this is where the principal investigator is located, along with the several members of the *Tumors in Space* team. We also have the support of the Norwegian Space Agency. Furthermore, Norway has identified China as a priority country for technical and scientific collaboration—for example, in satellite and broadband communication, as well as medicine. Norway and China are united by an underlying cooperative effort for the *Tumors in Space* collaboration. China is the host country for the Norwegian *Tumors in Space* experiment that has been selected for a 31-day space mission on board the China Space Station.

**Figure 5.** Tumors in Space *collaborating countries.*

## *Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

more complex tissue samples that mimic the structure and function of the source tissue (in this case, the human colon). Organoids are much smaller than the organ they mimic but can be up to 5 mm in size. These organoids are the most advanced and physiologically relevant bio-samples available for medical research today. This means that we can conduct cutting-edge cancer research using human organoids

Although monolayer cancer cell lines are easily accessible, well understood, and used in all previous cancer experiments in space, they lack spatial cellular organization and physiological relevance [16]. Cell lines cannot mimic conditions found in real humans, whereas organoids can. Using human organoids ensures that our results are applicable to patient diagnosis and treatment. This is crucial since we need to get as close as possible to studying the real human body in space. Organoid technology bridges the gap between cell lines and humans, or in vitro and in vivo. In 2017, organoids were named Method of the Year by *Nature Methods* for their unparalleled potential to advance our understanding of human biology, especially cancer [17]. We are the first team to send healthy and cancer human organoids

Before we dig into the seven steps of preparation for our spaceflight experiment, a quick note on international collaboration is warranted. Success of such a complex space medicine experiment requires world experts across many different fields. **Figure 5** is color coded to show collaborating countries, not only according to levels

of responsibility with regard to the project but also according to geopolitical boundaries. You can see from **Figure 5** that Norway (in light gray) and China (in dark green) stand apart from the other collaborating countries like the Netherlands,

Norway, the center of the figure, is the coordinating country because this is where the principal investigator is located, along with the several members of the *Tumors in Space* team. We also have the support of the Norwegian Space Agency. Furthermore, Norway has identified China as a priority country for technical and scientific collaboration—for example, in satellite and broadband communication, as well as medicine. Norway and China are united by an underlying cooperative effort for the *Tumors in Space* collaboration. China is the host country for the Norwegian *Tumors in Space* experiment that has been selected for a 31-day space mission on

rather than cell lines.

*Preparation of Space Experiments*

into space.

**4. International collaboration**

Belgium, and France (in dark blue).

board the China Space Station.

Tumors in Space *collaborating countries.*

**Figure 5.**

**218**

Our additional collaborators are based in Europe and are part of the European Union. The Netherlands is a collaborating country where two critical organizations are located. Firstly, our patient-derived organoids come from Hubrecht Organoid Technology (the HUB) in Utrecht, and some of our ground-based experiments will take place at the European Space Research and Technology Center of ESA in Noordwijk. Our radiation biology experts are located in Belgium at the Belgian Nuclear Research Center where our ground radiation experiments in preparation for spaceflight will be conducted. Not only do they house safe infrastructure for radiation biology experiments, our Belgian colleagues have years of space experience and substantial experience in the safe design of nuclear reactors. For the latter reason, our Belgian colleagues will also participate in the design and development of our spaceflight hardware in close collaboration with Norway and China. Finally, France is the location of the International Space University, to which the principal investigator and other *Tumors in Space* team members are affiliated. Our aircraft parabolic flight campaigns will also be flown from France.

This brief summary shows how important international collaboration is. Without all of these partners, realizing the most advanced cancer experiment ever conducted in space would not be possible. It is because of the unique strengths and expertise of all collaborating partners that the *Tumors in Space* project has become a reality.

## **5. The** *Tumors in Space* **experiment in seven steps**

The step-by-step preparation of our spaceflight experiment is now described. Each one of these steps could be a chapter in itself. Certainly, some details must be omitted. The success of this entire project relies heavily on the success of each step, both independently and collectively.

## **5.1 Ethics, regulatory affairs, and information management: step 1**

This step ensures proper procedures for the selection, release, and use of intraindividual healthy and cancer organoids according to biobanking protocols and informed consent procedures. Procedures and consents are subject not only to the approval of the HUB biobank in the Netherlands but also from the host institute in Norway, as well as our collaborating institutes where our organoid research will be conducted (the European Space Agency in the Netherlands, the Belgian Nuclear Research Center in Belgium, parabolic flight laboratory in France, and the China Manned Space Agency in China for our spaceflight). Each institute has its own procedure, and each country or set of countries have their own legislations. Shipping bio-samples involves material transfer agreements and customs paperwork, and since human organoids contain human DNA, special ethical requirements apply.

First and foremost, all patients provide written, informed consent to the HUB for future use of their bio-samples in medical research according to very specific guidelines. Research ethics approval for each experiment is required from the HUB and from the host institute in Norway. No experimentation can begin without these ethics approvals that assess our intended use of the samples across all of our collaborating sites in Europe and China. Since the HUB biobank is located in Europe, many of the intricacies with regard to ethics and legislation across our European collaborating sites are already in line. However, human DNA can be imported, but not exported from China. This means that we can ship our organoid samples into China in preparation for our spaceflight launch, but after our

## *Preparation of Space Experiments*

spaceflight experiment has been conducted and safely returned to Earth, those same samples cannot be exported to our genomics laboratory in Norway. Consequently, laboratory analysis of our spaceflight samples must be conducted in China, after which time the organoid samples will need to be destroyed and the data will need to be transferred to Norway for bioinformatic and statistical analysis.

Although polymerase chain reaction (PCR<sup>4</sup>

Analysis of Whole Genome [21] will be conducted.

measure radiation levels for the duration of our experiment.

ture and survival.

study.

**221**

**5.3 Spaceflight hardware readiness: step 3**

as consistent changes within each copy.

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

cannot use this approach for the current study. Even if we increase the sequencing depth to 5x the number of cells, we would get PCR-amplified copies of the DNA in a single organoid sample and would not be able to distinguish PCR errors from mutations. However, with rolling circle amplification, we would always use the same original (circularized) DNA fragment as a template for amplification. In this way, we will have a long DNA fragment with multiple copies of the original DNA fragment, and by sequencing these copies in a single read, we will detect mutations

*Bioinformatics and statistical analysis pipeline*: All somatic changes in whole genome data will be analyzed with mutation calling pipelines developed in Norway. Healthy tissue more than 5 cm from the tumor and consisting of epithelial and connective tissue [18] will be used as the "germline" reference. Mutations considered to be germline will be removed. Mutational signature extraction using nonnegative matrix factorization will be performed [20]. Signature attribution in reference to the Catalogue of Somatic Mutations in Cancer [10] and the Pan-Cancer

In this step, we are designing, developing, testing, and validating an automated cell culture experimental unit and six-paneled tantalum cover for cosmic radiation protection. Several characteristics make tantalum a good choice for radiation protection, all of which will be presented below. We will use our ground-based platforms, a sounding rocket, and aircraft parabolic flights to test the impact of environmental stressors and operational constraints (e.g., vibration, changes in gravity, changes in temperature, experimental lag time) on the fidelity of our organoid culture technique and the readiness of our spaceflight hardware. Our spaceflight hardware must fit according to requirements and constraints of the biological research module on the China Space Station. Other experimental equipment that we need for our experiment will be hosted by the China Space Station including a temperature-controlled incubator/cooler and a 1 g centrifuge or variable gravity rack. We also need dosimeters to

*Experimental unit*: We are designing and developing a biocompatible-automated organoid culture experimental unit. An essential first step is to complete biocompatibility testing for each material required for the construction of the unit. Direct contact between organoid culture medium and experimental unit materials must not attenuate organoid growth. More specifically, for our experimental unit to be deemed biocompatible, 75% of all organoid samples must survive. Our unit is required to be operational for the full duration of the 31-day experiment, including automated nutrient solution exchange on days 6, 12, 18, and 24. Our unit must adhere to a standard temperature profile (18–38°C for organoid growth, 4°C for fixed samples) and CO2 saturation level (5%). Finally, our unit must be capable of enduring physical stressors (vibration, g-forces) while maintaining organoid cul-

We cannot procure market-ready experimental units because all previous cancer experiments in space have used 2D monolayer cancer cell lines and *Tumors in Space* uses 3D human organoids. However, units used for previous cancer experiments in

<sup>4</sup> PCR is a widely used molecular biology method that makes millions to billions of copies from a DNA sample, allowing amplification of a very small amount of DNA to a larger amount of DNA for a detailed

) is a widely used approach, we

## **5.2 Laboratory, bioinformatics, and statistical analysis pipelines: step 2**

In this step, we go from human organoids in a lab that have been exposed to radiation (ground and space) and/or (micro)gravity (ground and space) to laboratory methods at the Genomics Core Facility (GCF) housed in Norway. We then treat the "big data" with bioinformatics tools to break down the noise and scale down the data for statistical analysis. It is important to note that the details in this step are given at the time of writing in March 2020. Genomics as a field is constantly advancing, sometimes exponentially, and so are the tools used to work with the samples and analyze the data. That being said, our *Tumors in Space* experimental methods are still being finalized, but this at least gives you a glimpse into our preparation for spaceflight.

*Organoid sample selection*: To account for tissue heterogeneity and differences in mutational signatures, we will use paired samples from three different patients. Assuming 7–10 subclones from each starting culture, we would have 21–30 paired samples which will give sufficient statistical power to detect consistent differences in mutational signatures. Subcloning is an essential step for this experimental strategy, as subcloning ensures that mutations occurring prior to subcloning will be fixed in subsequent culture, allowing these mutations to be detected by whole genome sequencing and somatic genotyping.

*Organoid cell culture and subcloning*: Laboratory procedures will be handled by the Genomics Core Facility at the Norwegian University of Science and Technology (NTNU) in accordance with previously published methods [18]. The CGF provides state-of-the-art high-throughput genomics technology including sequencing and genotyping microarray analysis. The major advantages of using the CGF include highly experienced staff and the use of robotics. Tumor organoids must be cultured in specifically designed medium [18]. After a fixed exposure time, subclones from each source organoid will be cultured for an additional fixed number of cell divisions to allow for the occurrence of random mutations before whole genome sequencing and somatic genotyping [19].

*Whole genome sequencing and somatic genotyping*: Classic sequencing-based whole genome analyses of mutational signatures (healthy vs tumor tissue) rely on mutations being clonally expanded due to tumor growth, i.e., the mutation occurred in an ancestor cell so that all daughter cells will have this mutation, and the mutation is detectable if a sufficiently large percentage of the tumor cells share this mutational ancestry. Clonal expansion ensures that within an organoid sample, there will be several DNA fragments and sequencing reads that share the same mutation. In this manner, mutations can be distinguished from sequencing errors. This also means that the mutations occurring within the tumor organoid source cells will not be detectable, as these mutations have not yet been amplified by cell division (unless the same mutation occurs at the exact same nucleotide within a sufficiently large subset of the current cells). The sequencing depth determines the percentage of cells that need to share mutations in order to be detected. For example, 100x sequencing depth can detect mutations if the mutations are present in 5% of cells, whereas 1000 sequencing depth can detect mutations present in only 0.5% of cells.

## *Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

spaceflight experiment has been conducted and safely returned to Earth, those same samples cannot be exported to our genomics laboratory in Norway. Consequently, laboratory analysis of our spaceflight samples must be conducted in China, after which time the organoid samples will need to be destroyed and the data will need to be transferred to Norway for bioinformatic and statistical

**5.2 Laboratory, bioinformatics, and statistical analysis pipelines: step 2**

In this step, we go from human organoids in a lab that have been exposed to radiation (ground and space) and/or (micro)gravity (ground and space) to laboratory methods at the Genomics Core Facility (GCF) housed in Norway. We then treat the "big data" with bioinformatics tools to break down the noise and scale down the data for statistical analysis. It is important to note that the details in this step are given at the time of writing in March 2020. Genomics as a field is constantly advancing, sometimes exponentially, and so are the tools used to work with the samples and analyze the data. That being said, our *Tumors in Space* experimental methods are still being finalized, but this at least gives you a glimpse into our

*Organoid sample selection*: To account for tissue heterogeneity and differences in mutational signatures, we will use paired samples from three different patients. Assuming 7–10 subclones from each starting culture, we would have 21–30 paired samples which will give sufficient statistical power to detect consistent differences in mutational signatures. Subcloning is an essential step for this experimental strategy, as subcloning ensures that mutations occurring prior to subcloning will be fixed in subsequent culture, allowing these mutations to be detected by whole

*Organoid cell culture and subcloning*: Laboratory procedures will be handled by the Genomics Core Facility at the Norwegian University of Science and Technology (NTNU) in accordance with previously published methods [18]. The CGF provides state-of-the-art high-throughput genomics technology including sequencing and genotyping microarray analysis. The major advantages of using the CGF include highly experienced staff and the use of robotics. Tumor organoids must be cultured in specifically designed medium [18]. After a fixed exposure time, subclones from each source organoid will be cultured for an additional fixed number of cell divisions to allow for the occurrence of random mutations before whole genome

*Whole genome sequencing and somatic genotyping*: Classic sequencing-based whole genome analyses of mutational signatures (healthy vs tumor tissue) rely on mutations being clonally expanded due to tumor growth, i.e., the mutation occurred in an ancestor cell so that all daughter cells will have this mutation, and the mutation is detectable if a sufficiently large percentage of the tumor cells share this mutational ancestry. Clonal expansion ensures that within an organoid sample, there will be several DNA fragments and sequencing reads that share the same mutation. In this manner, mutations can be distinguished from sequencing errors. This also means that the mutations occurring within the tumor organoid source cells will not be detectable, as these mutations have not yet been amplified by cell division (unless the same mutation occurs at the exact same nucleotide within a sufficiently large subset of the current cells). The sequencing depth determines the percentage of cells that need to share mutations in order to be detected. For example, 100x sequencing depth can detect mutations if the mutations are present in 5% of cells, whereas 1000 sequencing depth can detect mutations present in only 0.5% of

analysis.

preparation for spaceflight.

*Preparation of Space Experiments*

genome sequencing and somatic genotyping.

sequencing and somatic genotyping [19].

cells.

**220**

Although polymerase chain reaction (PCR<sup>4</sup> ) is a widely used approach, we cannot use this approach for the current study. Even if we increase the sequencing depth to 5x the number of cells, we would get PCR-amplified copies of the DNA in a single organoid sample and would not be able to distinguish PCR errors from mutations. However, with rolling circle amplification, we would always use the same original (circularized) DNA fragment as a template for amplification. In this way, we will have a long DNA fragment with multiple copies of the original DNA fragment, and by sequencing these copies in a single read, we will detect mutations as consistent changes within each copy.

*Bioinformatics and statistical analysis pipeline*: All somatic changes in whole genome data will be analyzed with mutation calling pipelines developed in Norway. Healthy tissue more than 5 cm from the tumor and consisting of epithelial and connective tissue [18] will be used as the "germline" reference. Mutations considered to be germline will be removed. Mutational signature extraction using nonnegative matrix factorization will be performed [20]. Signature attribution in reference to the Catalogue of Somatic Mutations in Cancer [10] and the Pan-Cancer Analysis of Whole Genome [21] will be conducted.

#### **5.3 Spaceflight hardware readiness: step 3**

In this step, we are designing, developing, testing, and validating an automated cell culture experimental unit and six-paneled tantalum cover for cosmic radiation protection. Several characteristics make tantalum a good choice for radiation protection, all of which will be presented below. We will use our ground-based platforms, a sounding rocket, and aircraft parabolic flights to test the impact of environmental stressors and operational constraints (e.g., vibration, changes in gravity, changes in temperature, experimental lag time) on the fidelity of our organoid culture technique and the readiness of our spaceflight hardware. Our spaceflight hardware must fit according to requirements and constraints of the biological research module on the China Space Station. Other experimental equipment that we need for our experiment will be hosted by the China Space Station including a temperature-controlled incubator/cooler and a 1 g centrifuge or variable gravity rack. We also need dosimeters to measure radiation levels for the duration of our experiment.

*Experimental unit*: We are designing and developing a biocompatible-automated organoid culture experimental unit. An essential first step is to complete biocompatibility testing for each material required for the construction of the unit. Direct contact between organoid culture medium and experimental unit materials must not attenuate organoid growth. More specifically, for our experimental unit to be deemed biocompatible, 75% of all organoid samples must survive. Our unit is required to be operational for the full duration of the 31-day experiment, including automated nutrient solution exchange on days 6, 12, 18, and 24. Our unit must adhere to a standard temperature profile (18–38°C for organoid growth, 4°C for fixed samples) and CO2 saturation level (5%). Finally, our unit must be capable of enduring physical stressors (vibration, g-forces) while maintaining organoid culture and survival.

We cannot procure market-ready experimental units because all previous cancer experiments in space have used 2D monolayer cancer cell lines and *Tumors in Space* uses 3D human organoids. However, units used for previous cancer experiments in

<sup>4</sup> PCR is a widely used molecular biology method that makes millions to billions of copies from a DNA sample, allowing amplification of a very small amount of DNA to a larger amount of DNA for a detailed study.

space do provide us with a good foundation for our own design and development. For example, Grimm's lab first tested their own cancer cell line experimental unit during the Shenzhou-8 mission [22], which was then further improved for the SpaceX CRS-8 ISS mission [23]. Grimm's unit sets the standard for safe cell culture and cell nourishment followed by fixation according to a pre-programmed timeframe. From this foundation, we are designing our experimental unit with built-in pre-programmed electronics to control temperature gradients for organoid cell culture medium, as well as automated oxygen, carbon dioxide, and nutrition cycles to keep our organoids alive for the duration of the 31-day space mission.

are being designed according to the size and shape of our experimental unit and the mass constraints of the payload. Several characteristics make tantalum a good choice for radiation shielding of biological experiments in space including very good biocompatibility, excellent corrosion resistance, and very high melting point.

*Dosimeters*: We also need electronic dosimeters to provide passive reading of radiation dosage for the duration of the 31-day experiment. The dosimeters must be sensitive enough to measure high-energy cosmic radiation, lightweight, and compact yet durable enough to be operational in the spaceflight environment [26]. The dosimeters must provide tissue equivalent readings. This means that the electronic data must provide the same information about cosmic radiation dose as we would

We will work in close collaboration with the China Manned Space Agency to develop, assemble, and test spaceflight hardware according to CMSA standards and quality controls with particular attention to interface compatibility, mass, size, power requirements, and duration of experiment. We will also test our spaceflight hardware during our ground, sounding rocket, and parabolic flight

In preparation for spaceflight, we are running several experiments on research platforms including ground-based space analogues that will produce effects on our organoid models similar to those experienced in space. This includes radiation facilities for ionizing and heavy ion irradiation and the random positioning machine (RPM) to simulate microgravity. It is important to remember that the spaceflight environment can be simulated but never replicated on the ground. We are also preparing for experiments on a sounding rocket and during aircraft parabolic flights. These preparatory experiments are essential to ensure a successful experiment in space. Not only will our organoid cultures and hardware development be tested, but we will also collect important baseline data for later comparison to our spaceflight data. In addition, we will have the opportunity to standardize our shipping, handling, and storage procedures for the organoids, as well as define our laboratory, bioinformatics, and statistical analysis pipelines. Finally, our ground radiation work does not only provide us with a mutational signature of different types of radiation for later comparison with cosmic radiation; it is designed to better understand the side effects of radiation therapy for cancer patients on Earth.

We are using ground-based facilities at the Belgian Nuclear Research Center and ESA's European Space Research and Technology Center to test our hypotheses and theoretical approach and to refine our methodology and operational procedures in preparation for spaceflight. We will collect baseline data on the mutational signature of ionizing and heavy ion radiation and use the random position machine (RPM) to test whether simulated microgravity stops or slows the growth of cancer. *Ionizing radiation*: To identify a unique mutational signature of cosmic radiation, we must verify the mutational signature of ionizing and heavy ion radiation for comparison. Using the gamma beam irradiator at the Belgian Nuclear Research

ii. Our samples exposed to simulated microgravity in the RPM

iii. Our experimental unit and tantalum cover

Tantalum can also be easily welded into the required shape and size.

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

expect if human tissue, rather than the dosimeter, was directly exposed.

experimental arms.

**5.4 Ground experiments: step 4**

Center, we will irradiate the following:

i. Our 1 g control samples

**223**

*A small temperature-controlled incubator/cooler with a rack insert*: Our experimental units will be fully automated removable inserts designed to interface with a small temperature-controlled incubator/cooler that serves as a miniature laboratory for self-contained, automatic microgravity experiments on cells/organoids similar to ESA's Kubik<sup>5</sup> on the International Space Station (**Figure 6**) [24]. This equipment will be hosted by the China Space Station. The rack insert of the incubator/cooler will provide passive structure to house our experimental unit. The incubator/cooler will operate between 4°C and 38°C and will permit a 1 g centrifuge insert to allow for simultaneous experiments with 1 g control samples and microgravity samples.

*Onboard 1 g centrifuge or variable gravity rack*: To thoroughly test our hypotheses and delineate compound spaceflight exposures (microgravity and cosmic radiation), we also require an onboard 1 g centrifuge or variable gravity rack that will allow us to expose our organoids to only cosmic radiation while maintaining the standard gravitational force felt on Earth (1 g). Our organoid medium can stay at 37°C, while in the centrifuge, our experimental unit will fit the standard payload boxes hosted by the China Space Station. A variable gravity rack which contains a centrifuge with rotating containers for biological and fluid experiments [25] may be larger than the standard 1 g centrifuge available within the incubator/cooler insert rack. Either the smaller 1 g centrifuge or the larger variable gravity rack will be used for our experiment, and this decision will be taken in close collaboration with the China Manned Space Agency during our hardware development phase.

*Tantalum cover for experimental unit*: In order to test whether exposure to microgravity slows or stops the growth of cancer, we need to protect our organoids from cosmic radiation exposure. To do so, we are designing a six-sided radiation shield to cover all sides of our organoid cultivation chamber in order to shield the organoids from cosmic radiation. The size, shape, and mass of the tantalum cover

**Figure 6.** *Kubik on the ISS (credit: ESA).*

<sup>5</sup> Kubik (from Russian for cube) is a miniaturized laboratory in a 40 cm cube, installed in the ESA orbital laboratory Columbus module on board the ISS, and is used for several kinds of biology experiments in space.

## *Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

space do provide us with a good foundation for our own design and development. For example, Grimm's lab first tested their own cancer cell line experimental unit during the Shenzhou-8 mission [22], which was then further improved for the SpaceX CRS-8 ISS mission [23]. Grimm's unit sets the standard for safe cell culture

incubator/cooler will provide passive structure to house our experimental unit. The incubator/cooler will operate between 4°C and 38°C and will permit a 1 g centrifuge

*Onboard 1 g centrifuge or variable gravity rack*: To thoroughly test our hypotheses and delineate compound spaceflight exposures (microgravity and cosmic radiation), we also require an onboard 1 g centrifuge or variable gravity rack that will allow us to expose our organoids to only cosmic radiation while maintaining the standard gravitational force felt on Earth (1 g). Our organoid medium can stay at 37°C, while in the centrifuge, our experimental unit will fit the standard payload boxes hosted by the China Space Station. A variable gravity rack which contains a centrifuge with rotating containers for biological and fluid experiments [25] may be larger than the standard 1 g centrifuge available within the incubator/cooler insert rack. Either the smaller 1 g centrifuge or the larger variable gravity rack will be used for our experiment, and this decision will be taken in close collaboration with the

insert to allow for simultaneous experiments with 1 g control samples and

China Manned Space Agency during our hardware development phase.

*Tantalum cover for experimental unit*: In order to test whether exposure to microgravity slows or stops the growth of cancer, we need to protect our organoids from cosmic radiation exposure. To do so, we are designing a six-sided radiation shield to cover all sides of our organoid cultivation chamber in order to shield the organoids from cosmic radiation. The size, shape, and mass of the tantalum cover

<sup>5</sup> Kubik (from Russian for cube) is a miniaturized laboratory in a 40 cm cube, installed in the ESA orbital laboratory Columbus module on board the ISS, and is used for several kinds of biology experiments in

microgravity samples.

*Preparation of Space Experiments*

**Figure 6.**

space.

**222**

*Kubik on the ISS (credit: ESA).*

and cell nourishment followed by fixation according to a pre-programmed timeframe. From this foundation, we are designing our experimental unit with built-in pre-programmed electronics to control temperature gradients for organoid cell culture medium, as well as automated oxygen, carbon dioxide, and nutrition cycles to keep our organoids alive for the duration of the 31-day space mission. *A small temperature-controlled incubator/cooler with a rack insert*: Our experimental units will be fully automated removable inserts designed to interface with a small temperature-controlled incubator/cooler that serves as a miniature laboratory for self-contained, automatic microgravity experiments on cells/organoids similar to ESA's Kubik<sup>5</sup> on the International Space Station (**Figure 6**) [24]. This equipment will be hosted by the China Space Station. The rack insert of the

are being designed according to the size and shape of our experimental unit and the mass constraints of the payload. Several characteristics make tantalum a good choice for radiation shielding of biological experiments in space including very good biocompatibility, excellent corrosion resistance, and very high melting point. Tantalum can also be easily welded into the required shape and size.

*Dosimeters*: We also need electronic dosimeters to provide passive reading of radiation dosage for the duration of the 31-day experiment. The dosimeters must be sensitive enough to measure high-energy cosmic radiation, lightweight, and compact yet durable enough to be operational in the spaceflight environment [26]. The dosimeters must provide tissue equivalent readings. This means that the electronic data must provide the same information about cosmic radiation dose as we would expect if human tissue, rather than the dosimeter, was directly exposed.

We will work in close collaboration with the China Manned Space Agency to develop, assemble, and test spaceflight hardware according to CMSA standards and quality controls with particular attention to interface compatibility, mass, size, power requirements, and duration of experiment. We will also test our spaceflight hardware during our ground, sounding rocket, and parabolic flight experimental arms.

In preparation for spaceflight, we are running several experiments on research platforms including ground-based space analogues that will produce effects on our organoid models similar to those experienced in space. This includes radiation facilities for ionizing and heavy ion irradiation and the random positioning machine (RPM) to simulate microgravity. It is important to remember that the spaceflight environment can be simulated but never replicated on the ground. We are also preparing for experiments on a sounding rocket and during aircraft parabolic flights. These preparatory experiments are essential to ensure a successful experiment in space. Not only will our organoid cultures and hardware development be tested, but we will also collect important baseline data for later comparison to our spaceflight data. In addition, we will have the opportunity to standardize our shipping, handling, and storage procedures for the organoids, as well as define our laboratory, bioinformatics, and statistical analysis pipelines. Finally, our ground radiation work does not only provide us with a mutational signature of different types of radiation for later comparison with cosmic radiation; it is designed to better understand the side effects of radiation therapy for cancer patients on Earth.

## **5.4 Ground experiments: step 4**

We are using ground-based facilities at the Belgian Nuclear Research Center and ESA's European Space Research and Technology Center to test our hypotheses and theoretical approach and to refine our methodology and operational procedures in preparation for spaceflight. We will collect baseline data on the mutational signature of ionizing and heavy ion radiation and use the random position machine (RPM) to test whether simulated microgravity stops or slows the growth of cancer.

*Ionizing radiation*: To identify a unique mutational signature of cosmic radiation, we must verify the mutational signature of ionizing and heavy ion radiation for comparison. Using the gamma beam irradiator at the Belgian Nuclear Research Center, we will irradiate the following:


Our 1 g control samples can be placed in direct line of the gamma ray to capture the mutational signature of ionizing radiation. By varying the dose and time of the beam, we will simulate radiation therapy given to cancer patients on Earth and examine whether healthy organoids from cancer patients turn cancerous after doseand time-dependent exposure to radiation. Next, our organoids will be contained in the RPM, and the RPM will then be placed in front of the irradiator. This will allow us to simultaneously expose our organoids to radiation and simulated microgravity. Finally, we will test the fidelity of our spaceflight hardware by exposing our organoids housed within the experimental unit and protected by the tantalum shielding to a predetermined dose rate of ionizing radiation.

**5.6 Parabolic flights: step 6**

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

**5.7 Spaceflight experiment: step 7**

for the duration of the experiment.

genotyping as described above.

and the China Manned Space Agency.

**6. Impact**

**225**

Similar to the sounding rocket, we will use aircraft parabolic flights to test the impact of environmental and operational factors on our experiment. The parabolic flight takes place on a specially designed commercial aircraft [30] that flies through a series of parabolas and gives 20–22 s of microgravity on each parabola. The aircraft flies up and down at 45° angles, and at the top of the curve, 20–22 s of microgravity is experienced. About 2 g is experienced during ascent and descent. A typical parabolic flight campaign involves 30 parabolas per flight and 3 flights over a 1-week period. This will be our final step before spaceflight and our last opportu-

According to standard operating procedures and in close collaboration with the China Manned Space Agency, we will finalize our preparation for spaceflight in the following manner. Our experimental units will be assembled prior to launch, including several backup units. All hardware will be sterilized and approved for launch. Our samples will be transported to the launch site on dry ice from our biobank in the Netherlands. Our organoids will arrive a minimum 7 days before the launch. After the initial phase of growth, our organoids will be placed into the validated and approved units, and the units will be inserted into the incubator/ cooler. The incubator/cooler housing our units will be loaded into the rocket prior to launch (-1 hour to -2 days). Once the payload is ready for flight, no additional

The experiment will start and end under constant environmental conditions (e.g., temperature, pressure, humidity) after our experimental units have been installed in the incubator/cooler on the China Space Station. The duration of our experiment is 31 days for organoids exposed to the natural spaceflight environment (microgravity and cosmic radiation), 31 days for organoids exposed to microgravity under the tantalum cover designed to protect our samples from cosmic radiation, and minimum 10 days for organoids exposed to cosmic radiation and Earth gravity with the use of the 1 g centrifuge. Dosimeters will be used to read the radiation level

The cell nourishment and fixation will be automated and pre-programmed according to experimental needs. Subsequent cold stowage of the fixed samples will be required at the end of the experiment. After completion of the mission and successful environmentally controlled return to the ground, our units will be transported to our laboratories for organoid subcloning, sequencing, and

*Tumors in Space* is a cancer research project at the intersection of stem-cell biology and space technology that will acquire new knowledge on cancer etiology due to the influence of the spaceflight environment. By conducting cutting-edge laboratory-based research in orbit and testing our two novel hypotheses, we are challenging the current scientific paradigm. We aim to spark curiosity and inspire the public by conducting excellent science at the forefront of international research in collaboration with the United Nations Office for Outer Space Affairs (UNOOSA)

nity to make any and all necessary improvements to our experiment.

handling of the experiment will be required before launch.

*The random position machine*: We are using the RPM at ESA-ESTEC to collect baseline data on the effect of simulated microgravity on the growth of cancer organoids. The RPM (**Figure 7**) uses two rotating axes, each with independent motor drives running at random speeds and generating random three-dimensional movements [27, 28] that change the direction of the gravitational vector felt by the organoids such that cumulative gravitational effects are cancelled over time. The maximum angular speed of the inner and outer frame of the RPM generally ranges from 20°/s to 120°/s. Thus, the geometry and size of the container within which the biological system is placed must be carefully considered [29]. The sample container will then be mounted to the inner axis. The RPM can run for the 31-day duration of the experiment, and cultural medium can be replaced when needed.

## **5.5 Sounding rocket: step 5**

We will use a sounding rocket to test the impact of environmental and operational factors (e.g., vibration, changes in gravity, changes in temperature, experimental lag time) on the fidelity of our models and readiness of our hardware. Sounding rockets carry experiments up to 750 km above the Earth's surface and offer up to 13 min of microgravity. After launch, the sounding rocket motor is shut down, and our experiment will be in free fall. Parachutes are deployed on the downward arc to return the rocket and experiments safely to the ground. The rocket and experimental unit will reach a peak gravitational force of 12 g which could impact our organoids, as can launch vibrations and other environmental factors. This will be our first microgravity experiment. Not only will we collect valuable data on the underlying gene expression of our organoids after exposure to microgravity, we will also have the opportunity to test our hardware as part of a rocket launch. At the end of this step, we will use the data to improve our logistics and operations, our organoid models, and the fidelity of our spaceflight hardware.

**Figure 7.** *The RPM at ESA-ESTEC (credit: ESA).*

## **5.6 Parabolic flights: step 6**

Our 1 g control samples can be placed in direct line of the gamma ray to capture the mutational signature of ionizing radiation. By varying the dose and time of the beam, we will simulate radiation therapy given to cancer patients on Earth and examine whether healthy organoids from cancer patients turn cancerous after doseand time-dependent exposure to radiation. Next, our organoids will be contained in the RPM, and the RPM will then be placed in front of the irradiator. This will allow us to simultaneously expose our organoids to radiation and simulated microgravity. Finally, we will test the fidelity of our spaceflight hardware by exposing our organoids housed within the experimental unit and protected by the tantalum

*The random position machine*: We are using the RPM at ESA-ESTEC to collect baseline data on the effect of simulated microgravity on the growth of cancer organoids. The RPM (**Figure 7**) uses two rotating axes, each with independent motor drives running at random speeds and generating random three-dimensional movements [27, 28] that change the direction of the gravitational vector felt by the organoids such that cumulative gravitational effects are cancelled over time. The maximum angular speed of the inner and outer frame of the RPM generally ranges from 20°/s to 120°/s. Thus, the geometry and size of the container within which the biological system is placed must be carefully considered [29]. The sample container will then be mounted to the inner axis. The RPM can run for the 31-day duration of

We will use a sounding rocket to test the impact of environmental and operational factors (e.g., vibration, changes in gravity, changes in temperature, experimental lag time) on the fidelity of our models and readiness of our hardware. Sounding rockets carry experiments up to 750 km above the Earth's surface and offer up to 13 min of microgravity. After launch, the sounding rocket motor is shut down, and our experiment will be in free fall. Parachutes are deployed on the downward arc to return the rocket and experiments safely to the ground. The rocket and experimental unit will reach a peak gravitational force of 12 g which could impact our organoids, as can launch vibrations and other environmental factors. This will be our first microgravity experiment. Not only will we collect valuable data on the underlying gene expression of our organoids after exposure to microgravity, we will also have the opportunity to test our hardware as part of a rocket launch. At the end of this step, we will use the data to improve our logistics and operations, our organoid models, and the fidelity of our spaceflight hardware.

shielding to a predetermined dose rate of ionizing radiation.

the experiment, and cultural medium can be replaced when needed.

**5.5 Sounding rocket: step 5**

*Preparation of Space Experiments*

**Figure 7.**

**224**

*The RPM at ESA-ESTEC (credit: ESA).*

Similar to the sounding rocket, we will use aircraft parabolic flights to test the impact of environmental and operational factors on our experiment. The parabolic flight takes place on a specially designed commercial aircraft [30] that flies through a series of parabolas and gives 20–22 s of microgravity on each parabola. The aircraft flies up and down at 45° angles, and at the top of the curve, 20–22 s of microgravity is experienced. About 2 g is experienced during ascent and descent. A typical parabolic flight campaign involves 30 parabolas per flight and 3 flights over a 1-week period. This will be our final step before spaceflight and our last opportunity to make any and all necessary improvements to our experiment.

## **5.7 Spaceflight experiment: step 7**

According to standard operating procedures and in close collaboration with the China Manned Space Agency, we will finalize our preparation for spaceflight in the following manner. Our experimental units will be assembled prior to launch, including several backup units. All hardware will be sterilized and approved for launch. Our samples will be transported to the launch site on dry ice from our biobank in the Netherlands. Our organoids will arrive a minimum 7 days before the launch. After the initial phase of growth, our organoids will be placed into the validated and approved units, and the units will be inserted into the incubator/ cooler. The incubator/cooler housing our units will be loaded into the rocket prior to launch (-1 hour to -2 days). Once the payload is ready for flight, no additional handling of the experiment will be required before launch.

The experiment will start and end under constant environmental conditions (e.g., temperature, pressure, humidity) after our experimental units have been installed in the incubator/cooler on the China Space Station. The duration of our experiment is 31 days for organoids exposed to the natural spaceflight environment (microgravity and cosmic radiation), 31 days for organoids exposed to microgravity under the tantalum cover designed to protect our samples from cosmic radiation, and minimum 10 days for organoids exposed to cosmic radiation and Earth gravity with the use of the 1 g centrifuge. Dosimeters will be used to read the radiation level for the duration of the experiment.

The cell nourishment and fixation will be automated and pre-programmed according to experimental needs. Subsequent cold stowage of the fixed samples will be required at the end of the experiment. After completion of the mission and successful environmentally controlled return to the ground, our units will be transported to our laboratories for organoid subcloning, sequencing, and genotyping as described above.

## **6. Impact**

*Tumors in Space* is a cancer research project at the intersection of stem-cell biology and space technology that will acquire new knowledge on cancer etiology due to the influence of the spaceflight environment. By conducting cutting-edge laboratory-based research in orbit and testing our two novel hypotheses, we are challenging the current scientific paradigm. We aim to spark curiosity and inspire the public by conducting excellent science at the forefront of international research in collaboration with the United Nations Office for Outer Space Affairs (UNOOSA) and the China Manned Space Agency.

**Acknowledgements**

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

Agency or the Norwegian Space Agency.

making this an open access publication.

accepted for implementation on board the CSS."

Institute of Health and Society, Oslo, Norway

provided the original work is properly cited.

\*Address all correspondence to: tricia.larose@flymed.uio.no

**Author note**

Simonetta Di Pippo.

**Author details**

Tricia L. Larose1,2

Strasbourg, France

**227**

*Tumors in Space* is represented by an exceptional research team with all necessary expertise and competence to ensure timely progress and ultimate success of the project: Norway, Tricia L. Larose (principal investigator and project coordinator), Carina Helle Berg, Ann-Iren Kittang Jost, Arve Jørgensen, Berge Solberg, Pål Sætrom; China, Mengyun Chen, Yang Yang; Belgium, Sarah Baatout, Bjorn Baselet, Vladimir Pletser, Roel Quintens; France, Ana Diaz Artiles, Ghislaine Scelo, Sergey Senkin, Chris Welch;

and the Netherlands, Annelien Bredenoord, Hans Clevers, Jack van Loon.

sity of Science and Technology, and is reused here with his permission.

Some text in this chapter subsection on laboratory methods was previously written by *Tumors in Space* collaborator, Pål Sætrom, from the Norwegian Univer-

*Tumors in Space* is under a programme of and funded by the European Space Agency with the support of the Norwegian Space Agency. The view expressed herein can in no way be taken to reflect the official opinion of the European Space

Thank you to my editor, Vladimir Pletser, and to the publisher, IntechOpen, for

"As a final outcome of the application and selection process in response to the first cycle of Announcement of Opportunity under the United Nations/China Cooperation on the Utilization of the China Space Station (CSS) initiative, being implemented by the Office for Outer Space Affairs (OOSA) and the China Manned Space Agency (CMSA) respectively, your proposal entitled "Tumors in Space: Signatures of early mutational events due to spaceflight conditions on 3D organoid cultures derived from intra-individual healthy and tumor tissue", has been fully

As announced on 12 June 2019 in Vienna, Austria, during the 62† Session of the

Committee on the Peaceful Uses Of Outer Space and signed on 9 July 2019 by Director of United Nations Office for Outer Space Affairs (UNOOSA), Ms.

1 Faculty of Medicine, Department of Community Medicine and Global Health,

2 Human Performance and Space Department, International Space University,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

#### **Figure 8.**

*The 17 Sustainable Development Goals (credit: United Nations).*

*The Sustainable Development Goals*: The UNOOSA works to promote the peaceful use of outer space for the benefit of humankind. This includes a collaboration with the CMSA to fly international experiments on board the China Space Station. The 17 Sustainable Development Goals (**Figure 8**) were adopted by the United Nations in 2015 [31] as a global call for action to improve environmental conditions on Earth and socioeconomic as well as health conditions for all humanity. *Tumors in Space* has a role to play in the attainment of the Sustainable Development Goals by 2030 through the peaceful and collaborative use of outer space to ensure healthy lives and promote well-being for all people of all ages.

*Science communication in the public domain*: Science funded by the people is meant to be delivered to the people, and this is one of the goals of *Tumors in Space.* We have designed a comprehensive science communication and research dissemination plan. The main feature of our plan is our project website that will include open access to our research results and scientific publications, research highlights, a color photo library, team bio-sketches, blog posts, and social media feeds (e.g., Twitter), as well as an upto-date list of all interviews and feature articles for the *Tumors in Space* project. Our efforts also include educational outreach focused on girls and marginalized youth.

## **7. Conclusions**

This chapter provides an overview of the *Tumors in Space* experiment beginning with the scientific evidence upon which the hypotheses are based. The seven main steps in our spaceflight preparation have been presented including ethics and regulatory affairs, laboratory methods, spaceflight hardware design, ground-based experiments (radiation and simulated microgravity), as well as sounding rocket and aircraft parabolic flight experiments. This takes us to the final preparations for our spaceflight experiment set to be launched to the China Space Station in 2025. Finally, some brief notes on the importance of the Sustainable Development Goals and communication of science in the public domain have been provided. To end this chapter, we provide a note of encouragement. Do share this chapter widely in the spirit of open access to scientific knowledge. We welcome you to follow our journey into orbit as we reach for our ultimate goal: to make novel contributions to cancer risk prediction, diagnostics, and therapeutics for human health during shortand long-duration space missions and public health on Earth.

## **Acknowledgements**

*Tumors in Space* is represented by an exceptional research team with all necessary expertise and competence to ensure timely progress and ultimate success of the project: Norway, Tricia L. Larose (principal investigator and project coordinator), Carina Helle Berg, Ann-Iren Kittang Jost, Arve Jørgensen, Berge Solberg, Pål Sætrom; China, Mengyun Chen, Yang Yang; Belgium, Sarah Baatout, Bjorn Baselet, Vladimir Pletser, Roel Quintens; France, Ana Diaz Artiles, Ghislaine Scelo, Sergey Senkin, Chris Welch; and the Netherlands, Annelien Bredenoord, Hans Clevers, Jack van Loon.

Some text in this chapter subsection on laboratory methods was previously written by *Tumors in Space* collaborator, Pål Sætrom, from the Norwegian University of Science and Technology, and is reused here with his permission.

*Tumors in Space* is under a programme of and funded by the European Space Agency with the support of the Norwegian Space Agency. The view expressed herein can in no way be taken to reflect the official opinion of the European Space Agency or the Norwegian Space Agency.

Thank you to my editor, Vladimir Pletser, and to the publisher, IntechOpen, for making this an open access publication.

## **Author note**

*The Sustainable Development Goals*: The UNOOSA works to promote the peaceful use of outer space for the benefit of humankind. This includes a collaboration with the CMSA to fly international experiments on board the China Space Station. The 17 Sustainable Development Goals (**Figure 8**) were adopted by the United Nations in 2015 [31] as a global call for action to improve environmental conditions on Earth and socioeconomic as well as health conditions for all humanity. *Tumors in Space* has a role to play in the attainment of the Sustainable Development Goals by 2030 through the peaceful and collaborative use of outer space to ensure healthy lives and

*Science communication in the public domain*: Science funded by the people is meant to be delivered to the people, and this is one of the goals of *Tumors in Space.* We have designed a comprehensive science communication and research dissemination plan. The main feature of our plan is our project website that will include open access to our research results and scientific publications, research highlights, a color photo library, team bio-sketches, blog posts, and social media feeds (e.g., Twitter), as well as an upto-date list of all interviews and feature articles for the *Tumors in Space* project. Our efforts also include educational outreach focused on girls and marginalized youth.

This chapter provides an overview of the *Tumors in Space* experiment beginning with the scientific evidence upon which the hypotheses are based. The seven main steps in our spaceflight preparation have been presented including ethics and regulatory affairs, laboratory methods, spaceflight hardware design, ground-based experiments (radiation and simulated microgravity), as well as sounding rocket and aircraft parabolic flight experiments. This takes us to the final preparations for our spaceflight experiment set to be launched to the China Space Station in 2025. Finally, some brief notes on the importance of the Sustainable Development Goals and communication of science in the public domain have been provided. To end this chapter, we provide a note of encouragement. Do share this chapter widely in the spirit of open access to scientific knowledge. We welcome you to follow our journey into orbit as we reach for our ultimate goal: to make novel contributions to cancer risk prediction, diagnostics, and therapeutics for human health during short-

and long-duration space missions and public health on Earth.

promote well-being for all people of all ages.

*The 17 Sustainable Development Goals (credit: United Nations).*

*Preparation of Space Experiments*

**7. Conclusions**

**226**

**Figure 8.**

"As a final outcome of the application and selection process in response to the first cycle of Announcement of Opportunity under the United Nations/China Cooperation on the Utilization of the China Space Station (CSS) initiative, being implemented by the Office for Outer Space Affairs (OOSA) and the China Manned Space Agency (CMSA) respectively, your proposal entitled "Tumors in Space: Signatures of early mutational events due to spaceflight conditions on 3D organoid cultures derived from intra-individual healthy and tumor tissue", has been fully accepted for implementation on board the CSS."

As announced on 12 June 2019 in Vienna, Austria, during the 62† Session of the Committee on the Peaceful Uses Of Outer Space and signed on 9 July 2019 by Director of United Nations Office for Outer Space Affairs (UNOOSA), Ms. Simonetta Di Pippo.

## **Author details**

Tricia L. Larose1,2

1 Faculty of Medicine, Department of Community Medicine and Global Health, Institute of Health and Society, Oslo, Norway

2 Human Performance and Space Department, International Space University, Strasbourg, France

\*Address all correspondence to: tricia.larose@flymed.uio.no

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Stewart BW, Wild CP, editors. World Cancer Report: Cancer Research for Cancer Prevention. Lyon, France: International Agency for Research on Cancer; 2014

[2] Wild CP, Weiderpass E, Stewart BW, editors. World Cancer Report: Cancer Research for Cancer Prevention. Lyon, France: International Agency for Research on Cancer; 2020

[3] Blackadar CB. Historical review of the causes of cancer. World Journal of Clinical Oncology. 2016;**7**(1):54-86. DOI: 10.5306/wjco.v7.i1.54

[4] Becker JL, Souza GR. Using spacebased investigations to inform cancer research on Earth. Nature Reviews Cancer. 2013;**13**(5):315-327. DOI: 10.1038/nrc3507

[5] Kanki B, Clervoy JF, Sandal G, editors. Space Safety and Human Performance. 1st ed. Oxford, United Kingdom: Butterworth-Heinemann; 2018

[6] European Space Agency. The Radiation Showstopper for Mars Exploration [Internet]. The Netherlands. Available from: https:// www.esa.int/Science\_Exploration/ Human\_and\_Robotic\_Exploration/The\_ radiation\_showstopper\_for\_Mars\_ exploration

[7] Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, et al. Patterns of somatic mutation in human cancer genomes. Nature. 2007; **446**(7132):153-158. DOI: 10.1038/ nature05610

[8] Behjati S, Gundem G, Wedge DC, Roberts ND, Tarpey PS, Cooke SL, et al. Mutational signatures of ionizing radiation in second malignancies. Nature Communications. 2016;**7**:12605. DOI: 10.1038/ncomms12605

[9] Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harbor Perspectives in Biology. 2010;**2**(1):a001008. DOI: 10.1101/cshperspect.a001008

platform. Frontiers in Bioengineering and Biotechnology. 2016;**4**:12. DOI:

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

> [24] European Space Agency. Kubik on the Space Station [Internet]. The Netherlands. 2020. Available from: https://www.esa.int/ESA\_Multimedia/ Images/2018/02/Kubik\_on\_Space\_

[25] Wang SK, Wang K, Zhou YL, Yan B, Li X, Zhang Y, et al. The development of the varying gravity rack (VGR) for the Chinese Space Station. Microgravity Science and Technology. 2018;**31**: 95-107. DOI: 10.1007/s12217-018-9670-1

[26] Benton E. Space radiation passive dosimetry. In: The Health Risks of Extraterrestrial Environments. United States of America: NASA; 2012

[27] Van Loon JJ. Some history and use of the random positioning machine, RPM, in gravity related research. Advances in Space Research. 2007;**39**(7):1161-1165.

DOI: 10.1016/j.asr.2007.02.016

and recommended terminology. Astrobiology. 2013;**13**(1):1-17. DOI:

[29] Leguy CA, Delfos R, Pourquie MJ, Poelma C, Krooneman J, Westerweel J, et al. Fluid motion for microgravity simulations in a random positioning machine. Gravitational and Space Biology Bulletin. 2011;**25**(1):36-39

[30] Pletser V, Rouquette S, Friedrich U, et al. The first European parabolic flight campaigns with the Airbus A310 ZERO-G. Microgravity Science and Technology. 2016;**28**(6):587-601. DOI: 10.1007/

[31] United Nations. Sustainable Development Goals. 2020. Available from: https://www.un.org/sustainablede velopment/sustainable-development-

10.1089/ast.2012.0876

s12217-016-9515-8

goals/

[28] Herranz R, Anken R, Boonstra J, Braun M, Christianen PC, de Geest M, et al. Ground-based facilities for simulation of microgravity: organismspecific recommendations for their use,

Station

[17] Method of the Year 2017: Organoids. Nature Methods. 2018;**15**(1). DOI:

[18] Roerink SF, Sasaki N, Lee-Six H, Young MD, Alexandrov LB, Behjati S, et al. Intra-tumour diversification in colorectal cancer at the single-cell level. Nature. 2018;**556**(7702):457-462. DOI:

10.3389/fbioe.2016.00012

10.1038/s41586-018-0024-3

[19] Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature Biotechnology. 2013;**32**(3):213-219. DOI:

[20] Qi Q, Zhao Y, Li M, Simon R. Nonnegative matrix factorization of gene expression profiles: A plug-in for BRB-Array tools. Bioinformatics. 2009;**25**(4): 545-547. DOI: 10.1093/bioinformatics/

[21] International Cancer Genome Consortium. The PanCancer Analysis of Whole Genome. [Internet]. 2019. Available from: https://omictools.

[22] Pietsch J, Ma X, Wehland M, Aleshcheva G, Schwarzwalder A, Segerer J, et al. Spheroid formation of human thyroid cancer cells in an automated culturing system during the Shenzhou-8 Space mission. Biomaterials. 2013;**34**(31):7694-7705. DOI: 10.1016/j.biomaterials.2013.06.054

[23] Pietsch J, Gass S, Nebuloni S, Echegoyen D, Riwaldt S, Baake C, et al. Three-dimensional growth of human endothelial cells in an automated cell culture experiment container during the SpaceX CRS-8 ISS space mission—The SPHEROIDS project. Biomaterials. 2017;

**124**:126-156. DOI: 10.1016/j. biomaterials.2017.02.005

**229**

10.1038/nmeth.4575

10.1038/nbt.2514

com/pcawg-tool

btp009

[10] Wellcome Sanger Institute. COSMIC: Catalogue of Somatic Mutations in Cancer [Internet]. United Kingdom; 2020. Available from: https:// cancer.sanger.ac.uk/cosmic

[11] Gridley DS, Slater JM, Luo-Owen X, Rizvi A, Chapes SK, Stodieck LS, et al. Spaceflight effects on T lymphocyte distribution, function and gene expression. Journal of Applied Physiology. 2009;**106**(1):194-202. DOI: 10/1152/japplphysiol.91126.2008

[12] Lewis ML. The cytoskeleton in spaceflown cells: an overview. Gravitational and Space Biology Bulletin. 2004;**17**:1-11

[13] Zhang ZJ, Tong YQ, Wang JJ, Yang C, Zhou GH, Li GH, et al. Spaceflight alters the gene expression profile of cervical cancer cells. Chinese Journal of Cancer. 2011;**30**(12):842-852. DOI: 10.5732/cjc.011.10174

[14] Lewis ML, Cubano LA, Zhao B, Dinh HK, Pabalan JG, Piepmeier EH, et al. cDNA microarray reveals altered cytoskeletal gene expression in spaceflown leukemic T lymphocytes (Jurkat). FASEB Journal. 2001;**15**(10):1783-1785. DOI: 10.1096/fj.00-0820fje

[15] Fatehullah A, Tan SH, Barker N. Organoids as an in vitro model of human development and disease. Nature Cell Biology. 2016;**18**(3): 246-254. DOI: 10.1038/ncb3312

[16] Katt ME, Placone AL, Wong AD, Xu ZS, Searson PC. In vitro tumor models: Advantages, disadvantages, variables, and selecting the right

*Tumors in Space: Preparation for Spaceflight DOI: http://dx.doi.org/10.5772/intechopen.93465*

platform. Frontiers in Bioengineering and Biotechnology. 2016;**4**:12. DOI: 10.3389/fbioe.2016.00012

**References**

Cancer; 2014

[1] Stewart BW, Wild CP, editors. World Cancer Report: Cancer Research for Cancer Prevention. Lyon, France: International Agency for Research on

*Preparation of Space Experiments*

[9] Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harbor Perspectives in Biology. 2010;**2**(1):a001008. DOI: 10.1101/cshperspect.a001008

[10] Wellcome Sanger Institute. COSMIC: Catalogue of Somatic

cancer.sanger.ac.uk/cosmic

Mutations in Cancer [Internet]. United Kingdom; 2020. Available from: https://

[11] Gridley DS, Slater JM, Luo-Owen X, Rizvi A, Chapes SK, Stodieck LS, et al. Spaceflight effects on T lymphocyte distribution, function and gene expression. Journal of Applied

Physiology. 2009;**106**(1):194-202. DOI: 10/1152/japplphysiol.91126.2008

[12] Lewis ML. The cytoskeleton in spaceflown cells: an overview. Gravitational and Space Biology

[13] Zhang ZJ, Tong YQ, Wang JJ, Yang C, Zhou GH, Li GH, et al. Spaceflight alters the gene expression profile of cervical cancer cells. Chinese Journal of Cancer. 2011;**30**(12):842-852.

[14] Lewis ML, Cubano LA, Zhao B, Dinh HK, Pabalan JG, Piepmeier EH, et al. cDNA microarray reveals altered cytoskeletal gene expression in spaceflown leukemic T lymphocytes (Jurkat). FASEB Journal. 2001;**15**(10):1783-1785.

[15] Fatehullah A, Tan SH, Barker N. Organoids as an in vitro model of human development and disease. Nature Cell Biology. 2016;**18**(3): 246-254. DOI: 10.1038/ncb3312

[16] Katt ME, Placone AL, Wong AD, Xu ZS, Searson PC. In vitro tumor models: Advantages, disadvantages, variables, and selecting the right

DOI: 10.5732/cjc.011.10174

DOI: 10.1096/fj.00-0820fje

Bulletin. 2004;**17**:1-11

[2] Wild CP, Weiderpass E, Stewart BW, editors. World Cancer Report: Cancer Research for Cancer Prevention. Lyon, France: International Agency for Research on Cancer; 2020

[3] Blackadar CB. Historical review of the causes of cancer. World Journal of Clinical Oncology. 2016;**7**(1):54-86.

[4] Becker JL, Souza GR. Using spacebased investigations to inform cancer research on Earth. Nature Reviews Cancer. 2013;**13**(5):315-327. DOI:

[5] Kanki B, Clervoy JF, Sandal G, editors. Space Safety and Human Performance. 1st ed. Oxford, United Kingdom: Butterworth-Heinemann;

[6] European Space Agency. The Radiation Showstopper for Mars Exploration [Internet]. The

Netherlands. Available from: https:// www.esa.int/Science\_Exploration/ Human\_and\_Robotic\_Exploration/The\_ radiation\_showstopper\_for\_Mars\_

[7] Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, et al. Patterns of somatic mutation in human

[8] Behjati S, Gundem G, Wedge DC, Roberts ND, Tarpey PS, Cooke SL, et al. Mutational signatures of ionizing radiation in second malignancies. Nature Communications. 2016;**7**:12605.

cancer genomes. Nature. 2007; **446**(7132):153-158. DOI: 10.1038/

DOI: 10.1038/ncomms12605

DOI: 10.5306/wjco.v7.i1.54

10.1038/nrc3507

2018

exploration

nature05610

**228**

[17] Method of the Year 2017: Organoids. Nature Methods. 2018;**15**(1). DOI: 10.1038/nmeth.4575

[18] Roerink SF, Sasaki N, Lee-Six H, Young MD, Alexandrov LB, Behjati S, et al. Intra-tumour diversification in colorectal cancer at the single-cell level. Nature. 2018;**556**(7702):457-462. DOI: 10.1038/s41586-018-0024-3

[19] Cibulskis K, Lawrence MS, Carter SL, Sivachenko A, Jaffe D, Sougnez C, et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nature Biotechnology. 2013;**32**(3):213-219. DOI: 10.1038/nbt.2514

[20] Qi Q, Zhao Y, Li M, Simon R. Nonnegative matrix factorization of gene expression profiles: A plug-in for BRB-Array tools. Bioinformatics. 2009;**25**(4): 545-547. DOI: 10.1093/bioinformatics/ btp009

[21] International Cancer Genome Consortium. The PanCancer Analysis of Whole Genome. [Internet]. 2019. Available from: https://omictools. com/pcawg-tool

[22] Pietsch J, Ma X, Wehland M, Aleshcheva G, Schwarzwalder A, Segerer J, et al. Spheroid formation of human thyroid cancer cells in an automated culturing system during the Shenzhou-8 Space mission. Biomaterials. 2013;**34**(31):7694-7705. DOI: 10.1016/j.biomaterials.2013.06.054

[23] Pietsch J, Gass S, Nebuloni S, Echegoyen D, Riwaldt S, Baake C, et al. Three-dimensional growth of human endothelial cells in an automated cell culture experiment container during the SpaceX CRS-8 ISS space mission—The SPHEROIDS project. Biomaterials. 2017; **124**:126-156. DOI: 10.1016/j. biomaterials.2017.02.005

[24] European Space Agency. Kubik on the Space Station [Internet]. The Netherlands. 2020. Available from: https://www.esa.int/ESA\_Multimedia/ Images/2018/02/Kubik\_on\_Space\_ Station

[25] Wang SK, Wang K, Zhou YL, Yan B, Li X, Zhang Y, et al. The development of the varying gravity rack (VGR) for the Chinese Space Station. Microgravity Science and Technology. 2018;**31**: 95-107. DOI: 10.1007/s12217-018-9670-1

[26] Benton E. Space radiation passive dosimetry. In: The Health Risks of Extraterrestrial Environments. United States of America: NASA; 2012

[27] Van Loon JJ. Some history and use of the random positioning machine, RPM, in gravity related research. Advances in Space Research. 2007;**39**(7):1161-1165. DOI: 10.1016/j.asr.2007.02.016

[28] Herranz R, Anken R, Boonstra J, Braun M, Christianen PC, de Geest M, et al. Ground-based facilities for simulation of microgravity: organismspecific recommendations for their use, and recommended terminology. Astrobiology. 2013;**13**(1):1-17. DOI: 10.1089/ast.2012.0876

[29] Leguy CA, Delfos R, Pourquie MJ, Poelma C, Krooneman J, Westerweel J, et al. Fluid motion for microgravity simulations in a random positioning machine. Gravitational and Space Biology Bulletin. 2011;**25**(1):36-39

[30] Pletser V, Rouquette S, Friedrich U, et al. The first European parabolic flight campaigns with the Airbus A310 ZERO-G. Microgravity Science and Technology. 2016;**28**(6):587-601. DOI: 10.1007/ s12217-016-9515-8

[31] United Nations. Sustainable Development Goals. 2020. Available from: https://www.un.org/sustainablede velopment/sustainable-developmentgoals/

## *Edited by Vladimir Pletser*

This book explains how researchers design, prepare, develop, test and fly their science experiments on microgravity platforms before sending them to space. All preparation phases are explained and presented, including aircraft parabolic flights as part of spaceflight preparation. Twenty international authors, all experts in their own microgravity research field, contribute to chapters describing their experience to prepare experiments before space flights. Fields covered are Physical Sciences and Life Sciences. Physical Sciences covers fluid physics (vibration effects on diffusion; red blood cell dynamics; cavitation in microgravity; capillary driven flows) and material sciences (electromagnetic levitator onboard International Space Station). Life Sciences includes human physiology (sampling earlobe blood; human cardiovascular experiments; tumours in space) and neurophysiology (dexterous manipulation of objects in weightlessness).

Published in London, UK © 2020 IntechOpen © bestdesigns / iStock

Preparation of Space Experiments

Preparation of

Space Experiments

*Edited by Vladimir Pletser*