Prospects of Cryopreservation as Applied in Blood and Plants

#### **Chapter 6**

## Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells (PBMCs)

*Nahla Afifi, Eiman Al-Khayat, Linda Hannigan, Monika Markovic Bordoski and Israa Khalaf*

#### **Abstract**

During cryopreservation of peripheral blood mononuclear cells (PBMCs), there are several recognized cooling methods, which include different cooling rates that might influence the stability of the PBMCs. This chapter will focus on three cooling methods trialled and will describe the different principles they are based on and the outcomes. One cooling method is based on repeatable −1°C/min cooling rate that requires only isopropyl alcohol (method A). The second cooling method is based on the cooling rate of −1° C/min solely (method B). The third cooling method is based on a user-predefined programmable controlled rate of freezing (method C). The first method was discontinued for safety reasons. A small comparative study was performed using 12 cell preparation tubes (CPT) using methods B and C. Cell Viability was measured based on the difference between pre-thaw and post-thaw viability percentages that were obtained from the flow cytometry. From our data, we conclude that although there were no significant differences in the outcomes of the comparative study of cooling methods, the use of either method B or C are the most suitable for long-term storage that will preserve the quality of the sample suitable for future research and clinical applications.

**Keywords:** cooling, PBMC's, stability, viability, pre-thaw, post-thaw

#### **1. Introduction**

PBMCs are white blood cells with round nuclei that includes lymphocytes (i.e. T cells, B cells, and NK cells), monocytes, and dendritic cells [1]. These cells are important biospecimens as researchers use them to recognize circulating disease biomarkers. While fresh viable cells are most often being used, the use of frozen viable PBMC's should be equally considered as they allow the screening of purified monocyte and lymphocyte populations. Denity gradient centrifugation has been utilized for isolating PBMC's because it is not expensive and needs very little specialised

#### **Figure 1***.*

equipment to implement in any lab [1], following cryopreservation, in addition to, functional studies, immunophenotyping, obtain lymphoblastoid cell lines (LCL) by Epstein Barr virus (EBV) transformation, and purification of CD34+ cells [2].

Using cell separation techniques, PBMCs can be isolated directly from whole blood. They are present in peripheral blood, and they have a crucial function of acting as the body's front line to defend against disease [3]. PBMC isolation is based mostly on the method of density gradient medium, and centrifugation [4] as shown in **Figure 1**.

Density gradient centrifugation has been utilized for isolating PBMCs because it is not expensive and needs very little specialized equipment, to implement to any lab [1, 5]. The aim of this chapter is to investigate the effectiveness of different approaches used in freezing rates to ensure that PBMCs are maintained within the optimal viability and functionality, and to preserve the cells with higher viability and biological activity, before and after the thawing processes.

#### **2. Literature review of different cooling rates on PBMCs stability**

Our research in this field highlighted that there is a lack of literature about the effects of different cooling rates during sample storage. Some studies provided data that shows the crucial steps of sample storage and handling in maintaining the viability of PBMCs, the recovery of PBMC and T-cell functionality [6]. As such, a review of the available studies will be discussed.

#### **2.1 Quality of frozen PBMCs**

Researchers have identified that cryopreservation affects the viability, recovery, and gene expression pattern of PBMCs, when compared to freshly isolated PBMCs [7, 8]. In addition, multiple factors impact the quality of PBMCs including preanalytical, analytical and post-analytical processes. Pre-analytical steps such as the time of sample collection, environmental conditions and calibration of equipment [9]. Analytical steps included the sample processing, type and time of exposure of the cryoprotectant media, viable cells manual mixing conditions, sterile environment and freezing conditions are all critically important for good sample cryopreservation [6]. Post-analytical factors included transportation of viable cells is of paramount consideration as they affect biological specimen viability and functionality [2]. Also, temperature fluctuations happen during retrieval or shipping of stored samples [8]. Nevertheless, an essential step is that the collected PBMCs are conserved in a natural state that renders them from being altered functionally. PBMCs need to be grown in cultures to show viability and to react to immune stimulation to show

*Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*

phenotypic capability [10] Best practices relating to the maintenance of PBMCs viability is obtained if they are stored below −132°C, the glass transition temperature of water (GTTW) [11]. At this temperature or below, the biological activities of cells are stopped [8]. Additional research suggested that quality control measures in cell repository should be adopted or based on their study findings. The separation of blood, and the storage using a controlled-rate freezer should be within six hours from collection. Environmental safety controls such as a temperature monitoring alarm system should be configured in the liquid nitrogen storage tanks. The study findings recommended to use liquid nitrogen vapor for maintaining a high cell viability through the storage for long-term purposes. However, using gasket threaded vials can also be used if storage is in the liquid nitrogen phase [12].

#### **2.2 Different cooling rates vs. cell viability**

PBMCs are being monitored through cell yield, viability, and cell population percentage using fluorescence flow cytometry. Research has concluded that it is important to disclose the temperature and time of processing when data from clinical trial of PBMCs is being published [13].

A study was done to show the impact of multiple temperature fluctuations on cell quality, PBMCs were stored under suboptimal storage condition from 10 different donors. The multiple temperature shifts were compared to optimal storage conditions without temperature shifts. Automated trypan blue dye exclusion and IFN-c ELISpot were used to measure cell viability, recovery, and functionality after cryopreservation in the standardized xeno-free cryomedium IBMT I and cell storage under 3 different conditions. The results were shown to minimize PBMC viability, PBMC recovery and T-cell functionality as measured by IFN-c ELISpot. Hence, temperature fluctuation has been shown to directly affect cell integrity, and the importance of carefully choosing optimal sample storage conditions [6, 14].

#### **2.3 Effects of slow cooling and super cooling on cell viability**

Since cooling rate is a major determinant of cell viability following cryopreservation, cryopreserved cells tend to die if it has been linked to intracellular ice formation (IIF) [15]. Research has shown that it has proved beneficial to avoid variable degrees of supercooling in multiple samples by deliberately inducing freezing (nucleation) at a point when the samples have cooled a few degrees below their equilibrium freezing point [16]. Research suggests that cell volume has a pivotal role in the occurrence of IIF than extracellular nucleation temperature or intracellular supercooling. Results indicated that larger cells were more likely to have IIF than smaller cells, and that smaller cells can withstand the supercooling effect before forming intracellular ice [15]. Other research has shown that in post-intracellular freezing, the plasma membrane lost its ability to act as a barrier for extracellular ice, which was similar to the damage caused by osmotic stresses [17].

Optimal slow cooling conditions resulting in retained cell viability are defined by the cooling rate that permits some cell dehydration without the formation of significant amounts of intracellular ice. Tolerances for cell shrinkage and intracellular ice formation vary between cell and tissue types. Ice formation in slowly cooled systems usually begins in the extracellular solution surrounding the biological material. Because ice is pure water, as ice formation occurs, the concentration of solute outside the cells increases and the cells begin to lose water by osmosis resulting in cell

**Figure 2***. Different cooling rates showing physical events occurring in cells as they start cooling.*

shrinkage. Cooling samples to their freezing point and beyond does not automatically result in freezing the samples at the equilibrium freezing point. Invariably, samples tend to under cool—often referred to as supercooling— to a varying degree that depends on the cooling rate, sample size, presence of nucleating agents, which are foreign particles in a solution that catalyze the formation of an ice nucleus, initiating the freezing process [16]. As samples start the cooling process and as it achieves approximately −5°C, the cells including the surrounding remain unfrozen. Ice starts to form as the temperature drops below −5 down to −15°C. At this stage, the cells and the external medium remain liquefied still, as the plasma membrane blocks the build of ice crystals into the cytoplasm. While the supercooled water in the cells has a greater chemical potential than the water in the partially frozen exterior solution; therefor, water flows out of the cells osmotically and freezes in the external medium. **Figure 2** shows the subsequent events occurring in the cells physically, depending on the cooling rates suggested [18].

Some studies were focused on the observation of the induced IIF which was described earlier in this chapter, which was a result of the water flux across the cells membrane during the freeze–thaw cycle [18]. According to a study model, researchers proposed that IIF may be induced by the plasma membrane through the effects of external ice on the plasma membrane, also known as surface-catalyzed nucleation (SCN), or by the intracellular particles, also known as volume-catalyzed nucleation (VCN). These different effects depend on the freezing conditions used. Also, they suggested that the effects of variables could be minimised if cells were cooled at rapid rates to avoid water flux during the process of freezing [19].

#### **2.4 Method A, B and C**

Different cooling methods are commercially available for PBMCs cryopreservation. Method A samples are placed in isopropanol chambers and into −80°C freezers, or into the vapor of liquid nitrogen at a temperature that varies between −135°C and − 190°C. This method is very simple and low in cost, but it does not provide any evidence for traceability or to verify the cooling rate. Therefore, this method would be avoided in clinical settings where a higher assurance of cell recovery and traceability of the freezing process is needed [20]. The freezing of samples using isopropanol filled devices (method A) requires long equilibration times and can introduce variability based on vial position, so the performance is dependent on vial position and continuous isopropanol replenishment. Method A was eventually excluded from the comparison study due to health and safety concerns relating to the use of the Isopropanol. Using programmable freezers (method C) can keep highly reproducible freeze rates, but are also costly, hard to maintain, susceptible to malfunction, and requires large spaces and energy [21]. Method B which is based mainly

#### *Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*

on an alcohol-free freezing at the rate of −1°C/minute combined with a − 80°C freezer [22]. This method ensures high thermal control and reproducibility while maintaining a small footprint [21]. A critical factor that influences the survival of cells during cryopreservation is the choice of an optimal cooling rate [20]. Several studies have been done to assess the cell viability for PBMCs, and it was shown to be consistently above 95% before freezing. An assay was blotted to show viability after freezing using either controlled-rate programmable freezer (method C), or the cell-freezing container (method B). In both methods, cells were frozen and stored at −80°C, then further stored at −150°C for 5 days. PBMCs were analyzed via flow cytometer using propidium iodide as a post-thaw viability. The viability rates were shown to be insignificant in the difference between both methods [21]. Nevertheless, the use of method C is thought to minimize two cell damage effects. The first effect, called solution effect which is extensive cell dehydration. The second effect, called mechanical damage which is intracellular ice crystallization. This is further explained as the continuous adjustments of the temperature reduces based on the temperature of the cells, therefore, compensating for fusion heat and reducing of supercooling effects [23]. This temperature compensation is provided by a programmed decrease in chamber temperature that both initiates nucleation and subsequently compensates for the release of the latent heat of fusion. The major variables involved are the rate of chamber temperature decrease, hold temperature and duration and the rate of temperature increase [16].

#### **2.5 Thawing processes**

As PBMCs survive cooling to ultra-low temperature, it is still challenging during the thawing processes at which it can exert effects on survival comparable with those of cooling [18]. So, determining how they survive both cooling and subsequent return to physiological condition is the consideration. An important question would be whether they freeze intracellularly or not, which occurs when cooling is too rapid as explained earlier (**Figure 2**). One study has developed equations that describe the kinetics of water loss and predict the likelihood of intracellular freezing as a function of cooling rate. Although it is necessary to avoid intracellular freezing to accomplish survival, but it is not sufficient. Slow freezing can introduce injury to the cell [24], as described earlier in Section 2.3. A study was developed to identify the risk in the addition of ice-chilled washing media to thawed cells at the same temperature, which was shown to be a high-risk practice that yielded significantly lower viability and functionality of recovered PBMC. This study also compared the previously mentioned outcome to the use of warm cryovials in temperatures of 37°C while adding a warm washing medium. The thawed PBMC in cryovials were kept up to 30minutes at 37°C in the presence of DMSO, and surprisingly showed that exposure to DMSO was a low-risk practice during the thawing process [25].

#### **2.6 Factors that impact PBMCs stability**

A major reason to use a freezing equipment or protocol rather than simply placing samples in cold environments is that the temperature compensation provided during controlled rate preservation for release of the latent heat results in improved post-cryopreservation cell viability [16]. In addition, research has focused on improving the interaction between cooling rates and the permeability of the plasma membrane to water and cryoprotectants [26]. The addressed interaction plays a

major role in PBMC stability. So, as the biological metabolism in cells dramatically diminishes at low temperatures, research has shown that cells were unable to endure the low temperatures. However, it is in fact the lethality of an intermediate zone of temperature (−15 to −60°C) that cells must traverse twice—once during cooling and once during warming [18]. Research studies recommend the induction of on-site training that facilitate a standardized method for cell counting, freezing, and thawing in order to maintain an environment with reduced variation in cell recovery. Nevertheless, external quality control programs can also enable the optimization of viability and cell recoveries with higher yields and viability to maximize the value of PBMC to be collected and stored for research studies [12].

#### **3. Materials and methods**

#### **3.1 Study sample preparation**

A total of 12 blood samples were collected and prepared from healthy adults. These samples were collected and processed according to Qatar Biobank (QBB) procedures. CPT closed sample collection kits with tubes containing additives of sodium citrate were used to collect whole blood [27]. A total of 24 viable cells in 1ml aliquot tubes were divided; every aliquot from the same parent CPT tube was placed in method B and method C, simultaneously. To obtain accurate measurements, viable cells were stored for a minimum of 24 hours in liquid nitrogen vapour.

#### **3.2 PBMC isolation and cryopreservation**

The procedure of PBMC separation was carried out in the laminar flow cabinet, which was turned on for at least 10 minutes before the work was started. The surface was disinfected using sodium hypochlorite followed by 70% ethanol and then type 1 water. CPT tubes were processed following a standard protocol [11] After centrifugation, the tubes appeared to have layers as shown previously in **Figure 1**. PBMCs were transferred using sterile tips into 15 ml sterile prelabelled intermediate tubes. This intermediate tube was connected to a parent tube by a Laboratory Information Management System tool for our labs. 100μl of PBMC were sub aliquoted from the intermediate tube into 5 ml prelabelled plain tubes that were also connected to the parent tube. The plain tube was processed in the cell counter to check the number of WBCs before running the samples in the flow cytometer (Section 3.4). In the 15 ml sterile intermediate tube, sterile phosphate-buffered saline (PBS) was added (in laminar flow cabinet) till 15 ml as first wash cycle. The cells were mixed gently by inverting the intermediate tube 5 times, then it was centrifuged for 15 min at 300 RCF at room temperature. The supernatant was disposed in an empty sterile waste bottle. The cell pellet was resuspended by gently vortexing or tapping tube with index finger. Sterile PBS solution was added until the 10 ml mark as second wash cycle. The tube was mixed by inverting 5 times, then it was centrifuged for 10 min at 300 RCF at room temperature. The supernatant was disposed in an empty sterile waste bottle. 1 ml of 10% DMSO was added to the tube and gently pipetted to mix with the cell suspension. 2 aliquots were created from each one parent tube in the corresponding sterile 1 ml tube. Using our laboratory information management system the parent tube was barcoded and scanned to be linked to 2 aliquots each. The aliquots were placed on a cooling shell to allow the cryoprotectant to enter the cells, and to prevent

*Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*

heat generation that can damage the cells. Keeping the specific timeline to allow stabilization, which is between 20 to 30 minutes to prevent the toxic effects of DMSO on the cells. 12 out of 24 created aliquots were transferred to method C, where they were gradually cooled in a user pre-defined temperature in the controlled rate freezer that is 1°C per 1 minute until −30°C, after that cooling rate is increased up to 5°C per 1 minute until −100°C is achieved, to ensure that the freezing process runs gradually to keep the cells, membranes, and cellular organelles safe and intact. Eventually, these aliquots were then stored in liquid nitrogen vapour. The remaining 12 created aliquots were transferred to method B, which uses a fridge temperature pre-cooled cool cell box, in which samples were then transferred to be cryopreserved within 4–24 hours of cool cell use time in the -80C freezer. This has been verified with an internally validated method in parallel with method C, in any contingency situation with the goal to cryopreserve high-quality quality PBMC samples.

#### **3.3 Thawing the PBMCs**

The cryopreserved PBMCs were retrieved from vapor phase of liquid nitrogen storage and placed directly in -80C portable freezer until samples were thawed. A standard thawing procedure for PBMCs was followed [28]. After thawing, cells were resuspended in PBS buffer as a preparation step for flow cytometer cell viability analysis explained in the next section. A standard thawing procedure is equally as or perhaps more important for obtaining maximum viability and recoveries of cryopreserved PBMC. The thawing procedure should also become part of the validation exercise to ensure reproducible sample preparation and cryopreservation.

#### **3.4 Assessing the cell viability using flow cytometry**

Prior to processing samples in the flow cytometer, a cleanse panel was run followed by a fluorescent microspheres suspension check. This step is mandatory as a routine quality control check prior to daily instrument operation. The PBMCs were extracted from the processing of CPT tubes that was previously explained in both sections 3.3 and 3.4. The cells were washed with 400 ul of PBS and centrifuged for 5 minutes at 500xg at 4°C. The supernatant was then discarded, and dyes were added to the cell suspension each prepared as follows, 10 μL of Annexin V-FITC, 20μL of 7-AAD viability dye and 10 μL CD45-APC750. The samples were mixed gently and kept for incubation in the fridge in the dark for 15 minutes. After incubation, 400 ul of binding buffer was added to each sample. Finally, the results were checked for the acceptable viability percentages for each sample as shown in **Figure 3**.

#### **Figure 3***.*

*Dot plot diagram from PBMCs showing cell population. D3. Double negative (Annexin V and 7-AAD negative) healthy cells. D4. Annexin V positive, 7-AAD negative apoptotic cell. D2. Annexin V & 7-AAD double positive necrotic cell.*

#### **3.5 Statistical analysis**

The temperature for method B was recorded at 10-second intervals over a 4-hour period, which was repeated twice. Also, the temperature for method C was recorded over 2-hour period. Thermocouple probes were calibrated and set up with a temperature data logger for method B to record the temperature every 10 seconds using temperature record data-logger software, while instrument-specific temperature record software was used to generate temperature curves for method C, in addition to, temperature record data-logger. Additionally, Student's t-test was generated based on the comparison of standard deviation and mean values of both method B and method C.

### **4. Results and discussion**

#### **4.1 Comparison between method B and method C -cooling rate temperatures**

Following manufacturer specification, method B had a cooling rate of 1°C per 1 minute. This fact was verified using probes as mentioned in Section 3.5, and the overall average rate was 0.98°C with slight differences in cooling rate from −15.5°C to −30°C, and from −30°C to −50°C, as shown in **Table 1** and temperature curve is shown in **Figure 4**. Cooling performance of method C was measured by instrumentspecific temperature record software configured with the instrument as shown in **Table 2** and **Figure 5**, to detect and record all temperatures and curves, in addition to temperature record data-logger as shown in **Table 3** and **Figure 6**.


#### **Table 1.**

*Method B temperature recorded over 4-hour interval.*

**Figure 4.** *Method B temperature curve for 4-hour interval.*

*Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*


#### **Table 2.**

*Method C freezing cycle program in instrument-specific temperature record.*

#### **Figure 5.**

*Curve of method C freezing cycle program in instrument-specific temperature record.*


#### **Table 3.**

*Method C temperature recorded over 2-hour interval.*

As shown in **Figure 4**, latent heat was generated during freezing, an exothermic process, or heat release, known as the latent heat of fusion or crystallization, during ice formation must be conducted away from the material being frozen.

Differences in temperature rate between method B and method C were not statistically significant as shown in **Table 4** below.

#### **4.2 Adjusting the cooling rate for better stability of PBMCs**

A critical variable factor that is the main scope of this chapter is the effect on the survival rate of cells by the cooling rate. During slow cooling the cells are exposed to DMSO, which is harmful for cell viability, and the concentration of external liquid is increasing, leading to dehydration as a consequence of efflux of water from cells due to change in osmotic status. Therefore, cells can shrink and become deformed. Using fast cooling rate, the dynamic characteristic of the cell membrane must be considered. The intracellular water cannot pass through membrane fast enough and freezes inside the cell. This process is lethal for the cell. During freezing, the following phases are appearing in the cell media: liquid phase cooling 1°C/min, supercooling (undercooling) is happening below the freezing point of media (DMSO freezing point is −5°C), this phase is followed by thermal increase as its exothermal reaction. Phase change-liquid to solid phase change followed immediately after supercooling. Solid phase I freezing is same 1C°/min. End solid phase I freezing is usually between −25°C to −50°C. Protocol of freezing can be shortened in the duration of the reaching the solid phase freezing, and cooling rate can be increased 5°C/min to the end of solid phase II freezing, which is between −80 and − 90°C. This provides adequate temperature security by preventing sample warming above End solid phase I. As the optimal cooling rate is essential for cell viability during cryopreservation, and specifically for that purpose, 4 control rate protocols were developed and compared in terms of temperature stability and viability. First program was following the control rate according to method B protocol with 1°C per 1 minute. Second program was designed to have cooling rate 1°C per 1 minute until −30°C, after that, cooling rate was increased up to 5°C per 1 minute until −100°C is achieved, as shown in **Figure 6** and **Table 5**.


A third programme was created to prevent the impact on cells by latent heat generation, which is clearly shown in **Figure 7** below.

#### **Table 4.**

*Comparison between method B and method C, program of 1°C/1 min.*

*Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*


#### **Table 5.**

*Method C freezing cycle program in instrument-specific temperature record, with addition to the program.*

#### **Figure 7.**

To improve the viability of the cells, an additional plot between −12°C and − 30°C was added (4°C/min), as shown in **Table 6** below.

The shape of the peak for latent heat generation in program 4 as shown in **Figures 8** and **9** was not as sharp as at the first program. Both of the programs were reaching the point of freezing media −5°C. And cooling rate was increasing for 4°C per minute to prevent the supercooling. To keep the cooling rate during crystallization as close to 1–2°C/min, a much greater difference between gas and sample had to be maintained as illustrated in **Table 7**. Heat of fusion was transferred through the wall of ampoule and heat capacity of the sample. After the intracellular phase transition was done (at −30°C), cooling rate can be increased to 5°C per minute.

The next improvement step was done at the temperature level of −30°C to achieve equal temperatures between the chamber and reference ampule (equal to sample) before increasing the cooling rate, as shown in **Table 8** below.


**Table 6.**

*Method C freezing cycle program in instrument-specific temperature record, with addition to the program.*

*Latent heat generation during the freezing of samples.*

#### **Figure 8.**

*Method C temperature curve for program 4, using temperature record data-logger.*

#### **Figure 9.**

*Curve of method C freezing cycle program in instrument-specific temperature record using program 4.*


#### **Table 7.**

*Difference between chamber and reference probe in ampule, and difference between reference probe in ampule and set temperature with regards to different programs.*

The instrument-specific temperature recording software has an option to define the ΔT between the set value and the actual value. If ΔT becomes smaller value, the program continues automatically. This is to assure that sample temperature has been stabilized at the chamber temperature before it is cooled down with the defined freezing rates of the freezing program.

*Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*


#### **Table 8.**

*Method C freezing cycle program in instrument-specific temperature record, with addition to the program.*

#### **4.3 PBMCs viability using method B and method C (comparative study)**

Viability was measured and compared before and after thawing [28]. Lowest result obtained was 95.9% and highest was 98.9% and the difference between each was between 0 and 1% which is in the acceptable range, shown in **Table 9**:

Then, method C protocol with first program that had the same cooling rate as in method B, with 1°C per 1 minute. Viability was measured and compared before and after thawing [28]. Lowest result obtained was 95.6% and the highest as 98.4% and difference between each was between 0 and 1.5% which is within an acceptable range, as shown in **Table 10** below:

Viability was measured based on the difference between pre-thaw and post-thaw viability percentages that were obtained from the flowcytometry. Viability 80% and 75% recoveries are recommended [28], and both methods are within acceptable ranges.

The initial hypothesis was that difference between method B and method C in terms of viability will not be statistically significant, which was proven by Student t test, as shown in **Table 11**. Statistical significance during this comparative study


**Table 9.** *Method B viability overview.*


#### **Table 10.**

*Method C viability overview.*


#### **Table 11.**

*Comparison between method B and method C, viability percentage.*

#### *Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*

was calculated using t test for temperature protocols (cooling rate variation): ItI < It tabI 0.003˂2.62 and post thaw viability detected on flow cytometer ItI < It tabI 0.03 < 3.06. Expected accuracy for post thaw viability interval was ±20% and obtained post thaw viability was 0.55%. Precision-reproducibility estimation during 5 different days was post thaw viability 0.66% when the acceptable interval was ±20%.

Precision estimation was designed through 4 days, 6 samples every day, for a total of 24 results from 12 CPT tubes. Between day variation was expected to be low as samples were in a stable frozen state. The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the reproducibility conditions. Statistical calculations were made in Microsoft Excel -Tools> Data Analysis> ANOVA, one factor test ordering Sw interserial standard deviation, and using Microsoft Excel functions to calculate Six, Sb and Stott. Results obtained are shown in **Table 12** below. Coefficient of variation (%) (Stot\*100/Xsr) was shown to be 0.79%. Hence, results of precision met the requested acceptable criteria. Viability percentages and difference in pre and post thaw viability was in acceptable criteria, lowest was 0.3 and highest was 4.1, as shown in **Figures 10** and **11**, respectively. **Table 13** illustrates all the validation study parameters that were taken and calculated.


**Table 12.** *CV % calculated.*

**Figure 10.** *Pre-thaw and post-thaw viability percentages.*

#### **Figure 11.**

*Pre-thaw and post-thaw viability differences.*


#### **Table 13.**

*Showing all values obtained through the validation study.*

### **5. Conclusions**

The presented study showed that there was no statistically significant difference in cooling methods. However, advantage of Method C is demonstrated in a major decrease in cooling time by reducing the PBMCs processing life cycle, without a need for intermediate storage space while sample traceability is enhanced by using the device software which can be integrated with our LIMS system. The risk of human error, which might occur with Method B, is minimized by reducing operator intervention. Both methods can be used in accordance with laboratory preferences, budget, and guidelines with integrated risk assessments and instrument downtime contingency plans.

*Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*

#### **Acknowledgements**

The authors would like to acknowledge the support that was received during the collection of these findings and the time given to produce this work. We would like to thank Professor Nahla Afifi for giving us this opportunity and believing in our work.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Nahla Afifi, Eiman Al-Khayat, Linda Hannigan, Monika Markovic Bordoski and Israa Khalaf\* Qatar Biobank for Medical Research, Qatar Foundation for Education, Science, and Community, Doha, Qatar

\*Address all correspondence to: ikhalaf@qf.org.qa

© 2022 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.

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[13] Jerram A, Guy T, Beutler L, Gunasegaran B, Sluyter R, Fazekas de St Groth B, et al. Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples. Bioscience Reports. 2022;**41**(2):BSR20203827

[14] Li Y, Mateu E, Díaz I. Impact of cryopreservation on viability, phenotype, *Impact of Different Cooling Methods on the Stability of Peripheral Blood Mononuclear Cells… DOI: http://dx.doi.org/10.5772/intechopen.107415*

and functionality of porcine PBMC. Frontiers in Immunology. 2022;**12**:765667

[15] Prickett R, Marquez-Curtis L, Elliott J, McGann L. Effect of supercooling and cell volume on intracellular ice formation. Cryobiology. 2022;**70**(2):156-163

[16] Internal validation report generated by Qatar Biobank [QBB]. 2018

[17] Muldrew K, McGann L. Mechanisms of intracellular ice formation. Biophysical Journal. 2022;**57**(3):525-532

[18] Gao D, Critser J. Mechanisms of cryoinjury in living cells. ILAR Journal. 2000;**41**(4):187-196

[19] Toner M, Cravalho E, Karel M. Thermodynamics and kinetics of intracellular ice formation during freezing of insrument-specific temperature recordical cells. Journal of Applied Physics. 2022;**67**(3):1582-1593

[20] Shu Z, Kang X, Chen H, Zhou X, Purtteman J, Yadock D, et al. Development of a reliable low-cost controlled cooling rate instrument for the cryopreservation of hematopoietic stem cells. Cytotherapy. 2022;**12**(2):161-169

[21] Drug Development and Delivery. Standardization Technology - Innovative Temperature Standardization Technology Supports Cell Therapy Clinical Trials [Internet]. 2022. Available from: https://drug-dev.com/standardizationtechnology-innovative-temperaturestandardization-technology-supportscell-therapy-clinical-trials/. [Accessed: July 19, 2022]

[22] Corning™ CoolCell™ LX Cell Freezing Vial Containers [Internet]. fishersci. 2022. Available from: https://www.fishersci.com/shop/ products/coolcell-lx-cell-freezing-vialcontainers/p-6936053. [Accessed: July 6, 2022]

[23] Buhl T, Legler T, Rosenberger A, Schardt A, Schön M, Haenssle H. Controlled-rate freezer cryopreservation of highly concentrated peripheral blood mononuclear cells results in higher cell yields and superior autologous T-cell stimulation for dendritic cell-based immunotherapy. Cancer Immunology, Immunotherapy. 2022;**61**(11):2021-2031

[24] Mazur P. Freezing of living cells: Mechanisms and implications. The American Journal of Physiology. 2022;**247**(3 Pt 1):C125-C142

[25] Ramachandran H, Laux J, Moldovan I, Caspell R, Lehmann P, Subbramanian R. Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in highthroughput human immune monitoring studies. Cell. 2022;**1**(3):313-324

[26] Holt W, Medrano A, Thurston L, Watson P. The significance of cooling rates and animal variability for boar sperm cryopreservation: Insights from the cryomicroscope. Theriogenology. 2022;**63**(2):370-382

[27] Stemcell.com. How to Cryopreserve PBMCs [Internet]. 2022. Available from: https://www.stemcell.com/how-tocryopreserve-pbmcs.html. [Accessed: July 4, 2022]

[28] Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved Guideline. CLSI Document MM13-a. Wayne, Pennsylvania, USA: Clinical and Laboratory Standards Institute; 2005

#### **Chapter 7**

## Plant Cryopreservation Importance, Approaches and Future Trends

*Victor Acheampong Amankwaah, Ruth Naa Ashiokai Prempeh and Marian Dorcas Quain*

#### **Abstract**

Plant cryopreservation is useful for long term storage of clonal germplasm and endangered species. Clonally propagated crops which produce recalcitrant seeds cannot be easily conserved using conventional methods. Preservation of plants *in vitro* is limited to two years and not ideal for germplasm storage for a very long time. The need to conserve plant genetic resources through cryopreservation techniques to mitigate the effects of climate change such as extinction of certain plant species cannot be underestimated. Different cryopreservation methods including dehydration, programmed freezing, vitrification and v cryo-plate are employed in the long-term storage of different plants. These methods are usually based on the principle of the removal of freezable water from tissues by physical or osmotic dehydration followed by ultra-rapid freezing. There have been several advancements in the identification and use of cryoprotective agents, nonetheless, its toxicity remains a challenge. To accelerate plant cryopreservation, there is the need for the development of global expertise. The current practice for the conservation of germplasm in the Biotechnology Laboratory in Ghana is through the use of slow growth media. Moving forward, there is the need to work on developing cryopreservation protocols for preservation of germplasm using liquid nitrogen and cryogenic refrigerators.

**Keywords:** cryopreservation, vitrification, conservation, gene bank, shoot tip

#### **1. Introduction**

As early as 2000 BC, archaeological findings has shown that icehouse were used throughout Mesopotamia to store foods [1]. Since time immemorial, the preservation of biological material has been known. The storage of biological material at ultralow temperatures is referred to as cryopreservation. In broad terms, cryopreservation refers to the study of life at low temperatures [2]. Plant cryopreservation is a conservation method that permits long-term storage of tissue samples at very low temperatures of −135°C to -196°C with little risk of causing variation. Cells can successfully be cryopreserved in liquid nitrogen when extracellular water has been removed to the extent that any remaining water form the so-called biological glass

(vitrification), thereby mitigating the adverse effect of ice crystal formation and growth [3]. Cryopreservation for storage of plant cells, tissues, and organs became operational in the 1960s till date. Long term storage of *in vitro* cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains a sine qua non for many scientists, organizations and companies [4]. In the case of clonally propagated crops which produce recalcitrant seeds and cannot be readily conserved by conventional methods through seed preservation, cryopreservation is important for long term conservation. Over the years, a lot of research on different crops to study the feasibility of the long-term storage of plant species has taken place. Prof. Akira Sakai, researched on mulberry twigs after exposing them to liquid nitrogen. This study is reported to be the pioneer in plant cryopreservation research [2, 5]. Research in cryopreservation in the twentieth century was devoted to basic studies of ice formation, vitrification of solutions and the beginnings of cryopreservation as a long-term storage technique [4]. In recent times, cryopreservation research has focused on practical procedures for gene bank storage, thereby enabling cells and meristems to be cryopreserved by direct transfer into liquid nitrogen. The development of simple and reliable methods for cryopreservation has led to cryo-banking [6].

#### **2. Importance of cryopreservation of plants**

A prerequisite for the short- and long-term survival of plant species in their natural habitat is genetic diversity [7]. Biological diversity conservation importance was recognized in 196 countries this led to the generation of a treaty that includes the sustainable use of its components, fair and equitable participation in the benefits derived from the use of plant resources [3]. The long-term conservation of tissues using cryopreservation has been increasingly used in recent years as it requires very little storage space, minimal upkeep, and eliminates the risk of contamination, makes the germplasm available for posterity, and its applicability to a wide range of plant tissues [6, 8].

#### **2.1 Advantages of cryopreservation over other methods**

Plant genetic resources are usually conserved in their natural habit (*in situ*) or other sites (*ex situ*). Preservation off site is partially used or for the entire population when preservation *in situ* is extremely challenging usually as a result of lack of complete control over many factors that influence the survival of plant materials and its genetic make-up [3]. Maintenance of plant genetic material *in vitro* is more efficient and secure than conservation in the field. *In vitro* conservation has been reported for the long-term conservation of germplasm for approximately two years without sub-culture such as in the case of potato. This notwithstanding, *in vitro* preservation is not ideal for long term germplasm conservation because it is labour consuming, costly, and carries risks of losing germplasm due to human error, such as contamination and mislabeling during sub-culturing [9]. Furthermore, erratic power supply, malfunctions in air-conditioning and lighting system could sometimes pose a challenge for *in vitro* conservation. Moreover, another setback of tissue culture for long term conservation is the induction of genetic variation or somaclonal variation during prolonged sub-culture [6]. Besides, mites, thrips, and other small arthropods can cause extensive fungal contaminations in tissue culture and are difficult to eliminate.

#### *Plant Cryopreservation Importance, Approaches and Future Trends DOI: http://dx.doi.org/10.5772/intechopen.108806*

In addition, tissue culture collections are constrained by the occurrence of cellular aging and senescence during prolonged cultivation. The effect of cellular aging may appear in parallel with slow growing endophytic microbes that can accumulate over time [10].

The only *ex situ* conservation method that allows long term survival of organisms at very low temperature and using reagents such as liquid nitrogen is cryopreservation. Plant materials stored in liquid nitrogen have indefinite lifespan in spite of the fact that no biological specimen is immortal [11]. Again, it is the only *ex situ* conservation method used for long-term conservation of plant materials that cannot be stored in seed banks, for instance clonal crops or species with a low number of progenies or recalcitrant seeds. Furthermore, it requires only a minimum space and maintenance efforts (**Figure 1a** and **b**). It has become a very important tool for the long-term storage of plant genetic material [11, 12]. Moreover, in addition to its use for the conservation of genetic resources, cryopreservation can also be applied for the safe storage of plant tissues with specific characteristics. Plant cells of different types, gametic cells, tissues and organs can be cryopreserved [13]. Due to the totipotency, various plant cells can be manipulated to enhance regrowth after cryopreservation, paying attention to genetic integrity.

#### **2.2 Food security, biotechnology and breeding**

The world's most important food crops for food, nutrition, and livelihoods most especially for the poorest people are vegetatively propagated crops. Examples of some of these crops include banana (*Musa sapientum*), plantain (*Musa paradisiaca*), sweetpotato (*Ipomoea batatas*), cassava (*Manihot esculenta*), yam (*Dioscorea* spp.), citrus (*Citrus* spp.) and coconut (*Cocos nucifera*) [14]. Plant genetic resources constitute the store of genome information and are important for world food security, crop improvement and conservation of genetic diversity [15]. It is important in breeding programs to obtain new or more productive plants that are resistant to biotic and abiotic stresses, due to the changing weather patterns [2]. Globally, food, feed and fiber utilization are restricted to very few species, hence, advanced biotechnology

#### **Figure 1.**

*Comparison of conservation of 150 plantain accessions on (a) RITA temporary immersion bioreactor system compared with the use of a (b) cryo-freezer in terms of space for storage at CSIR-CRI, Kumasi-Ghana.*

techniques such as cryopreservation represents an efficient alternative method for *ex situ* conservation of germplasm of various crop species. It helps in overcoming several challenges of storage by conventional means. In recent times over 10,000 accessions through initial *in vitro* introduction and subsequent preservation using cryogenic methods have been used for several crops. Above 80% of these crops belong to crops that are widely consumed such as potato, cassava, bananas, mulberry and garlic [16].

In modern breeding programs, cryopreservation is important for providing long-term storage and international access to various genetic materials. The genetic materials accessible internationally include seeds, pollen and meristematic apices and buds. Plant breeders and horticulturists involved in fruit and forest tree improvement are very much particular about pollen storage. Techniques for pollen culture have been used for decades to obtain haploids or homozygous diploid plants from various plant species such as maize and rice. Regular supply of viable pollen provided by pollen banks takes away seasonal, geographical or physiological limitations of hybridization programs and supports hybrid development between genera and species. Large field areas are required by traditional pollen banks at different stages to synchronize flowering both of which are very labour intensive and needs a lot of funds to be operational. Other methods of pollen banking for the purposes of breeding for shortterm storage such as freeze drying, freeze storage, vacuum drying and cold storage in organic solvents lead to frequently observed sharp reduction in pollen viability. The most efficient means of pollen storage is cryopreservation that does not require any expensive cryostats. This is so because pollen grains can be directly immersed in liquid nitrogen for long-term storage [16].

Advanced biotechnology application such as cryopreservation is a very good efficient method for *ex-situ* conservation of plant germplasm. This method supersedes the challenges and limitations of conventional methods seed banks and conventional orchards [2]. Preservation of plant germplasm for plant breeding and biotechnology has long been recognized and it is very essential for enhancing breeding activities. It has been reported that easy access to diverse plant germplasm is a pre-requisite for breeding more productive cultivars. This in the long run ensures food security [16, 17]. With respect to biotechnological interventions, the consistently evolving area of phyto-chemical production via biotechnological methods is supported by cryobanking of root cultures, embryogenic and non-embryogenic cell lines to ensure their genetic and biochemical stability [16, 18].

#### **2.3 Agrobiodiversity**

Plants are recognized as a vital component of biodiverse ecosystems (the carbon cycle, food production and bio-economy) [19]. An important issue concerning human population worldwide is the conservation of plant biodiversity. Plant biodiversity is a natural source of products to the food industries. Provision of basic raw materials is its hallmark. Maintenance of plant biodiversity in their natural habitat, as well as domesticated and cultivated species on the farm or in the surroundings where they have developed their distinctive characteristics, represent the *in situ* strategies. Due to heavy loss of species, populations and ecosystem composition leading to loss of biodiversity, *ex situ* conservation is a viable way for saving plants from extinction, and in some instances, it is the only possible strategy to conserve certain species [17]. Plant genetic resources are highly important for agro-biodiversity because they can be used to breed new or more productive crops that can withstand biological and environmental stresses [12, 13].

#### **2.4 Cryotherapy for virus elimination**

Systemic pathogens such as viruses, phytoplasmas and bacteria could be eliminated by treating shoot tips with liquid nitrogen using cryopreservation protocols. It is a novel approach for pathogen eradication in plants. The uneven distribution of viruses and other pathogens in shoot tips allows the elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from surviving pathogen-free meristematic cells. Cryopreservation methods have been useful in pathogen eradication by means of shoot tips cryotherapy [17]. The use of cryotherapy to remove viruses from vegetatively propagated crops has been reported [4]. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Consequently, it has the potential to replace more traditional methods like meristem culture, chemo- and thermo-therapies. This method has been utilized for eradication of severe pathogens in banana, citrus, grapevine, raspberry, potato and sweetpotato [17].

#### **2.5 Importance of cryopreservation in the era of climate change**

Greater risks of extreme weather and changes in climate variables such as prolonged drought and storms are events that biomes will have to adapt as one of the measures to prevent extinction [11, 20]. Effects of climate change on biodiversity, agricultural production and food security have been a matter of great concern [21]. The need to adopt strategies to conserve plant genetic resources to mitigate the effects of climate change that has a potential of causing the extinction of certain plant species cannot be underestimated. One strategy to address the issues of climate change in order not to lose endangered species is cryopreservation. For instance, critically endangered species growing in the wild in Finland has been successfully cryopreserved to enable its long-term conservation through the use of droplet vitrification protocol. Additionally, protocol development for cryogenic preservation of plant species is an additional tool to *ex situ* conservation toolbox for the maintenance of plants to avert the effects of climate change [11].

#### **3. Stages in cryopreservation**

Depending on the selected technique, cryopreservation is made up of different stages which includes preparation and explant excision, preculture, cryoprotection, vitrification/dehydration, fast cooling in liquid nitrogen, rewarming, cryoprotector elimination, regeneration and plant culture [8, 22].

In an effort to preserve biological materials for cryopreservation, the following steps are followed. The first step involves harvesting or selection of material, the growth stage has to be considered where applicable. Much attention should be paid to volume or size, density, pH and morphology. The second stage has to do with addition of cryo-protectant agents that include glycerol, salts, sugars, glycols that are added to samples. This stage is then followed by the application of different methods of freezing to protect cells from damage and cell death by their exposure to the warm solutions of cryoprotective agents (CPA). After all these have taken place, the cryopreserved samples are stored in −80°C in a freezer for at least 24 hours before transferring it to storage vessels. Finally, the process of thawing is initiated which involves warming the biological samples in order to control the rate of cooling and prevention of cell damage caused by crystallization [23].

#### **4. Plant material used for cryopreservation and cryopreservation agents**

The state of mother plant especially with regards to physiological state is a key factor for the success of cryopreservation. For cryopreservation techniques, any totipotent tissue may be used. Most commonly used tissues are shoot tips, and to a lesser extent, somatic embryos and embryonic axes. Shoot tips and somatic embryos for cryopreservation require tissue culture systems with established micropropagation regimes [24].

Decisions concerning the choice of a plant material for cryopreservation are dependent on plant type and reason for storage. Based on knowledge of plant vulnerability, curators need to make decisions on which plants to store based on their knowledge of plant vulnerability. The decision to select a plant part for cryopreservation technique depends on growth conditions. Generally, practice shows that plants that are diseased or not thriving for any reason are generally poor candidates for cryopreservation. Plant materials should be in an optimal growth phase, dormant materials should fully break dormancy, and where appropriate fully cold acclimated [25]. The question thus remains about how amenable plants indigenous to the tropical regions can respond successfully to cryopreservation.

Meristems and embryos are explants normally conserved using encapsulation techniques. Alginate beads which contain mineral salts and organic substances are used for the encapsulation of meristems and embryos. Cryopreservation agents are used for the treatment of plant genetic material as in the case of vitrification methods. The most commonly applied vitrification solutions include vitrification solution number 2, which contains glycerol, ethylene glycol and sucrose. These reagents are used by synseeds during regrowth so that they quickly grow to prevent loss of viability. Vitrification solutions contain penetrating and non-penetrating cryoprotective substances to preserve both inside and outside of plant genetic material and prevent the formation of lethal ice crystal so that cells remain viable for a long period of time [26].

Pollens are cryopreserved for breeding purposes. Viability of pollen after cryopreservation depends on a number of factors. Pollen moisture content, freezing and thawing procedure, physiological stage of mother plant, flowering stage, plant vigor and genotypic differences are the factors that determine pollen viability [16, 24].

#### **5. Methods of cryopreservation and application**

Different techniques are employed in the long-term storage of different plants. These techniques include dehydration, controlled-rate cooling and vitrification [4]. Cryopreservation technique is based on the principle of the removal of freezable water from tissues by physical or osmotic dehydration followed by ultra-rapid freezing. In cryopreservation procedures, water plays a central role in preventing freezing injury and in maintaining post-thaw viability of cryopreserved cells stored in a small volume, requiring a very limited maintenance. Classical freezing procedures encapsulates the use of different cryoprotective solutions combined with pre-growth of material followed by slow cooling (0.5–2.0°C/min) to a determined pre-freezing temperature (usually around −40°C), rapid immersion of samples in liquid nitrogen, storage, rapid thawing and recovery [17]. Cells with low water content which includes pollen, seeds, and dormant tissues of stress-tolerant species, may be introduced to low temperatures such as in the case of using liquid nitrogen without lethal damage. On the other hand, plant cells with higher water content present considerable problem as a result of ice crystal growth causing cell bursting [3].

*Plant Cryopreservation Importance, Approaches and Future Trends DOI: http://dx.doi.org/10.5772/intechopen.108806*

For any cryopreservation to be successful, it is important to avoid the lethal intracellular freezing that occurs during rapid cooling. Consequently, in any cryogenic procedure, the cells and shoot tips must be sufficiently dehydrated in order to preclude freezing and to allow vitrification in liquid nitrogen [12].

In the past 25 years, many cryopreservation techniques have been established based on the conventional slow freezing techniques. The different approaches used include vitrification, droplet vitrification, dehydration and pre-growth and pregrowth dehydration [19]. Cryopreservation methods are commonly used globally. It has been reported that new cryogenic methods using cryo-plates (the V cryo-plate and D cryo-plate) are advantageous over early developed methods. Advantages are manifested in ease of handling during the procedure and high regrowth rates after cryopreservation [12].

#### **5.1 Dimethyl sulfoxide droplet**

Since 1866, dimethyl sulfoxide (DMSO) has been commonly used for the cryopreservation of tissues because of its low cost and relatively low level of cytotoxicity [27]. DMSO acts by reducing the electrolyte concentration in the residual unfrozen solution in and around a cell at any given temperature. With this method, plant materials are treated with a 10% DMSO in liquid Murashige Skoog (MS) medium with 30 g sucrose, 0.5 mg/l zeatin riboside, 0.2 mg/l GA3 and 0.5 mg/l IAA. This method appears to be simple because only 10% DMSO in liquid medium is used as cryoprotectant solution. The explants (shoot tips of 2–3 mm) are then incubated in MSTo medium overnight at 22°C and treated with cryoprotectant solution (10% DMSO in MSTo medium) for 1–3 h at RT followed by transfer into droplets of 2.5 μl cryoprotectant solution one by one on aluminium foil. The aluminium foil is then immersed directly into cryotube filled with liquid nitrogen [6]. DMSO droplet has been routinely used for by the for safe and long-term conservation storage of shoot tips of sweetpotato by the International Potato Center (CIP) [9].

#### **5.2 Dehydration**

It involves dehydration of samples by either air current, silica gels, or incubation with cryoprotectant followed by rapid freezing or two-step freezing. It usually results in 100% recovery rate after liquid nitrogen drying in a laminar flow hood until 5–15% moisture content. For this technique, shoot tips or embryo are precultured on 0.3–0.6 M sucrose medium for 1–3 days. This is followed by encapsulation into alginate beads and treated with highly concentrated sucrose solution (approx. 0.8 M) for 16 h. These treatments induce tolerance in the samples. Following the pretreatment, plant genetic materials are dehydrated on silica gels or in a laminar flow cabinet to reach their optimal hydration levels. The advantage of this method is that it eliminates the need for other cryoprotectants that have been implicated in inducing genetic changes after cryopreservation such as DMSO and ethylene glycol [12].

#### **5.3 Programmed freezing**

With this method, samples are pretreated in cryoprotectants. The cryoprotectant agents used include DMSO, ethylene glycol and sucrose alone or in low concentration mixtures. Pretreated samples are dehydrated while frozen slowly (0.3–1°C/min) between −40°C and −70°C, then plunged directly into liquid nitrogen. This method

is based on the principle of free-induced dehydration. Programmed freezer that is expensive is required and it is a major disadvantage. Additionally, relatively long exposure of samples to subzero temperatures, which can be deleterious for coldsensitive species is also a disadvantage [16].

#### **5.4 Vitrification**

The physical process by which a highly concentrated cryoprotective solution super cools to very low temperatures and finally solidifies into a metastable glass without undergoing crystallization at a practical rate. It was proposed as a method for the cryopreservation of biological materials because it avoid the potentially detrimental effects of extracellular and intracellular freezing [25]. This process involves pre-culturing of plant tissues on basal medium supplemented with cryoprotectants, pre-treatment with loading solution, dehydration with PVS, and rapid freezing rewarming. In general, vitrification protocols have been very useful for cryopreserving complex organs like shoot tips, and somatic embryos that could not be effectively frozen following classical protocols. The vitrification method uses a highly concentrated solution. This solution sufficiently dehydrates tissues and does not lead to injury. This leads to the formation of a stable glass along with the surrounding highly concentrated solution plunged in liquid nitrogen. Cells or shoot tips must be sufficiently dehydrated with highly concentrated vitrification solution at 0°C or 25°C and should not lead to injury. Recovery rate is 74.5% with 5-day pre-culture on 0.5 M sucrose followed by PVS2 treatment for 1 h at 0°C. This method has been applied to several plants that includes tropical and subtropical species. It has been applied in the cocoa industry through cocoa somatic embryos [12].

#### **5.5 Droplet-vitrification**

Droplet-vitrification is a protocol derived from combination of droplet procedure with droplet freezing technique. With regards to all the steps, droplet-vitrification is similar to vitrification method but the only difference is that materials are cryopreserved on foil strips in drops of vitrification solution. It has been successfully used for rubber shoot tips. It has a relatively lower recovery rate of 43% regrowth with preculture on basal + proline (0.193 M) for 24 h in the dark at 25°C and PVS2 15 minutes at 0°C.

#### **5.6 V cryo-plate**

This method involves the culturing of plant material such as nodal segments in the case of potato on solid MS medium containing 30 g/l sucrose and 0.3 g/l CaCl2 at 20°C for 2 weeks. The shoot tips are then excised from the *in vitro* grown shoots and pre-cultured on MS medium containing sucrose at 25°C overnight. Pre-cultured shoot tips are then placed on aluminium cryo-plates with ten wells and embedded with calcium alginate gel. The next step is the performance of osmo-protection by immersing the cryo-plates for 30 minutes at 25°C in 25 ml pipetting reservoirs filled with MS medium with 2 M glycerol and 0.8 M sucrose. For dehydration step, the cryo-plates are transferred and immersed in another reservoirs filled with PVS2 for 30 minutes at 25°C. This is followed by the transfer of the cryo-plate in an uncapped 2 ml cryotube

in liquid nitrogen and immersed in a 2 ml cryotube and directly plunged into liquid nitrogen. The cryo-plate is then retrieved for rewarming in the in liquid nitrogen and immersed in a 2 ml cryotube containing 2 ml MS basal medium with 1 M sucrose, in which it is incubated for 15 minutes at room temperature. Rewarmed shoot tips are placed on solid MS medium and cultured under standard conditions [6].

#### **6. Future trends and challenges**

There have been several advancements in the identification and use of CPA in cryopreservation procedures. However, CPA toxicity remains a challenge in cryopreservation techniques. Mechanisms of the toxicity of CPA has not been understood fully [1]. Researchers are still working to better understand how different protective chemicals work to protect cells from the rigid temperature of liquid nitrogen.

Cryopreservation protocols have been developed for several crops. However, the number of crops represented in cryo-banks is still limited. Also the ability to successfully repeat the protocol in another laboratory has been a challenge. There is still more room for improvement for the cryopreservation of vegetatively propagated crops and the system requires a lot of optimisation. There is also the need for the development of efficient protocols, challenges related to cryo-banking capacities such as insufficient funding, lack of equipment and infrastructure, inadequate skilled personnel with knowledge on plant genetic resources [10]. The need for the acceleration of plant cryopreservation procedures especially for vegetatively propagated crops requires the development of global expertise. There should be a community of practice initiative involving curators of gene banks, researchers, advocacy organizations, academic institutions and other stakeholders to address the unmet need for cryopreservation advances. Other challenges should be outlined, underinvestment and untapped opportunities should also be identified [14]. There is the need for the establishment of pollen cryo-banks to facilitate a regular supply of pollen to support breeding programs for anther culture activities. The Biotechnology Laboratory at the Council for Scientific and Industrial Research (CSIR)-Crops Research Institute (CRI) in Kumasi, Ghana in sub-Sahara Africa receives germplasm of vegetatively propagated crops such as sweetpotato and cassava from research scientists in Ghana, CIP, International Institute of Tropical Agriculture (IITA) and other centers of the Consultative Group for International Agricultural Research (CGIAR). The current practice for preservation of these plant materials is conservation using slow growth media. Moving forward, the Biotechnology Laboratory in Ghana, should work on developing cryopreservation protocols for preservation of germplasm using liquid nitrogen and cryogenic refrigerators. Also, there is the need for the development of cryotherapy protocols for virus elimination of vegetatively propagated crops sent to the laboratory for *in vitro* propagation and long-term conservation.

A new threat for conserving global biodiversity in addition to other human activities that could lead to global mass extinction of germplasm is climate change. Recent approaches to *in situ* conservation are not very reliable to address anticipated changes. Therefore, there is the urgent need for the creation of new cryogenic models, protocols and technologies to mitigate the threats of climate change. Since cryopreservation is the safest and most cost-effective strategy for long-term conservation of germplasm of economically important plant species as well as endangered species [16].

#### **7. Conclusions**

Cryopreservation has been in existence since 2000 BC as demonstrated in archaeological findings that icehouse were used throughout Mesopotamia to store foods. In simple terms cryopreservation refers to the study of life at low temperatures. Plant cryopreservation on the other hand refers to conservation method for long-term storage of tissue samples at very low temperatures of −135°C to −196°C. The risk of causing variation is usually less. Cryopreservation methods are suitable are very useful for long-term storage of in vitro cultures of secondary metabolite cell cultures, embryonic cultures, clonal germplasm, endangered species, and transgenic products. The advantages of cryopreservation of plant genetic materials are enormous with several advantages of cryopreservation over other methods. Cryotherapy for virus elimination hold a lot of potential for crop germplasm dissemination.

The development of cryopreservation protocols are enormous. However, the number of crops represented in cryo-banks are still limited. For the acceleration of cryopreservation, there is the need for the development of global expertise. It is recommended that a community of practice initiative involving gene banks, researchers, advocacy organizations, academic institutions and other stakeholders come together to address the gaps in cryopreservation advances

#### **Acknowledgements**

The authors wish to express their sincere thanks to scientists and technicians at the CSIR-Crops Research Biotechnology Laboratory who contributed in one way or the other towards the preparation of this book chapter.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Victor Acheampong Amankwaah\*, Ruth Naa Ashiokai Prempeh and Marian Dorcas Quain Biotechnology, Seed Science and Postharvest Division, CSIR-Crops Research Institute, Kumasi, Ghana

\*Address all correspondence to: va.amankwaah@gmail.com

© 2022 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.

*Plant Cryopreservation Importance, Approaches and Future Trends DOI: http://dx.doi.org/10.5772/intechopen.108806*

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### *Edited by Marian Quain*

Cryopreservation is the preservation of cells at sub-zero temperatures. Although useful, the process is not without its challenges. One of the major challenges is the formation of ice crystals in the cells to be preserved. However, vitrification, where glass is formed instead of crystals, can be achieved using cryoprotection agents (CPAs). This book provides a comprehensive overview of cryopreservation and its applications. It discusses advancements in the field, challenges, guidelines, and recommendations for successful germplasm conservation. Chapters discuss the use of CPAs, cryopreservation of fish sperm, cryopreservation of oocytes and sperms, female fertility preservation, cryopreservation of large structures and tissues, and much more.

### *Robert Koprowski, Biomedical Engineering Series Editor*

Published in London, UK © 2023 IntechOpen © blackdovfx / iStock

Cryopreservation - Applications and Challenges

IntechOpen Series

Biomedical Engineering, Volume 18

Cryopreservation

Applications and Challenges

*Edited by Marian Quain*