Cryobiology Aiding Organ Transplant

*Cryopreservation - Current Advances and Evaluations*

semen. Research in Veterinary Science.

[155] Bucak MN, Sarıözkan S, Tuncer PB,

[156] Bucak M, Ataman M, Başpınar N, Uysal O, Taşpınar M, Bilgili A, et al. Lycopene and resveratrol improve post-thaw bull sperm parameters: Sperm motility, mitochondrial activity and DNA integrity. Andrologia.

Sakin F, Ateşşahin A, Kulaksız R, et al. The effect of antioxidants on post-thawed angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research.

2009;**87**:468-472

2010;**89**:24-30

2015;**47**:545-552

**116**

**119**

**Chapter 6**

**Abstract**

**1. Introduction**

Techniques

Current Advancements in

ments that have led to better outcomes to islet health.

Pancreatic Islet Cryopreservation

*Samuel Rodriguez, David Whaley, Michael Alexander,* 

*Mohammad Rezaa Mohammadi and Jonathan R.T. Lakey*

There have been significant advancements in the research of pancreatic islet transplantations over the past 50 years as a treatment for Type 1 Diabetes Mellitus (T1DM). This work has resulted in hundreds of clinical islet transplantation procedures internationally. One limitation of the procedure includes effective storage techniques during donor-recipient cross-matching following islet isolation from deceased donor. Cryopreservation, which is heavily used in embryology research, has been proposed as a prospective method for pancreatic islet banking to bridge the temporal intervals between donor-recipient matching. The cryopreservation methods currently involve the freezing of islets to subzero (−80/−196°C) temperatures for storage followed by a thawing and warming period, which can be increasingly harmful to islet viability and insulin secretion capabilities. Recent advances in islet cryopreservation technologies have improved outcomes for islet health and survivability during this process. The aim of this chapter is to characterize aspects of the islet cryopreservation method while reviewing current procedural improve-

**Keywords:** cryopreservation, islet, cryoprotectant, alginate, vitrification, diabetes

Pancreatic islet transplantations are currently used in human clinical studies to treat Type 1 Diabetes Mellitus (T1DM); however, one of the major limitations of this therapy remains efficient and effective storage of islet prior to transplant, during donor-recipient cross-matching [1]. Islet cryopreservation has a distinctly vigorous research history as the storage, transportation, and overall preservation are critical steps in islet transportation. The first human islet allotransplantation took place at the Washington University in the 1980s, which provided proof of insulin independence following the procedure [2]. Following these early successes, clinical trials at the University of Pittsburgh observed further prolonged insulin independence using an improved steroid-free immunosuppressive regimen [3]. These initial clinical islet transplantations demonstrated the need for a preservation process to bridge a temporal gap between islet isolation from donor and transplantation of islet graft into recipients. In 1989, clinical trials at the University of Alberta were able to use both freshly isolated islets supplemented with cryopreserved islets

## **Chapter 6**

## Current Advancements in Pancreatic Islet Cryopreservation Techniques

*Samuel Rodriguez, David Whaley, Michael Alexander, Mohammad Rezaa Mohammadi and Jonathan R.T. Lakey*

## **Abstract**

There have been significant advancements in the research of pancreatic islet transplantations over the past 50 years as a treatment for Type 1 Diabetes Mellitus (T1DM). This work has resulted in hundreds of clinical islet transplantation procedures internationally. One limitation of the procedure includes effective storage techniques during donor-recipient cross-matching following islet isolation from deceased donor. Cryopreservation, which is heavily used in embryology research, has been proposed as a prospective method for pancreatic islet banking to bridge the temporal intervals between donor-recipient matching. The cryopreservation methods currently involve the freezing of islets to subzero (−80/−196°C) temperatures for storage followed by a thawing and warming period, which can be increasingly harmful to islet viability and insulin secretion capabilities. Recent advances in islet cryopreservation technologies have improved outcomes for islet health and survivability during this process. The aim of this chapter is to characterize aspects of the islet cryopreservation method while reviewing current procedural improvements that have led to better outcomes to islet health.

**Keywords:** cryopreservation, islet, cryoprotectant, alginate, vitrification, diabetes

## **1. Introduction**

Pancreatic islet transplantations are currently used in human clinical studies to treat Type 1 Diabetes Mellitus (T1DM); however, one of the major limitations of this therapy remains efficient and effective storage of islet prior to transplant, during donor-recipient cross-matching [1]. Islet cryopreservation has a distinctly vigorous research history as the storage, transportation, and overall preservation are critical steps in islet transportation. The first human islet allotransplantation took place at the Washington University in the 1980s, which provided proof of insulin independence following the procedure [2]. Following these early successes, clinical trials at the University of Pittsburgh observed further prolonged insulin independence using an improved steroid-free immunosuppressive regimen [3]. These initial clinical islet transplantations demonstrated the need for a preservation process to bridge a temporal gap between islet isolation from donor and transplantation of islet graft into recipients. In 1989, clinical trials at the University of Alberta were able to use both freshly isolated islets supplemented with cryopreserved islets

in two T1DM patients, which resulted in partial graft function [4]. The international trial on Edmonton protocol reported success after some of over 43 Type 1 diabetic patients achieved either partial or complete insulin independence for up to 3–5 years post-transplantation [5, 6]. After the establishment of the Edmonton protocol, the Clinical Islet Transplantation Registry (CITR) has recorded more than 1500 human islet allotransplant recipients, which is projected to increase steadily in the future [7]. The Edmonton method of islet isolation was shown to improve islet survival during islet cryopreservation as well. A study comparing cryopreserved islets before the establishment of the Edmonton protocol to human islets treated via Edmonton method observed a 24-hour survival rate increase of 19.3% (50.1 versus 69.4% respectively) with added increases recorded after 7 days of culture [8]. Although the impact of the Edmonton protocol on cryopreservation is significant, there is still vast room for improvement in the islet cryopreservation process.

A major problem of the transplantation field is the lack of human donors' sources; the islet transplantation process is particularly hard-hit from this problem since each transplant recipient must be infused with islets from multiple pancreases [9]. One recipient may also require multiple infusions of donor islets, which can further strain the donor source [10, 11]. With the epidemiologic increase in IDDM diagnosis, islet allotransplantation islet supply will be increasingly strained by the growing demand for islet replacement therapy with projected increases in the population of IDDM individuals [12]. The islet donor supply problems can be partially addressed from improvements in human islet yield, purity, and function. Although an average human cadaveric pancreas contains over 1 million islets, the human islet isolation process can be especially harsh on the isolation yield resulting in loses of up to 50% depending on the degree of success of the isolation [13, 14]. Cryopreservation techniques have been employed to address some of these islet isolation and preservation issues before islets are implanted into the recipient.

The human islet isolation and culturing process involves several steps that vary in temperature, each of which has its own benefits and deficits regarding the health of the islets [13]. Islet procurement from a whole donor pancreas first exposes islet to 4°C (histidine-tryptophan ketoglutarate solution) during sterile transportation to an approved clinical islet center. During the pancreas digestion and purification, the islets are exposed to varying temperatures between 4°C and room temperature. Preservation of islet currently involves the cooling of isolated islets in a temperature-regulated solution at 4°C prior to culturing as prolonged warm-ischemia will increase islet death and subsequent decrease in islet yield [13, 15, 16]. The final step of islet preparation involves a 24- to72-hour culture in approved islet media at 37°C, which has been shown to improve islet yield and functions with reductions in dead/apoptotic islet cell mass [17, 18]. Isolated human islets are preserved while donor islets of similar cross-matching biocompatibility are compiled and matched to an islet recipient [13]. During this critical time period, cryopreservation has been suggested as a preservation method for use during the pre-transplantation period to improve islet health. Improvements in the islet cryopreservation field can translate directly to improvements in human islet cyro-banking as well. This book chapter outlines the history of islet cryopreservation, current techniques in freezing/thawing periods, and cryoprotective additives.

## **1.1 History of islet cryopreservation**

Following the discovery of the microscope, Spallanzani observed that sperm could maintain mobility even when exposed to cold temperature conditions in

**121**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

1776 [19]. Research into the effects of cryopreservation on live tissue had its roots in late 1800s when scientists used this technology to preserve both spermatozoa and red blood cells (RBCs). During this time, research demonstrated weaknesses in the process which caused inconsistent results and frequent infertility caused by early embryonic death. A breakthrough occurred in the 1950s when James Lovelock discovered that the cryopreservation process caused osmotic stress in the cell by instantly freezing the liquid and causing the formation of ice crystals in RBCs. In 1963, Mazur et al. were able to characterize that process when they demonstrated that the rate of temperature change within a cell-containing medium controlled the movement of water across a cell membrane and thus the degree of intracellular freezing [20]. This together helped to improve the overall understanding of the mechanism associated with the cryoprotective process. During the 1980s, research surrounding the cryopreservation process revealed that the speed at which the freezing and thawing process occurred was the most important factor in determining the survivability of the cells [21, 22]. It was demonstrated that small, slow increments in both the freezing and thawing processes prevented the rapid formation of ice crystals and increased membrane-bound solutes associated with early cell death [23]. Another initial advance in cryopreservation occurred in the late 1940s when researchers discovered that the use of glycerol as a medium increased the survivability of spermatozoa in subfreezing (−70°C) temperatures [24]. Using glycerol as a medium effectively served to protect the cells from rapid formation of ice crystal during the preservation process. A commonly used cyroprotective agent currently employed is dimethyl sulfoxide (DMSO), which is added to cell media prior to the freezing process [25, 26]. DMSO (10%) when added to the cell media, commonly at 2 M concentration, increases the porosity of the cellular membrane, which allows water to flow more freely through the membrane [27, 28]. In addition, early research has demonstrated that nucleation is another way to prevent the rapid formation of ice crystals during freezing [28, 29]. During the freezing process, a metal rod supercooled with liquid nitrogen is applied to the meniscus of the medium containing islets wherein the liquid molecules begin to nucleate. These nucleation reactions are due to the release of latent heat of fusion from the medium, which causes the

During the 1970s, cryopreservation technology was applied to rat islet preservation in both storage and transportation, which demonstrated maintenance of high viability and function, which showed no significant difference when compared to non-treated islets [30, 31]. In one study, T1DM rats received allogeneic islet transplants cryopreserved using 2 M DMSO (Freezing rate = 0.25°C/min; Thawing rate = 7.5°C), which caused normoglycemia for up to 3 months (**Figure 1**). Additionally, when a modified cryopreservation protocol was applied to canine allotransplantations (freezing rate = 0.25°C/min; thawing rate = 3.4°C/min), T1DM canine recipients demonstrated prolonged glycemic control for up to 18 months [32]. These results highlighted the potential use of cryopreservation for islet transplantations in both small and large animal models in addition to differences required when cryofreezing small and large animal pancreases. Recent advances in islet quality control like oxygen consumption rate (OCR), qPCR, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay allow researchers to study islet cryopreservation technology more effectively [33]. Islet cryopreservation research is a very active research topic with many studies aiming to characterize and improve cryopreservation freezing/thawing processing, benefits of potential cyroprotective additives, and the effects of encapsulation on islet function during cryopreservation. The following section will discuss the islet

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

temperature to decrease more homogenously.

cryopreservation process (**Figure 2**).

## *Current Advancements in Pancreatic Islet Cryopreservation Techniques DOI: http://dx.doi.org/10.5772/intechopen.89363*

*Cryopreservation - Current Advances and Evaluations*

in two T1DM patients, which resulted in partial graft function [4]. The international trial on Edmonton protocol reported success after some of over 43 Type 1 diabetic patients achieved either partial or complete insulin independence for up to 3–5 years post-transplantation [5, 6]. After the establishment of the Edmonton protocol, the Clinical Islet Transplantation Registry (CITR) has recorded more than 1500 human islet allotransplant recipients, which is projected to increase steadily in the future [7]. The Edmonton method of islet isolation was shown to improve islet survival during islet cryopreservation as well. A study comparing cryopreserved islets before the establishment of the Edmonton protocol to human islets treated via Edmonton method observed a 24-hour survival rate increase of 19.3% (50.1 versus 69.4% respectively) with added increases recorded after 7 days of culture [8]. Although the impact of the Edmonton protocol on cryopreservation is significant, there is still vast room for improvement in the islet cryopreservation process. A major problem of the transplantation field is the lack of human donors' sources; the islet transplantation process is particularly hard-hit from this problem since each transplant recipient must be infused with islets from multiple pancreases [9]. One recipient may also require multiple infusions of donor islets, which can further strain the donor source [10, 11]. With the epidemiologic increase in IDDM diagnosis, islet allotransplantation islet supply will be increasingly strained by the growing demand for islet replacement therapy with projected increases in the population of IDDM individuals [12]. The islet donor supply problems can be partially addressed from improvements in human islet yield, purity, and function. Although an average human cadaveric pancreas contains over 1 million islets, the human islet isolation process can be especially harsh on the isolation yield resulting in loses of up to 50% depending on the degree of success of the isolation [13, 14]. Cryopreservation techniques have been employed to address some of these islet isolation and preservation issues before islets are implanted into the recipient. The human islet isolation and culturing process involves several steps that vary in temperature, each of which has its own benefits and deficits regarding the health of the islets [13]. Islet procurement from a whole donor pancreas first exposes islet to 4°C (histidine-tryptophan ketoglutarate solution) during sterile transportation to an approved clinical islet center. During the pancreas digestion and purification, the islets are exposed to varying temperatures between 4°C and room temperature. Preservation of islet currently involves the cooling of isolated islets in a temperature-regulated solution at 4°C prior to culturing as prolonged warm-ischemia will increase islet death and subsequent decrease in islet yield [13, 15, 16]. The final step of islet preparation involves a 24- to72-hour culture in approved islet media at 37°C, which has been shown to improve islet yield and functions with reductions in dead/apoptotic islet cell mass [17, 18]. Isolated human islets are preserved while donor islets of similar cross-matching biocompatibility are compiled and matched to an islet recipient [13]. During this critical time period, cryopreservation has been suggested as a preservation method for use during the pre-transplantation period to improve islet health. Improvements in the islet cryopreservation field can translate directly to improvements in human islet cyro-banking as well. This book chapter outlines the history of islet cryopreservation, current techniques in freezing/thawing periods, and cryopro-

**120**

tective additives.

**1.1 History of islet cryopreservation**

Following the discovery of the microscope, Spallanzani observed that sperm could maintain mobility even when exposed to cold temperature conditions in

1776 [19]. Research into the effects of cryopreservation on live tissue had its roots in late 1800s when scientists used this technology to preserve both spermatozoa and red blood cells (RBCs). During this time, research demonstrated weaknesses in the process which caused inconsistent results and frequent infertility caused by early embryonic death. A breakthrough occurred in the 1950s when James Lovelock discovered that the cryopreservation process caused osmotic stress in the cell by instantly freezing the liquid and causing the formation of ice crystals in RBCs. In 1963, Mazur et al. were able to characterize that process when they demonstrated that the rate of temperature change within a cell-containing medium controlled the movement of water across a cell membrane and thus the degree of intracellular freezing [20]. This together helped to improve the overall understanding of the mechanism associated with the cryoprotective process. During the 1980s, research surrounding the cryopreservation process revealed that the speed at which the freezing and thawing process occurred was the most important factor in determining the survivability of the cells [21, 22]. It was demonstrated that small, slow increments in both the freezing and thawing processes prevented the rapid formation of ice crystals and increased membrane-bound solutes associated with early cell death [23]. Another initial advance in cryopreservation occurred in the late 1940s when researchers discovered that the use of glycerol as a medium increased the survivability of spermatozoa in subfreezing (−70°C) temperatures [24]. Using glycerol as a medium effectively served to protect the cells from rapid formation of ice crystal during the preservation process. A commonly used cyroprotective agent currently employed is dimethyl sulfoxide (DMSO), which is added to cell media prior to the freezing process [25, 26]. DMSO (10%) when added to the cell media, commonly at 2 M concentration, increases the porosity of the cellular membrane, which allows water to flow more freely through the membrane [27, 28]. In addition, early research has demonstrated that nucleation is another way to prevent the rapid formation of ice crystals during freezing [28, 29]. During the freezing process, a metal rod supercooled with liquid nitrogen is applied to the meniscus of the medium containing islets wherein the liquid molecules begin to nucleate. These nucleation reactions are due to the release of latent heat of fusion from the medium, which causes the temperature to decrease more homogenously.

During the 1970s, cryopreservation technology was applied to rat islet preservation in both storage and transportation, which demonstrated maintenance of high viability and function, which showed no significant difference when compared to non-treated islets [30, 31]. In one study, T1DM rats received allogeneic islet transplants cryopreserved using 2 M DMSO (Freezing rate = 0.25°C/min; Thawing rate = 7.5°C), which caused normoglycemia for up to 3 months (**Figure 1**). Additionally, when a modified cryopreservation protocol was applied to canine allotransplantations (freezing rate = 0.25°C/min; thawing rate = 3.4°C/min), T1DM canine recipients demonstrated prolonged glycemic control for up to 18 months [32]. These results highlighted the potential use of cryopreservation for islet transplantations in both small and large animal models in addition to differences required when cryofreezing small and large animal pancreases. Recent advances in islet quality control like oxygen consumption rate (OCR), qPCR, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay allow researchers to study islet cryopreservation technology more effectively [33]. Islet cryopreservation research is a very active research topic with many studies aiming to characterize and improve cryopreservation freezing/thawing processing, benefits of potential cyroprotective additives, and the effects of encapsulation on islet function during cryopreservation. The following section will discuss the islet cryopreservation process (**Figure 2**).

## **Figure 1.**

*Sample image of encapsulated human islets stained with dithizone for 15 min, taken at 2× magnification with objective lens 20/40 PH. Scale bar represents 2 mm. Imaging performed at UCI laboratory under supervision of Dr. Jonathan Lakey PhD.*

## **Figure 2.**

*Flowchart of cryopreservation. This chart describes the range of temperature, rate of temperature change, and the procedure involved during cryopreservation.*

## **2. Characteristics of the islet cryopreservation process**

## **2.1 Background**

A crucial aspect of cryofreezing islets is the rate of freezing and thawing, which can have major effects on the islet health and morphology. The freezing process describes the process of cooling the islet-containing medium to around −196°C. If the freezing process is done too rapidly (>0.25°C/min), the liquid in the medium will freeze too quickly and crystal ice structure will form within cell membranes. Conversely, if the freezing process is performed too slowly (<0.1°C/min), then innate/adaptive immune cells, such as macrophages, dendritic cells, and lymphocytes which are present within the islet medium survive in greater numbers and can contribute to foreign body response (FBR)-mediated graft rejection upon transplantation [3, 34]. Taylor et al. demonstrated increase in macrophage viability (91%) cryofreezing is done at a rate of 0.1°C/min compared to 72–75% viable macrophage when the rate between 0.1–20°C/min [35]. Therefore, a key aspect of cryofreezing is the use of an optimal freezing rate based on islet type and volume to prevent ice crystal formation and immune cell survival. Over the years, many studies have described varying optimal freezing rates, which has made it difficult to compare between freezing/thawing methods. A consistent freezing/thawing

**123**

ing CPAs.

**2.3 Thawing**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

protocol uses slow freezing from −40 to −196°C followed by a rapid thawing starting from −196°C [36]. An early study aimed at characterizing differences between cooling and thawing rates exposed islets to several freezing rates between 0.3 and 100°C/min and thawing rates of 10 or 50°C/min. Highest survival rates were detected at 0.3°C/min rate with slight decreases observed between 60 and 1000°C/min rates [37]. The study also demonstrated the critical nature of using DMSO as a medium to protect the islet viability and function from cooling process. Cyroprotective agents (CPAs) like DMSO are neutral solutes of both low toxicity and molecular weight that replace up to 30% of the cell water and provide optimal conditions for subzero temperatures [38]. While a variety of DMSO concentrations have been tested, the most popular one used is 2 M DMSO, which is added in a stepwise fashion (1 M DMSO to 2 M DMSO) during pre-freezing [36]. One study found that when islets were exposed to 1 M DMSO for 30 min followed by incubation in 2 M DMSO for 10 min before cooling phase, then the islet insulin secretory patterns were improved after thawing [39]. More recently, studies have shown that the rate of cooling is much less important than the use of cryoprotective additives

The process of exposure and equilibration of permeating cyroprotective additives to islets is known as vitrification, which was first described by Rall et al. [38, 40]. While the use of CPAs reduces the risk of rapid ice crystal formation during the cooling phase, cryoprotective agents, such as DMSO and ethylene glycol, have been shown to be toxic to islet viability and function when concentrated in the medium [38, 41, 42]. Vitrification is used to slow the exposure of islets to CPAs by adding the CPAs in a stepwise fashion, usually in ascending concentrations of CPA, thereby allowing the CPAs to slowly permeate and form a solute equilibrium across the cell membrane. The vitrification process also causes water to flow extracellularly where, during cooling, vitreous water crystals slowly form outside the islet cells [38]. This vitreous medium exists in a solid-liquid transition state that is maintained at a supercooled temperature (≤100°C), thus having the structure of a liquid but behaving mechanically like a solid [43]. In addition to reducing ice crystal formation, vitrification involves exposing the CPAs in a stepwise fashion (1 M DMSO followed by 2 M DMSO), thus reducing the toxicity of the CPAs as the cooling process proceeds. Once the cooling process is finished, the cryopreserved islets will be stored at

When the supercooled islet is in −196°C storage, the vitreous medium is still locked in a liquid-solid transition state; however, once the warming process begins, there is risk of ice recrystallization within the medium, which can damage the islets [36, 43]. A widely accepted procedure includes the use of rapid thawing from −196°C at a rate of 150–200°C/min. Mechanical agitation is applied to thawing samples in a 37°C water bath until all ice is gone (0°C). At 0°C, the samples are then placed in an ice slush bath (0°C) until thawing is completed [36]. Following the warming phase, it is important to remove the cryoprotectant from the medium. After thawing is complete, samples are spun down at 1500 RPM and supernatants containing most of the cryoprotectants are removed. The pellet is then resuspended in an isotonic buffer (0.75 M sucrose) to dilute out the remain-

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

during the pre-freezing phase [36, 39].

−196°C liquid nitrogen freezer until use.

**2.2 Vitrification**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques DOI: http://dx.doi.org/10.5772/intechopen.89363*

protocol uses slow freezing from −40 to −196°C followed by a rapid thawing starting from −196°C [36]. An early study aimed at characterizing differences between cooling and thawing rates exposed islets to several freezing rates between 0.3 and 100°C/min and thawing rates of 10 or 50°C/min. Highest survival rates were detected at 0.3°C/min rate with slight decreases observed between 60 and 1000°C/min rates [37]. The study also demonstrated the critical nature of using DMSO as a medium to protect the islet viability and function from cooling process. Cyroprotective agents (CPAs) like DMSO are neutral solutes of both low toxicity and molecular weight that replace up to 30% of the cell water and provide optimal conditions for subzero temperatures [38]. While a variety of DMSO concentrations have been tested, the most popular one used is 2 M DMSO, which is added in a stepwise fashion (1 M DMSO to 2 M DMSO) during pre-freezing [36]. One study found that when islets were exposed to 1 M DMSO for 30 min followed by incubation in 2 M DMSO for 10 min before cooling phase, then the islet insulin secretory patterns were improved after thawing [39]. More recently, studies have shown that the rate of cooling is much less important than the use of cryoprotective additives during the pre-freezing phase [36, 39].

## **2.2 Vitrification**

*Cryopreservation - Current Advances and Evaluations*

**2. Characteristics of the islet cryopreservation process**

A crucial aspect of cryofreezing islets is the rate of freezing and thawing, which can have major effects on the islet health and morphology. The freezing process describes the process of cooling the islet-containing medium to around −196°C. If the freezing process is done too rapidly (>0.25°C/min), the liquid in the medium will freeze too quickly and crystal ice structure will form within cell membranes.

*Flowchart of cryopreservation. This chart describes the range of temperature, rate of temperature change, and* 

*Sample image of encapsulated human islets stained with dithizone for 15 min, taken at 2× magnification with objective lens 20/40 PH. Scale bar represents 2 mm. Imaging performed at UCI laboratory under supervision of* 

Conversely, if the freezing process is performed too slowly (<0.1°C/min), then innate/adaptive immune cells, such as macrophages, dendritic cells, and lymphocytes which are present within the islet medium survive in greater numbers and can contribute to foreign body response (FBR)-mediated graft rejection upon transplantation [3, 34]. Taylor et al. demonstrated increase in macrophage viability (91%) cryofreezing is done at a rate of 0.1°C/min compared to 72–75% viable macrophage when the rate between 0.1–20°C/min [35]. Therefore, a key aspect of cryofreezing is the use of an optimal freezing rate based on islet type and volume to prevent ice crystal formation and immune cell survival. Over the years, many studies have described varying optimal freezing rates, which has made it difficult to compare between freezing/thawing methods. A consistent freezing/thawing

**122**

**2.1 Background**

*the procedure involved during cryopreservation.*

**Figure 2.**

**Figure 1.**

*Dr. Jonathan Lakey PhD.*

The process of exposure and equilibration of permeating cyroprotective additives to islets is known as vitrification, which was first described by Rall et al. [38, 40]. While the use of CPAs reduces the risk of rapid ice crystal formation during the cooling phase, cryoprotective agents, such as DMSO and ethylene glycol, have been shown to be toxic to islet viability and function when concentrated in the medium [38, 41, 42]. Vitrification is used to slow the exposure of islets to CPAs by adding the CPAs in a stepwise fashion, usually in ascending concentrations of CPA, thereby allowing the CPAs to slowly permeate and form a solute equilibrium across the cell membrane. The vitrification process also causes water to flow extracellularly where, during cooling, vitreous water crystals slowly form outside the islet cells [38]. This vitreous medium exists in a solid-liquid transition state that is maintained at a supercooled temperature (≤100°C), thus having the structure of a liquid but behaving mechanically like a solid [43]. In addition to reducing ice crystal formation, vitrification involves exposing the CPAs in a stepwise fashion (1 M DMSO followed by 2 M DMSO), thus reducing the toxicity of the CPAs as the cooling process proceeds. Once the cooling process is finished, the cryopreserved islets will be stored at −196°C liquid nitrogen freezer until use.

## **2.3 Thawing**

When the supercooled islet is in −196°C storage, the vitreous medium is still locked in a liquid-solid transition state; however, once the warming process begins, there is risk of ice recrystallization within the medium, which can damage the islets [36, 43]. A widely accepted procedure includes the use of rapid thawing from −196°C at a rate of 150–200°C/min. Mechanical agitation is applied to thawing samples in a 37°C water bath until all ice is gone (0°C). At 0°C, the samples are then placed in an ice slush bath (0°C) until thawing is completed [36]. Following the warming phase, it is important to remove the cryoprotectant from the medium. After thawing is complete, samples are spun down at 1500 RPM and supernatants containing most of the cryoprotectants are removed. The pellet is then resuspended in an isotonic buffer (0.75 M sucrose) to dilute out the remaining CPAs.

## **3. Factors that affect islet cryopreservation process**

## **3.1 Background**

While the use of CPAs is a critical step, there are several factors that play a significant role in the success of an islet cryopreservation process. Technological advances in these areas can help reduce the stress of the process on the islets while more effectively processing the islets tissue during cryopreservation.

## *3.1.1 Oxygen treatment*

Changes in ambient atmospheric oxygen concentration can have a negative effect on islet viability and function via ATP metabolite depletion. In hypoxic conditions, β-cells are put under oxidative stress followed by ROS production, which facilitates islet death [44]. The thawing and rewarming phases have been shown to place hypoxic-related oxidative stress on cryopreserved tissues [45]. During the thawing/rewarming period, as islets are brought back from subfreezing temperature, cellular enzymes begin to function and increase oxygen consumption. This process can cause a reduction in adenosine 3-phosphate (ATP), a cellular metabolite, which can lead to islet death [45]. To address this issue, researchers hypothesized that hyperbaric conditions might improve islet recovery. Human islets were exposed to both normal oxygen conditions (21% O2, 74% N2, and 5% CO2) and hyperbaric conditions (50% O2, 45% N2, and 5% CO2) at 22°C for 45 min followed by 37°C for 45 min. Short-term (post-rewarming) and long-term (2-day culture) islet function analysis was conducted via GSIS, qPCR ischemia-gene analysis, and islet metabolism via oxygen consumption rate (OCR) assay. Long-term culture also compared normal and hyperbaric culture conditions. No significant short-term function and metabolic differences were observed between conditions and non-treated islets. However, the hyperbaric conditions were shown to suppress the increases of inflammation detected in untreated cryopreserved islets. Islet recovery after long-term culture was significantly better under hyperbaric conditions and was shown to increase after hyperbaric 2-day culture. A recent advancement in organ oxygenation known as persufflation involves the perfusion of humidified oxygen into the vasculature of the pancreas before and after cryopreservation. This technique could potentially mitigate the islet loss from hypoxia and ischemia during cryofreezing, thawing, and rewarming phase.

## *3.1.2 Cryopreservation storage duration*

Development of an islet tissue bank will require islets to be cryopreserved for long periods of time, years if necessary. Generally, islet cryopreservation studies will only cryopreserve islets for short periods of time (1–90 days) [45–47]. For human islet cryopreservation, the upper limit of storage duration was set by Fox et al. in 2015. Human islets were cryopreserved at −196°C for an average of 17.6 ± 0.4 years. Between 2012 and 2014, human islets were then thawed and warmed, after which islet electrophysiology and function were analyzed. After measurement of β-cell excitability via path-clamp assay, similar Ca2+-influx conductance patterns were observed between cryopreserved and fresh islets [48]. However, insulin stimulation index was significantly lower for cryopreserved (1.90 ± 0.24) compared to fresh islets (9.53 ± 0.92). However, after cryopreserved islets were transplanted into STZ-induced mice, partial normoglycemia was observed for 60 days with improvements to glucose tolerance [48]. More research

**125**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

is needed to infer a limit to cryopreservation storage duration to ensure that islets

Difficulties with the cryopreservation of whole organs are partially due to the non-homogenous temperature distribution within the large tissue structure of organs. Pancreatic islets exist as a spherical cluster, with an average diameter of 100 μm, of several thousands of cells connected by a dense network of connective tissue [49]. The cells that lay within the islet core are susceptible to hypoxia-related stress, particularly during the cryopreservation process [50, 51]. A recent study aimed to address this structural problem by reducing islets to single cells followed by cryopreservation [52]. Islets were reduced to single cells and cryopreserved with 10% DMSO and stored at −196°C (1°C/min) for four or more weeks. After a rapid thawing and warming phase, islets were reaggregated at 37°C [52]. Reaggregated islets were recovered at a rate of 80% and had similar diameter to intact cryopreserved islets. The viability of reaggregated islets was significantly higher than intact islets (80 versus 25% respectively) post-thawing. No significant differences in GSIS function were detected between reaggregated islets and intact islets. Upon allotransplantation of reaggregated islets into omentum of STZ-induced diabetic rats, normoglycemia was achieved in 24-hours and was sustained for 10-months. Intact cryopreserved islets failed to achieve normoglycemia. Graft volume necessary to achieve diabetic correction was lower for reaggregated islets (5–8500 IE/kg) than

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

remain functional post-thawing.

fresh islets (10–12,000 IE/kg) [52].

and insulin secretory behavior of islets.

**4.1 Principles background**

**4. Advances in islet cryoprotective additive technology**

CPAs can be divided into two major types, namely permeating and nonpermeating additives [53]. The main difference between the two major sub-types is whether the substance can penetrate the intracellular space during vitrification [54, 55]. Since the accidental discovery of glycerol in the 1940s [24], penetrating CPAs, such as DMSO and ethylene glycol, have shown significant benefits for islet survival in many studies [36]. These penetrating cryoprotectants, usually lowmolecular weight polar aprotic solvents, penetrate the cell membrane and increase the inner volume of the cell. An equilibrium is reached across the cell membrane when the intracellular water content is lower than physiological normal range, thus reducing the probability of intracellular ice crystal formation [56, 57]. Nonpenetrating cryoprotective additives like saccharides, which have a large molecular weight, remain in the extracellular space during the freezing process [56]. A buildup of these molecules in the extracellular space induces an osmotic gradient across the cell membrane, which causes water to move out of the cell. Water movement into the extracellular space helps to reduce the risk of intracellular ice crystal formation in addition to depressing the freezing point of intracellular water [58–60]. The mechanism of non-penetrating CPAs has been demonstrated to be temperature sensitive and suboptimal for certain cell types; therefore, penetrating CPAs have been traditionally favored over non-penetrating CPAs even though penetrating CPAs have increased the risk of toxicity [61, 62]. Recently, the integration of both permeating and non-permeating CPAs (e.g., DMSO with University of Wisconsin Solution (UW)) has shown improvements to post-cryopreservation islet recovery

*3.1.3 Islet structure limitations*

is needed to infer a limit to cryopreservation storage duration to ensure that islets remain functional post-thawing.

## *3.1.3 Islet structure limitations*

*Cryopreservation - Current Advances and Evaluations*

**3.1 Background**

*3.1.1 Oxygen treatment*

and rewarming phase.

*3.1.2 Cryopreservation storage duration*

**3. Factors that affect islet cryopreservation process**

While the use of CPAs is a critical step, there are several factors that play a significant role in the success of an islet cryopreservation process. Technological advances in these areas can help reduce the stress of the process on the islets while

Changes in ambient atmospheric oxygen concentration can have a negative effect on islet viability and function via ATP metabolite depletion. In hypoxic conditions, β-cells are put under oxidative stress followed by ROS production, which facilitates islet death [44]. The thawing and rewarming phases have been shown to place hypoxic-related oxidative stress on cryopreserved tissues [45]. During the thawing/rewarming period, as islets are brought back from subfreezing temperature, cellular enzymes begin to function and increase oxygen consumption. This process can cause a reduction in adenosine 3-phosphate (ATP), a cellular metabolite, which can lead to islet death [45]. To address this issue, researchers hypothesized that hyperbaric conditions might improve islet recovery. Human islets were exposed to both normal oxygen conditions (21% O2, 74% N2, and 5% CO2) and hyperbaric conditions (50% O2, 45% N2, and 5% CO2) at 22°C for 45 min followed by 37°C for 45 min. Short-term (post-rewarming) and long-term (2-day culture) islet function analysis was conducted via GSIS, qPCR ischemia-gene analysis, and islet metabolism via oxygen consumption rate (OCR) assay. Long-term culture also compared normal and hyperbaric culture conditions. No significant short-term function and metabolic differences were observed between conditions and non-treated islets. However, the hyperbaric conditions were shown to suppress the increases of inflammation detected in untreated cryopreserved islets. Islet recovery after long-term culture was significantly better under hyperbaric conditions and was shown to increase after hyperbaric 2-day culture. A recent advancement in organ oxygenation known as persufflation involves the perfusion of humidified oxygen into the vasculature of the pancreas before and after cryopreservation. This technique could potentially mitigate the islet loss from hypoxia and ischemia during cryofreezing, thawing,

Development of an islet tissue bank will require islets to be cryopreserved for long periods of time, years if necessary. Generally, islet cryopreservation studies will only cryopreserve islets for short periods of time (1–90 days) [45–47]. For human islet cryopreservation, the upper limit of storage duration was set by Fox et al. in 2015. Human islets were cryopreserved at −196°C for an average of 17.6 ± 0.4 years. Between 2012 and 2014, human islets were then thawed and warmed, after which islet electrophysiology and function were analyzed. After measurement of β-cell excitability via path-clamp assay, similar Ca2+-influx conductance patterns were observed between cryopreserved and fresh islets [48]. However, insulin stimulation index was significantly lower for cryopreserved (1.90 ± 0.24) compared to fresh islets (9.53 ± 0.92). However, after cryopreserved islets were transplanted into STZ-induced mice, partial normoglycemia was observed for 60 days with improvements to glucose tolerance [48]. More research

more effectively processing the islets tissue during cryopreservation.

**124**

Difficulties with the cryopreservation of whole organs are partially due to the non-homogenous temperature distribution within the large tissue structure of organs. Pancreatic islets exist as a spherical cluster, with an average diameter of 100 μm, of several thousands of cells connected by a dense network of connective tissue [49]. The cells that lay within the islet core are susceptible to hypoxia-related stress, particularly during the cryopreservation process [50, 51]. A recent study aimed to address this structural problem by reducing islets to single cells followed by cryopreservation [52]. Islets were reduced to single cells and cryopreserved with 10% DMSO and stored at −196°C (1°C/min) for four or more weeks. After a rapid thawing and warming phase, islets were reaggregated at 37°C [52]. Reaggregated islets were recovered at a rate of 80% and had similar diameter to intact cryopreserved islets. The viability of reaggregated islets was significantly higher than intact islets (80 versus 25% respectively) post-thawing. No significant differences in GSIS function were detected between reaggregated islets and intact islets. Upon allotransplantation of reaggregated islets into omentum of STZ-induced diabetic rats, normoglycemia was achieved in 24-hours and was sustained for 10-months. Intact cryopreserved islets failed to achieve normoglycemia. Graft volume necessary to achieve diabetic correction was lower for reaggregated islets (5–8500 IE/kg) than fresh islets (10–12,000 IE/kg) [52].

## **4. Advances in islet cryoprotective additive technology**

## **4.1 Principles background**

CPAs can be divided into two major types, namely permeating and nonpermeating additives [53]. The main difference between the two major sub-types is whether the substance can penetrate the intracellular space during vitrification [54, 55]. Since the accidental discovery of glycerol in the 1940s [24], penetrating CPAs, such as DMSO and ethylene glycol, have shown significant benefits for islet survival in many studies [36]. These penetrating cryoprotectants, usually lowmolecular weight polar aprotic solvents, penetrate the cell membrane and increase the inner volume of the cell. An equilibrium is reached across the cell membrane when the intracellular water content is lower than physiological normal range, thus reducing the probability of intracellular ice crystal formation [56, 57]. Nonpenetrating cryoprotective additives like saccharides, which have a large molecular weight, remain in the extracellular space during the freezing process [56]. A buildup of these molecules in the extracellular space induces an osmotic gradient across the cell membrane, which causes water to move out of the cell. Water movement into the extracellular space helps to reduce the risk of intracellular ice crystal formation in addition to depressing the freezing point of intracellular water [58–60]. The mechanism of non-penetrating CPAs has been demonstrated to be temperature sensitive and suboptimal for certain cell types; therefore, penetrating CPAs have been traditionally favored over non-penetrating CPAs even though penetrating CPAs have increased the risk of toxicity [61, 62]. Recently, the integration of both permeating and non-permeating CPAs (e.g., DMSO with University of Wisconsin Solution (UW)) has shown improvements to post-cryopreservation islet recovery and insulin secretory behavior of islets.

## **4.2 Permeating cryoprotective additives**

## *4.2.1 Dimethyl sulfoxide (DMSO)*

DMSO toxicity toward islets has been shown to be minimal at concentrations used during the freezing phase and has even demonstrated protective capabilities against selective β-cell necrosis antagonist alloxan [38, 41, 63]. DMSO is considered the gold standard in islet cryoprotective additives and has been heavily used in research for the prevention of intracellular ice crystal formation. A 1999 study sought to compare the effect of DMSO-mediated cryopreservation on the recovery and function of canine islets [64]. Islets from seven consecutive canine isolations were dissociated into single cells and cryopreserved in 2 M DMSO medium using a slow stepwise cooling method (0.25°C/min) to 40°C followed by storage in −196°C. Following rapid thawing (200°C/min), 81.5% of cryopreserved islets were recovered with no significant difference in insulin stimulation index (SI) when compared to non-treated canine islets (10.5 A.U. versus 12.4 A.U. respectively) [64]. Another study sought to standardize the critical removal process of DMSO from islet medium during the thawing phase. This protocol involves the slow stepwise addition of sucrose solution to dilute out the DMSO post-thawing [64]. Overall, DMSO will continue to play an important role in islet cryopreservation research.

## *4.2.2 Ethylene/polyethylene glycol*

A common constituent of car antifreeze, other permeating CPAs include both ethylene and polyethylene glycol, which have been studied for islets cryopreservation [53]. These low-molecular weight substances easily penetrate the cell membrane, much like DMSO, and cause solute equilibrium, which osmotically drives water toward the extracellular space [38]. Once the use of DMSO as a CPA was established in islet cryofreezing, studies in rat islets began to suggest potential toxicity issues when DMSO was exposed to rat islets [41, 65]. One study comparing DMSO and EG CPAs resulted in DMSO islets that exhibited lower cellular DNA, insulin, glucagon, and impaired insulin secretory patterns compared to EG, which was more like non-frozen islets. Upon transplantation of each islet group, normal glycemic control was achieved in 100% of EG-treated and non-frozen islets but only 92% of DMSO-treated islets recipients, which also experienced delays in diabetes correction [65]. When islets cryopreserved with varying concentrations (1, 2, and 3 M) of DMSO, EG, and PG were exposed to islets from canine and human sources, the permeability (Ps = μm/s) was quantified. The highest Ps was achieved in canine islets when 2 M EG (2.47 μm/s) was used while 2 M PG showed the highest Ps in human islets (3.48 μm/s) suggesting potential use of EG and PG in islet cryopreservation [66].

## *4.2.3 Permeating CPA mixtures*

Attempts have been made to produce mixtures of DMSO and EP (30% EP, 20% DMSO) for use during vitrification phase, which can help reduce the toxicity risks of using DMSO alone. A mixture of ethylene glycol (EG) and DMSO, classified as EDT324, was used as a cryoprotectant during the cooling phase with rat islets. EDT324-treated cryopreserved islets showed significant increases in islet viability and insulin secretory capability compared to use of DMSO (10%) alone [67]. EDT324-treated islets were then transplanted into allogenic rat recipients and diabetic correction was achieved after 2 days. Similar results were observed after islets

**127**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

were treated with one of two EG/DMSO mixtures (1 M ME2SO + 1 M EG, or 1 M ME2SO + 0.5 M EG). Islets treated with permeating CPA mixtures achieved significantly higher yield and viability compared to islets treated with DMSO only. When transplanted into STZ-induced mice, islets treated with DMSO/EG mixtures caused normoglycemia 12 days faster on average than DMSO only-treated islets [68].

Although permeating cryoprotectants have been mainly used during mammalian cell cryopreservation, saccharides have demonstrated survival advantages when added to the vitrification medium. When adult human islets were treated with 300 mmol/L trehalose, a 92% recovery rate was achieved compared to 58% recovery of DMSO-treated islets in addition to 14-fold increase in insulin content within islet grafts. More prominent differences in recovery were observed in fetal human islets treated with trehalose compared to islet only treated with DMSO [69]. More recently, an antifreeze glycoprotein (AFGP) was included to DMSO slow cooling phase medium during cryofreezing of rat islets. When compared to DMSO only protocol, AFGP-treated islets demonstrated significant increases in recovery rate (85 ± 6.25 versus 63.3 ± 14.2%) and insulin stimulation index (3.86 ± 0.43 versus 2.98 ± 0.22) were observed [70]. These results demonstrate that saccharides and saccharide-containing substances can be used in conjunction with lower DMSO

High-molecular weight polymeric compounds such as polyvinylpyrrolidone (PVP) and dextran have been shown to be effective at formation of amorphous glass matrix during cryofreezing phase [71]. When 10% PVP was added to cryopreservation medium before cooling phase, rat islet recovery and function were significantly higher than when islets were treated with 2 M DMSO and 3 M glycerol. Islets treated with 2 M PG demonstrated comparable islet recovery and function to PVP-treated group [72]. Although, the use of high-molecular weight cryoprotectants has been observed, past studies suggest that these compounds are ineffective at slow cooling temperature transitions, which is a crucial step of the

One of the most important causes of cell damage/death during cryopreservation is due to ice crystal formation. However, there are unavoidable damaging consequences to islet health when islet cells, especially when in multicellular tissues fragments, are exposed to subfreezing temperatures (≥100°C), which can lead to apoptosis and/or necrosis after the post-thawing phase of cryopreservation [73]. Due to its fragile multicellular tissue structure, islet fragments are susceptible to various stresses including oxidative stress, osmotic stress, hypoxia, hypothermia, and inflammation induced by the cryopreservation process, which can have acute and/or long-term effects on islet graft viability and function [74, 75]. As research into islet cryoprotection has become more nuanced in recent years, studies have started to target CPAs, which reduce stress-induced cell death associated with the

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

**4.3 Non-permeating cryoprotective additives**

concentrations and help reduce islet toxicity.

*4.3.2 Polymeric compounds*

cryofreezing process [71].

**4.4 Other potential CPAs**

cryopreservation process (**Table 1**).

*4.3.1 Saccharides*

were treated with one of two EG/DMSO mixtures (1 M ME2SO + 1 M EG, or 1 M ME2SO + 0.5 M EG). Islets treated with permeating CPA mixtures achieved significantly higher yield and viability compared to islets treated with DMSO only. When transplanted into STZ-induced mice, islets treated with DMSO/EG mixtures caused normoglycemia 12 days faster on average than DMSO only-treated islets [68].

## **4.3 Non-permeating cryoprotective additives**

## *4.3.1 Saccharides*

*Cryopreservation - Current Advances and Evaluations*

DMSO toxicity toward islets has been shown to be minimal at concentrations used during the freezing phase and has even demonstrated protective capabilities against selective β-cell necrosis antagonist alloxan [38, 41, 63]. DMSO is considered the gold standard in islet cryoprotective additives and has been heavily used in research for the prevention of intracellular ice crystal formation. A 1999 study sought to compare the effect of DMSO-mediated cryopreservation on the recovery and function of canine islets [64]. Islets from seven consecutive canine isolations were dissociated into single cells and cryopreserved in 2 M DMSO medium using a slow stepwise cooling method (0.25°C/min) to 40°C followed by storage in −196°C. Following rapid thawing (200°C/min), 81.5% of cryopreserved islets were recovered with no significant difference in insulin stimulation index (SI) when compared to non-treated canine islets (10.5 A.U. versus 12.4 A.U. respectively) [64]. Another study sought to standardize the critical removal process of DMSO from islet medium during the thawing phase. This protocol involves the slow stepwise addition of sucrose solution to dilute out the DMSO post-thawing [64]. Overall, DMSO will continue to play an important role in islet cryopreservation research.

A common constituent of car antifreeze, other permeating CPAs include both ethylene and polyethylene glycol, which have been studied for islets cryopreservation [53]. These low-molecular weight substances easily penetrate the cell membrane, much like DMSO, and cause solute equilibrium, which osmotically drives water toward the extracellular space [38]. Once the use of DMSO as a CPA was established in islet cryofreezing, studies in rat islets began to suggest potential toxicity issues when DMSO was exposed to rat islets [41, 65]. One study comparing DMSO and EG CPAs resulted in DMSO islets that exhibited lower cellular DNA, insulin, glucagon, and impaired insulin secretory patterns compared to EG, which was more like non-frozen islets. Upon transplantation of each islet group, normal glycemic control was achieved in 100% of EG-treated and non-frozen islets but only 92% of DMSO-treated islets recipients, which also experienced delays in diabetes

correction [65]. When islets cryopreserved with varying concentrations (1, 2, and 3 M) of DMSO, EG, and PG were exposed to islets from canine and human sources, the permeability (Ps = μm/s) was quantified. The highest Ps was achieved in canine islets when 2 M EG (2.47 μm/s) was used while 2 M PG showed the highest Ps in human islets (3.48 μm/s) suggesting potential use of EG and PG in

Attempts have been made to produce mixtures of DMSO and EP (30% EP, 20% DMSO) for use during vitrification phase, which can help reduce the toxicity risks of using DMSO alone. A mixture of ethylene glycol (EG) and DMSO, classified as EDT324, was used as a cryoprotectant during the cooling phase with rat islets. EDT324-treated cryopreserved islets showed significant increases in islet viability and insulin secretory capability compared to use of DMSO (10%) alone [67]. EDT324-treated islets were then transplanted into allogenic rat recipients and diabetic correction was achieved after 2 days. Similar results were observed after islets

**4.2 Permeating cryoprotective additives**

*4.2.1 Dimethyl sulfoxide (DMSO)*

*4.2.2 Ethylene/polyethylene glycol*

islet cryopreservation [66].

*4.2.3 Permeating CPA mixtures*

**126**

Although permeating cryoprotectants have been mainly used during mammalian cell cryopreservation, saccharides have demonstrated survival advantages when added to the vitrification medium. When adult human islets were treated with 300 mmol/L trehalose, a 92% recovery rate was achieved compared to 58% recovery of DMSO-treated islets in addition to 14-fold increase in insulin content within islet grafts. More prominent differences in recovery were observed in fetal human islets treated with trehalose compared to islet only treated with DMSO [69]. More recently, an antifreeze glycoprotein (AFGP) was included to DMSO slow cooling phase medium during cryofreezing of rat islets. When compared to DMSO only protocol, AFGP-treated islets demonstrated significant increases in recovery rate (85 ± 6.25 versus 63.3 ± 14.2%) and insulin stimulation index (3.86 ± 0.43 versus 2.98 ± 0.22) were observed [70]. These results demonstrate that saccharides and saccharide-containing substances can be used in conjunction with lower DMSO concentrations and help reduce islet toxicity.

## *4.3.2 Polymeric compounds*

High-molecular weight polymeric compounds such as polyvinylpyrrolidone (PVP) and dextran have been shown to be effective at formation of amorphous glass matrix during cryofreezing phase [71]. When 10% PVP was added to cryopreservation medium before cooling phase, rat islet recovery and function were significantly higher than when islets were treated with 2 M DMSO and 3 M glycerol. Islets treated with 2 M PG demonstrated comparable islet recovery and function to PVP-treated group [72]. Although, the use of high-molecular weight cryoprotectants has been observed, past studies suggest that these compounds are ineffective at slow cooling temperature transitions, which is a crucial step of the cryofreezing process [71].

## **4.4 Other potential CPAs**

One of the most important causes of cell damage/death during cryopreservation is due to ice crystal formation. However, there are unavoidable damaging consequences to islet health when islet cells, especially when in multicellular tissues fragments, are exposed to subfreezing temperatures (≥100°C), which can lead to apoptosis and/or necrosis after the post-thawing phase of cryopreservation [73]. Due to its fragile multicellular tissue structure, islet fragments are susceptible to various stresses including oxidative stress, osmotic stress, hypoxia, hypothermia, and inflammation induced by the cryopreservation process, which can have acute and/or long-term effects on islet graft viability and function [74, 75]. As research into islet cryoprotection has become more nuanced in recent years, studies have started to target CPAs, which reduce stress-induced cell death associated with the cryopreservation process (**Table 1**).


## **Table 1.**

*Cryoprotection: A quick summary of the parameters that had the best outcome for the islets during cryopreservation.*

## *4.4.1 Butylated hydroxyanisole (BHA)*

Oxidative stress in cells produces endogenous reactive oxygen species (ROS), such as superoxide (O2 <sup>−</sup>) and free hydroxyl (OH<sup>−</sup>), which leads to an increase in free radical concentration intracellularly [76]. Elevated internal levels of free radicals can cause cellular damage and lead to cellular process disruptions. To combat cryopreservation-related oxidative stress, one early study added butylated hydroxyanisole (BHA) to islet cryomedium while monitoring oxidative stress via glutathione redox state (GSH/GSSG). Islets treated with BHA demonstrated enhanced insulin secretory behavior (2.2-fold increase) when compared to untreated islets. In addition, exposure to alloxan, a highly damaging free radical generating agent, did not induce significant oxidative stress [77].

## *4.4.2 Ascorbic acid-2 glucoside (AA2G)*

Ascorbic acid-2 glucoside (AA2G), a derivative of Vitamin C, is a potent antioxidant and can deliver stable antioxidant activity into culture media [78]. AA2G (100 μg/mL) in combination with the UW islet preservation solution was used as the cryopreservation medium [79, 80]. Following 3 months of storage (−80°C), the islets treated with UW/AA2G demonstrated viability maintenance (68.3 ± 5.6%) and significantly increased insulin stimulation via glucose-stimulated insulin secretion (GSIS) test, when compared to treatment with UW alone (1.93 ± 0.5 and 1.17 ± 0.6 respectively). Transplantation of thawed AAG2/UW-treated into liver of nude mice produced engraftment with insulin-positive cells observed.

## *4.4.3 Curcumin*

Curcumin, the main component of turmeric spice, has demonstrated antioxidant and anti-inflammatory effects in multiple cell types [81]. Curcumin has not been shown to increase insulin stimulation; however, it has demonstrated upregulation of oxidative stress-reducing genes Hsp70 and HO-1 [47]. To evaluate cyroprotective abilities of curcumin, Kanitkar et al. compared the effect of 10% DMSO with and without 10 μM curcumin on islets treated with slow cooled cryopreservation (−196°C) for 7 days. Curcumin-treated islets showed increases in SI compared to non-treated cryopreserved islets but no difference from fresh islets.

**129**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

islets were transplanted into immunocompetent mice.

*4.4.5 Γ-aminobutyric acid (GABA)*

non-treated cryopreserved islets [46].

*4.4.6 Metformin*

chicken islets [88].

*4.4.7 Sericin*

incrementally increase as the cryopreservation process unfolded [47].

In curcumin-treated medium, over-expression of HO-1 and Hsp70 was observed to

An amino sulfonic acid, taurine has been suggested as a CPA because of chemical properties that allow antioxidant and osmoregulatory properties [82]. An addition of taurine to the cryopreservation medium demonstrated cyroprotective effects during islet cryopreservation (Hardikar 2001). Pretreatment of taurine prior to cryopreservation freezing phase at 0.3 mM and 3.0 mM resulted in high maintained viability of 91.9 ± 2.3 and 94.6 ± 1.58% respectively. Lipid peroxidation, which is a known indicator of oxidative stress, was reduced significantly compared to controls. Finally, normoglycemia was achieved when taurine-treated cryopreserved

GABA neurotransmitters facilitate the inhibitory neuronal pathways within the central nervous system and have demonstrated regulatory and protective effects on β-cells [83]. GABA has been shown to produce membrane polarization (Ca2+-influx) which activates survival and growth pathways (PI3-K/Akt) and can restore β-cells' mass in severely diabetic mice [83, 84]. Islets were treated with 50 or 100 μM GABA and were cryopreserved to 196°C for up to 30 days. At both 15 and 30 days post-thawing, islets treated with GABA exhibited similar insulin secretory behavior compared to fresh islets. When oxidative stress was measured via MTT assay, reduced ROS content was observed in GABA-treated islets in comparison to

Metformin is a standard-of-care drug used for the treatment of Type 2 Diabetes and has been identified as an "essential medicine" by the WHO [85]. While the complete mechanism of action is unknown for metformin, it has demonstrated insulin-sensitizing properties and reduces unfettered liver gluconeogenesis [86, 87]. When used as a cryoprotective agent at ultralow temperatures, metformin produced membrane stabilizing effects. Cryopreserved islets treated with metformin-containing cyromixtures exhibited comparable viability (90 versus 100%) to non-treated fresh islets. Improved insulin secretion was observed at 15 days post-thawing (8 ng/mL) with a stimulation index value of >5, suggesting islets were highly functional [46]. Recently, similar cyroprotective results were observed in

Produced by *Bombyx mori* silkworm, this gel-like protein has previously demonstrated oxidative stress reduction properties induced by freezing temperatures in both rat islets and various other mammalian cell lines [89, 90]. When added to media, Ahnishi et al. showed no significant differences between GSIS results between the FBS + DMSO and the sericin + DMSO groups. This study demonstrated that reduction in DMSO content in cyromixtures is possible with addition of 1% sericin, which could reduce the toxic risk posed by high concentrations of DMSO.

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

*4.4.4 Taurine*

In curcumin-treated medium, over-expression of HO-1 and Hsp70 was observed to incrementally increase as the cryopreservation process unfolded [47].

## *4.4.4 Taurine*

*Cryopreservation - Current Advances and Evaluations*

*4.4.1 Butylated hydroxyanisole (BHA)*

not induce significant oxidative stress [77].

*4.4.2 Ascorbic acid-2 glucoside (AA2G)*

insulin-positive cells observed.

*4.4.3 Curcumin*

such as superoxide (O2

**Table 1.**

*cryopreservation.*

Oxidative stress in cells produces endogenous reactive oxygen species (ROS),

Encapsulation 1.75% Alginate encapsulation prior to cryopreservation [64]

**Parameter Method Reference** Cryoprotectant EPA + DHA + Metformin [40] Cooling rate Rapid (50–70°C/min) [27–29] Thawing rate Rapid (150–200°C/min) [32] Oxygen environment 50% during thawing [54]

3D structure Freeze as individual cells, re-aggregate into spheroids after

*Cryoprotection: A quick summary of the parameters that had the best outcome for the islets during* 

*Assessment methods include islet viability, glucose sensitivity, and GSIS values after thawing.*

thaw

free radical concentration intracellularly [76]. Elevated internal levels of free radicals can cause cellular damage and lead to cellular process disruptions. To combat cryopreservation-related oxidative stress, one early study added butylated hydroxyanisole (BHA) to islet cryomedium while monitoring oxidative stress via glutathione redox state (GSH/GSSG). Islets treated with BHA demonstrated enhanced insulin secretory behavior (2.2-fold increase) when compared to untreated islets. In addition, exposure to alloxan, a highly damaging free radical generating agent, did

Ascorbic acid-2 glucoside (AA2G), a derivative of Vitamin C, is a potent antioxidant and can deliver stable antioxidant activity into culture media [78]. AA2G (100 μg/mL) in combination with the UW islet preservation solution was used as the cryopreservation medium [79, 80]. Following 3 months of storage (−80°C), the islets treated with UW/AA2G demonstrated viability maintenance (68.3 ± 5.6%) and significantly increased insulin stimulation via glucose-stimulated insulin secretion (GSIS) test, when compared to treatment with UW alone (1.93 ± 0.5 and 1.17 ± 0.6 respectively). Transplantation of thawed AAG2/UW-treated into liver of nude mice produced engraftment with

Curcumin, the main component of turmeric spice, has demonstrated antioxidant and anti-inflammatory effects in multiple cell types [81]. Curcumin has not been shown to increase insulin stimulation; however, it has demonstrated upregulation of oxidative stress-reducing genes Hsp70 and HO-1 [47]. To evaluate cyroprotective abilities of curcumin, Kanitkar et al. compared the effect of 10% DMSO with and without 10 μM curcumin on islets treated with slow cooled cryopreservation (−196°C) for 7 days. Curcumin-treated islets showed increases in SI compared to non-treated cryopreserved islets but no difference from fresh islets.

<sup>−</sup>) and free hydroxyl (OH<sup>−</sup>), which leads to an increase in

[51]

**128**

An amino sulfonic acid, taurine has been suggested as a CPA because of chemical properties that allow antioxidant and osmoregulatory properties [82]. An addition of taurine to the cryopreservation medium demonstrated cyroprotective effects during islet cryopreservation (Hardikar 2001). Pretreatment of taurine prior to cryopreservation freezing phase at 0.3 mM and 3.0 mM resulted in high maintained viability of 91.9 ± 2.3 and 94.6 ± 1.58% respectively. Lipid peroxidation, which is a known indicator of oxidative stress, was reduced significantly compared to controls. Finally, normoglycemia was achieved when taurine-treated cryopreserved islets were transplanted into immunocompetent mice.

## *4.4.5 Γ-aminobutyric acid (GABA)*

GABA neurotransmitters facilitate the inhibitory neuronal pathways within the central nervous system and have demonstrated regulatory and protective effects on β-cells [83]. GABA has been shown to produce membrane polarization (Ca2+-influx) which activates survival and growth pathways (PI3-K/Akt) and can restore β-cells' mass in severely diabetic mice [83, 84]. Islets were treated with 50 or 100 μM GABA and were cryopreserved to 196°C for up to 30 days. At both 15 and 30 days post-thawing, islets treated with GABA exhibited similar insulin secretory behavior compared to fresh islets. When oxidative stress was measured via MTT assay, reduced ROS content was observed in GABA-treated islets in comparison to non-treated cryopreserved islets [46].

## *4.4.6 Metformin*

Metformin is a standard-of-care drug used for the treatment of Type 2 Diabetes and has been identified as an "essential medicine" by the WHO [85]. While the complete mechanism of action is unknown for metformin, it has demonstrated insulin-sensitizing properties and reduces unfettered liver gluconeogenesis [86, 87]. When used as a cryoprotective agent at ultralow temperatures, metformin produced membrane stabilizing effects. Cryopreserved islets treated with metformin-containing cyromixtures exhibited comparable viability (90 versus 100%) to non-treated fresh islets. Improved insulin secretion was observed at 15 days post-thawing (8 ng/mL) with a stimulation index value of >5, suggesting islets were highly functional [46]. Recently, similar cyroprotective results were observed in chicken islets [88].

## *4.4.7 Sericin*

Produced by *Bombyx mori* silkworm, this gel-like protein has previously demonstrated oxidative stress reduction properties induced by freezing temperatures in both rat islets and various other mammalian cell lines [89, 90]. When added to media, Ahnishi et al. showed no significant differences between GSIS results between the FBS + DMSO and the sericin + DMSO groups. This study demonstrated that reduction in DMSO content in cyromixtures is possible with addition of 1% sericin, which could reduce the toxic risk posed by high concentrations of DMSO.

## **4.5 Recent advances in islet cryopreservation technology**

## *4.5.1 Cryopreservation with alginate-based microencapsulation technology*

Alginate-based microencapsulation technologies have developed in concert with islet transplantation research where the alginate polymer forms a semipermeable immune-isolating barrier around islet fragments [91–93]. Alginate-microencapsulation has recently been applied to the field of islet cryopreservation in order to characterize its effect on islet survivability [33]. Chen et al. reported the development of an oxygen-sensing alginate coating to encapsulate islets prior to cryopreservation [94, 95]. Islets were encapsulated with alginate coating containing ruthenium-based oxygen-sensitive fluorophore (ROF) after which the encapsulated islets were subjected to a 10% DMSO with or without 50x10<sup>−</sup><sup>3</sup> M trehalose and stored for 1–7 days. Encapsulated islets undergoing cryopreservation showed significantly higher insulin stimulation behavior than bare islets at Day 1 and 7 [94]. In addition to cyroprotective abilities, the microcapsules treated with ROS demonstrated viable oxygen sensitivity during OCR measurements. This study demonstrates the use of multiple cyroprotective parameters to mitigate potential damage during cryopreservation [94]. Other studies have demonstrated the benefits of alginate microencapsulation use during cryopreservation as the 3D barrier is porous and can resist stress/strain associated with ice formation [94, 96, 97].

## *4.5.2 Hollow fiber vitrification*

Previously described for use in embryological studies, vitrification scaffolds have been suggested as a medium for cryopreservation of islets to improve the islet survival [98, 99]. This technique involves the loading of islets into a hollow fiber chamber (HFV) composed of cellulose-triacetate, which is permeable to CPAs [100]. Researchers used a combination of permeating CPAs like DMSO and EG during cooling phase of both non-vitrified islets and islets vitrified using HFV method. *In Vitro* assays demonstrated similar islet structure and insulin gene promoter expression (NeuroD, Pdx1, MafA) to non-vitrified islets; however, the insulin stimulation index was significantly decreased for islets undergoing HFV compared to non-vitrified islets (27.8 ± 8.2 and 3.5 ± 0.6 respectively). Nonetheless, after HVF and non-vitrified islets were transplanted into kidney subcapsular space, all mice were euglycemic within 4–8 days and remained so for 1 month until nephrectomy, which induced hyperglycemia.

## **5. Conclusion**

Islet cryopreservation has come a long way in the last 40 years. Many parameters of cryopreserving islets are being actively researched because of the high demand for long-term storage. Currently, entire organ cryopreservation is not entirely feasible, and is only shown possible after a few hours of storage [101]. Based on experiments performed in our lab along with results from other research groups, we predict that future improvements in islet cryopreservation will rely on the use of a mixture of cyroprotective additives along with the use of secondary technologies like alginate encapsulation [96]. We hope that with a standardized cryopreservation protocol, islet banking would be more feasible, and ultimately, transplantation would no longer be throttled by the donorrecipient mismatch.

**131**

**Author details**

Samuel Rodriguez1

Irvine, CA, USA

CA, USA

Mohammad Rezaa Mohammadi2

, David Whaley1

\*Address all correspondence to: jlakey@uci.edu

provided the original work is properly cited.

, Michael Alexander1

1 Department of Surgery, University of California Irvine, Orange, CA, USA

2 Department of Materials Science and Engineering, University of California Irvine,

3 Department of Biomedical Engineering, University of California Irvine, Irvine,

© 2019 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,

and Jonathan R.T. Lakey1,3\*

,

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

The authors gratefully acknowledge the support from the Department of Surgery and the Department of Biomedical Engineering at University of California, Irvine, and the Sue and Bill Gross Stem Cell Research Center, for all the support in

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

**Acknowledgements**

the writing of this chapter.

*Current Advancements in Pancreatic Islet Cryopreservation Techniques DOI: http://dx.doi.org/10.5772/intechopen.89363*

## **Acknowledgements**

*Cryopreservation - Current Advances and Evaluations*

associated with ice formation [94, 96, 97].

*4.5.2 Hollow fiber vitrification*

which induced hyperglycemia.

**5. Conclusion**

recipient mismatch.

without 50x10<sup>−</sup><sup>3</sup>

**4.5 Recent advances in islet cryopreservation technology**

*4.5.1 Cryopreservation with alginate-based microencapsulation technology*

Alginate-based microencapsulation technologies have developed in concert with islet transplantation research where the alginate polymer forms a semipermeable immune-isolating barrier around islet fragments [91–93]. Alginate-microencapsulation has recently been applied to the field of islet cryopreservation in order to characterize its effect on islet survivability [33]. Chen et al. reported the development of an oxygen-sensing alginate coating to encapsulate islets prior to cryopreservation [94, 95]. Islets were encapsulated with alginate coating containing ruthenium-based oxygen-sensitive fluorophore (ROF) after which the encapsulated islets were subjected to a 10% DMSO with or

going cryopreservation showed significantly higher insulin stimulation behavior than bare islets at Day 1 and 7 [94]. In addition to cyroprotective abilities, the microcapsules treated with ROS demonstrated viable oxygen sensitivity during OCR measurements. This study demonstrates the use of multiple cyroprotective parameters to mitigate potential damage during cryopreservation [94]. Other studies have demonstrated the benefits of alginate microencapsulation use during cryopreservation as the 3D barrier is porous and can resist stress/strain

Previously described for use in embryological studies, vitrification scaffolds have been suggested as a medium for cryopreservation of islets to improve the islet survival [98, 99]. This technique involves the loading of islets into a hollow fiber chamber (HFV) composed of cellulose-triacetate, which is permeable to CPAs [100]. Researchers used a combination of permeating CPAs like DMSO and EG during cooling phase of both non-vitrified islets and islets vitrified using HFV method. *In Vitro* assays demonstrated similar islet structure and insulin gene promoter expression (NeuroD, Pdx1, MafA) to non-vitrified islets; however, the insulin stimulation index was significantly decreased for islets undergoing HFV compared to non-vitrified islets (27.8 ± 8.2 and 3.5 ± 0.6 respectively). Nonetheless, after HVF and non-vitrified islets were transplanted into kidney subcapsular space, all mice were euglycemic within 4–8 days and remained so for 1 month until nephrectomy,

Islet cryopreservation has come a long way in the last 40 years. Many parameters of cryopreserving islets are being actively researched because of the high demand for long-term storage. Currently, entire organ cryopreservation is not entirely feasible, and is only shown possible after a few hours of storage [101]. Based on experiments performed in our lab along with results from other research groups, we predict that future improvements in islet cryopreservation will rely on the use of a mixture of cyroprotective additives along with the use of secondary technologies like alginate encapsulation [96]. We hope that with a standardized cryopreservation protocol, islet banking would be more feasible, and ultimately, transplantation would no longer be throttled by the donor-

M trehalose and stored for 1–7 days. Encapsulated islets under-

**130**

The authors gratefully acknowledge the support from the Department of Surgery and the Department of Biomedical Engineering at University of California, Irvine, and the Sue and Bill Gross Stem Cell Research Center, for all the support in the writing of this chapter.

## **Author details**

Samuel Rodriguez1 , David Whaley1 , Michael Alexander1 , Mohammad Rezaa Mohammadi2 and Jonathan R.T. Lakey1,3\*

1 Department of Surgery, University of California Irvine, Orange, CA, USA

2 Department of Materials Science and Engineering, University of California Irvine, Irvine, CA, USA

3 Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA

\*Address all correspondence to: jlakey@uci.edu

© 2019 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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[80] Southard J, Ametani M, Vreugdenhil P, Lindell S, Pienaar B, Belzer F. Important components of the UW solution. Transplantation. 1990;**49**(2):251-257

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[82] Huxtable R. Physiological actions of taurine. Physiological Reviews. 1992;**72**(1):101-163

[83] Soltani N, Qiu H, Aleksic M, et al. GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. Proceedings of the National Academy of Sciences. 2011;**108**(28):11692-11697

[84] Braun M, Ramracheya R, Bengtsson M, et al. γ-Aminobutyric acid (GABA) is an autocrine excitatory transmitter in human pancreatic β-cells. Diabetes. 2010;**59**(7):1694-1701

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**137**

*Current Advancements in Pancreatic Islet Cryopreservation Techniques*

functionalized hydrogel microcapsules.

[96] Kojayan GFA, Li S, Alexander M, Lakey JR. Cryopreserved alginate encapsulated islets can restore euglycemia in diabetic animal model better than cryopreserved non. Journal

Advanced Healthcare Materials.

[95] Fraker C, Timmins MR, Guarino RD, et al. The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: Oxygen consumption as a potential measure of islet potency. Cell Transplantation.

2016;**5**(2):223-231

2006;**15**(8-9):745-758

of Cell Medicine. 2019;**II**:1

2010;**16**(5):965-977

[97] Malpique R, Osório LM, Ferreira DS, et al. Alginate

[98] Matsunari H, Maehara M, Nakano K, et al. Hollow fiber vitrification: A novel method for vitrifying multiple embryos in a single device. Journal of Reproduction and Development. 2012;**58**:2011-2051

[99] Maehara M, Matsunari H, Honda K, et al. Hollow fiber

fertilization-derived porcine embryos. Biology of Reproduction.

procedure for pancreatic islets using hollow fiber vitrification. Hormone and Metabolic Research.

[101] Giwa S, Lewis JK, Alvarez L, et al. The promise of organ and tissue preservation to transform medicine. Nature Biotechnology. 2017;**35**(6):530

2012;**87**(6):131-138

2016;**48**(08):540-549

vitrification provides a novel method for cryopreserving in vitro maturation/

[100] Nagaya M, Matsunari H, Kanai T, et al. An effective new cryopreservation

encapsulation as a novel strategy for the cryopreservation of neurospheres. Tissue Engineering Part C: Methods.

*DOI: http://dx.doi.org/10.5772/intechopen.89363*

[86] Hundal RS, Inzucchi SE. Metformin.

Geneva, Switzerland: World Health

Drugs. 2003;**63**(18):1879-1894

[87] Sirtori CR, Franceschini G, Galli-Kienle M, et al. Disposition of metformin (N, N-dimethylbiguanide) in man. Clinical Pharmacology & Therapeutics. 1978;**24**(6):683-693

[88] Chandravanshi B, Datar S, Bhonde R. Response of chick B islets to insulin secretagogues is comparable to those of human islet equivalents. JOP Journal of the Pancreas.

[89] Morikawa M, Kimura T,

Murakami M, Katayama K, Terada S, Yamaguchi A. Rat islet culture in serumfree medium containing silk protein sericin. Journal of Hepato-Biliary-Pancreatic Surgery. 2009;**16**(2):223

[90] Sasaki M, Kato Y, Yamada H, Terada S. Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin. Biotechnology and Applied Biochemistry. 2005;**42**(2):183-188

[91] de Vos P, Faas MM, Strand B,

[92] Fritschy WM, Wolters GH, Van Schilfgaarde R. Effect of alginatepolylysine-alginate microencapsulation

on in vitro insulin release from rat pancreatic islets. Diabetes.

1991;**40**(1):37-43

Calafiore R. Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials. 2006;**27**(32):5603-5617

[93] O'Shea GM, Sun AM. Encapsulation of rat islets of Langerhans prolongs xenograft survival in diabetic mice. Diabetes. 1986;**35**(8):943-946

[94] Chen W, Shu Z, Gao D, Shen AQ. Sensing and sensibility: Single-isletbased quality control assay of cryopreserved pancreatic islets with

2015;**16**(3):318-323

Organization; 2015

*Current Advancements in Pancreatic Islet Cryopreservation Techniques DOI: http://dx.doi.org/10.5772/intechopen.89363*

Geneva, Switzerland: World Health Organization; 2015

*Cryopreservation - Current Advances and Evaluations*

[78] Yamamoto I, Suga S, Mitoh Y, Tanaka M, Muto N. Antiscorbutic activity of L-ascorbic acid 2-glucoside and its availability as a vitamin C supplement in normal rats and Guinea pigs. Journal of Pharmacobio-Dynamics.

[79] Arata T, Okitsu T, Fukazawa T, et al. Maintenance of glucose-sensitive insulin secretion of cryopreserved human islets with University of Wisconsin solution and ascorbic acid-2 glucoside. Artificial

1990;**13**(11):688-695

Organs. 2004;**28**(6):529-536

[80] Southard J, Ametani M,

[81] Hsuuw YD, Chang CK,

1990;**49**(2):251-257

2005;**205**(3):379-386

1992;**72**(1):101-163

Vreugdenhil P, Lindell S, Pienaar B, Belzer F. Important components of the UW solution. Transplantation.

Chan WH, Yu JS. Curcumin prevents methylglyoxal-induced oxidative stress and apoptosis in mouse

embryonic stem cells and blastocysts. Journal of Cellular Physiology.

[82] Huxtable R. Physiological actions of taurine. Physiological Reviews.

[83] Soltani N, Qiu H, Aleksic M, et al. GABA exerts protective and regenerative effects on islet beta cells and reverses diabetes. Proceedings of the National Academy of Sciences.

2011;**108**(28):11692-11697

[84] Braun M, Ramracheya R, Bengtsson M, et al. γ-Aminobutyric acid (GABA) is an autocrine excitatory transmitter in human pancreatic β-cells.

Diabetes. 2010;**59**(7):1694-1701

[85] Organization WH. The Selection and Use of Essential Medicines: Report of the WHO Expert Committee, 2015 (Including the 19th WHO Model List of Essential Medicines and the 5th WHO Model List of Essential Medicines for Children). Vol. 994.

recovery and preserves function of human pancreatic islets after Long-term storage. Diabetes. 1997;**46**(3):519-523

[70] Matsumoto S, Matsusita M, Morita T, et al. Effects of synthetic antifreeze glycoprotein analogue on islet cell survival and function during cryopreservation. Cryobiology.

[71] Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology.

[72] El-Shewy HM, William FK, Darrabie M, Collins BH, Opara EC. Polyvinyl Pyrrolidone: A novel Cryoprotectant in islet cell

cryopreservation. Cell Transplantation.

[73] Baust JM. Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell Preservation and Technology.

[74] Bissoyi A, Nayak B, Pramanik K, Sarangi SK. Targeting cryopreservation-

induced cell death: A review. Biopreservation and Biobanking.

[75] Modak MA, Parab PB,

Ghaskadbi SS. Pancreatic islets are very poor in rectifying oxidative DNA damage. Pancreas. 2009;**38**(1):23-29

[76] Robertson RP. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. Journal of Biological Chemistry. 2004;**279**(41):42351-42354

[77] Janjic D, Andereggen E, Deng S, et al. Improved insulin secretion of cryopreserved human islets by antioxidant treatment. Pancreas.

2006;**52**(1):90-98

2017;**76**:74-91

2004;**13**(3):237-243

2002;**1**(1):17-31

2014;**12**(1):23-34

**136**

1996;**13**(2):166-172

[86] Hundal RS, Inzucchi SE. Metformin. Drugs. 2003;**63**(18):1879-1894

[87] Sirtori CR, Franceschini G, Galli-Kienle M, et al. Disposition of metformin (N, N-dimethylbiguanide) in man. Clinical Pharmacology & Therapeutics. 1978;**24**(6):683-693

[88] Chandravanshi B, Datar S, Bhonde R. Response of chick B islets to insulin secretagogues is comparable to those of human islet equivalents. JOP Journal of the Pancreas. 2015;**16**(3):318-323

[89] Morikawa M, Kimura T, Murakami M, Katayama K, Terada S, Yamaguchi A. Rat islet culture in serumfree medium containing silk protein sericin. Journal of Hepato-Biliary-Pancreatic Surgery. 2009;**16**(2):223

[90] Sasaki M, Kato Y, Yamada H, Terada S. Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin. Biotechnology and Applied Biochemistry. 2005;**42**(2):183-188

[91] de Vos P, Faas MM, Strand B, Calafiore R. Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials. 2006;**27**(32):5603-5617

[92] Fritschy WM, Wolters GH, Van Schilfgaarde R. Effect of alginatepolylysine-alginate microencapsulation on in vitro insulin release from rat pancreatic islets. Diabetes. 1991;**40**(1):37-43

[93] O'Shea GM, Sun AM. Encapsulation of rat islets of Langerhans prolongs xenograft survival in diabetic mice. Diabetes. 1986;**35**(8):943-946

[94] Chen W, Shu Z, Gao D, Shen AQ. Sensing and sensibility: Single-isletbased quality control assay of cryopreserved pancreatic islets with

functionalized hydrogel microcapsules. Advanced Healthcare Materials. 2016;**5**(2):223-231

[95] Fraker C, Timmins MR, Guarino RD, et al. The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: Oxygen consumption as a potential measure of islet potency. Cell Transplantation. 2006;**15**(8-9):745-758

[96] Kojayan GFA, Li S, Alexander M, Lakey JR. Cryopreserved alginate encapsulated islets can restore euglycemia in diabetic animal model better than cryopreserved non. Journal of Cell Medicine. 2019;**II**:1

[97] Malpique R, Osório LM, Ferreira DS, et al. Alginate encapsulation as a novel strategy for the cryopreservation of neurospheres. Tissue Engineering Part C: Methods. 2010;**16**(5):965-977

[98] Matsunari H, Maehara M, Nakano K, et al. Hollow fiber vitrification: A novel method for vitrifying multiple embryos in a single device. Journal of Reproduction and Development. 2012;**58**:2011-2051

[99] Maehara M, Matsunari H, Honda K, et al. Hollow fiber vitrification provides a novel method for cryopreserving in vitro maturation/ fertilization-derived porcine embryos. Biology of Reproduction. 2012;**87**(6):131-138

[100] Nagaya M, Matsunari H, Kanai T, et al. An effective new cryopreservation procedure for pancreatic islets using hollow fiber vitrification. Hormone and Metabolic Research. 2016;**48**(08):540-549

[101] Giwa S, Lewis JK, Alvarez L, et al. The promise of organ and tissue preservation to transform medicine. Nature Biotechnology. 2017;**35**(6):530

**139**

**Chapter 7**

**Abstract**

Cryopreservation in

*Yuting Shao, Chao Chen, Qi Zhou, Jun Yang, Xiao Lv,* 

**Keywords:** amnion, cornea, cryopreservation, indication, ophthalmology

Ever since Davis [1] first used human amniotic membrane (**Figure 1**) (AM) for skin transplantation, people have been exploring this remarkable biomaterial. AM is located in the innermost layer of the fetal membranes [2]. It is 0.02–0.05 mm thick, lightweight, elastic, almost transparent, and avascular membrane classically composed of three layers: the epithelium, the basement membrane and the stroma layer [2]. There are two types of cells with stemness properties in AM: amniotic epithelial cells (AECs) and amniotic mesenchymal cells (AMSCs) [3], which are responsible for its unique biological properties including anti-inflammatory, anti-scarring, anti-microbial, angio-modulating, immunomodulatory, and anti-cancer effects [4–10]. Due to these properties, AM has become an ideal material for ocular reconstruction including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries. However, its use span is short and many viruses (such as HIV-1/2, hepatitis B, hepatitis C, human T-cell lymphotropic virus, syphilis, and cytomegalovirus)

**1. Cryopreservation of amniotic membranes**

Amniotic membranes (AMs) and corneas are critical materials in ocular surface reconstruction. AM has specific structures (e.g., basement and two types of cells with stemness characteristics: amniotic epithelial cells and amniotic mesenchymal cells), which contribute to its attractive physical and biological properties that make it fundamental to clinical application. The corneal endothelial cell is a vital part of the cornea, which can influence postoperative vision directly. However, widespread use of fresh AM and cornea has been limited due to their short use span and safety concerns. To overcome these concerns, different preservation methods have been introduced. Cryopreservation is distinguished from many preservation methods for its attractive advantages of prolonged use span, optimally retained tissue structure, and minimized infection risk. This review will focus on recent advances of cryopreserved AM and cornea, including different cryopreservation methods and their

Ophthalmology

*Mingyue Lin and Yanlong Bi*

indications in ophthalmology.

**1.1 Introduction**

## **Chapter 7**

## Cryopreservation in Ophthalmology

*Yuting Shao, Chao Chen, Qi Zhou, Jun Yang, Xiao Lv, Mingyue Lin and Yanlong Bi*

## **Abstract**

Amniotic membranes (AMs) and corneas are critical materials in ocular surface reconstruction. AM has specific structures (e.g., basement and two types of cells with stemness characteristics: amniotic epithelial cells and amniotic mesenchymal cells), which contribute to its attractive physical and biological properties that make it fundamental to clinical application. The corneal endothelial cell is a vital part of the cornea, which can influence postoperative vision directly. However, widespread use of fresh AM and cornea has been limited due to their short use span and safety concerns. To overcome these concerns, different preservation methods have been introduced. Cryopreservation is distinguished from many preservation methods for its attractive advantages of prolonged use span, optimally retained tissue structure, and minimized infection risk. This review will focus on recent advances of cryopreserved AM and cornea, including different cryopreservation methods and their indications in ophthalmology.

**Keywords:** amnion, cornea, cryopreservation, indication, ophthalmology

## **1. Cryopreservation of amniotic membranes**

## **1.1 Introduction**

Ever since Davis [1] first used human amniotic membrane (**Figure 1**) (AM) for skin transplantation, people have been exploring this remarkable biomaterial. AM is located in the innermost layer of the fetal membranes [2]. It is 0.02–0.05 mm thick, lightweight, elastic, almost transparent, and avascular membrane classically composed of three layers: the epithelium, the basement membrane and the stroma layer [2]. There are two types of cells with stemness properties in AM: amniotic epithelial cells (AECs) and amniotic mesenchymal cells (AMSCs) [3], which are responsible for its unique biological properties including anti-inflammatory, anti-scarring, anti-microbial, angio-modulating, immunomodulatory, and anti-cancer effects [4–10]. Due to these properties, AM has become an ideal material for ocular reconstruction including the treatment of persistent epithelial defects and non-healing corneal ulcers, corneal perforations and descemetoceles, bullous keratopathy, as well as corneal disorders with associated limbal stem cell deficiency, pterygium, conjunctival reconstruction, corneoscleral melts and perforations, and glaucoma surgeries. However, its use span is short and many viruses (such as HIV-1/2, hepatitis B, hepatitis C, human T-cell lymphotropic virus, syphilis, and cytomegalovirus)

**Figure 1.** *Amniotic membrane (AM).*

can be in their "window period" and escape detection, further limiting the use of fresh AM. To overcome these concerns, different preservation methods have emerged, such as freezing, lyophilization, and cryopreservation. However, most methods result in the destruction of endogenous cells and cause varying degrees of extracellular matrix (ECM) damage, which can affect the functionality of AM and its clinical benefits for wound treatment [11, 12]. Cryopreservation was first introduced by Lee and Tseng and has been proven to achieve high success rate in AM transplantation, which has been distinguished from many methods for its attractive advantages of prolonging use span, optimally retaining tissue structure, and minimizing infection risk [13, 14].

In this part, we classify the cryopreservation methods applied to amnion by commonly used cryoprotectant and analyze the influence of cryopreservation on AM combined with specific clinical trials.

## **1.2 General cryopreservation techniques**

The AM is normally washed using balanced saline solution containing antibiotics such as streptomycin, penicillin, neomycin, and amphotericin prior to storage. Pieces of AM resting on a carrier are placed in a vial containing cryoprotectant solution at a controlled cooling rate. Storage temperatures of −80°C are often utilized, with the maximum storage times ranging between 1 and 2 years [1, 11, 12].

The main disadvantage of cryopreservation is the requirement of a deepfreezing facility, which is expensive, cumbersome, and frequently unavailable, especially in underdeveloped countries. In addition, maintaining stable storage temperatures during transportation is also relatively difficult.

## **1.3 Cryopreservation methods on AM**

## *1.3.1 Glycerol-cryopreservation*

Glycerol storage was first introduced in the Netherlands in 1984 to preserve donor skin for transplantation [13]. Positive results over subsequent decades have led to its clinical acceptance, including in the preservation of AM. Glycerol has led to higher cell viability and higher bFGF secretion for up to three months of AM storage [14]. After strict preservation and sterilization processes, pieces of AM resting on a carrier are placed in a vial containing storage solution. Tseng's laboratory introduced a methodology of glycerol (86%) in Dulbecco's Modified Eagle Medium at a ratio of 1:1 [15, 16]. The most common

**141**

**Figure 2.**

*Pathways of cellular injury during freeing.*

*Cryopreservation in Ophthalmology*

glycerol is responsible for that.

glycerol cryopreservation on AMs.

age method.

*DOI: http://dx.doi.org/10.5772/intechopen.91312*

been reported to be clinically effective [15].

cryopreservation protocol reported in the literature involves the use of 50% glycerol and storage at −80°C [17–21]. Undiluted and 98% glycerol have also

In 2011, Thomasen et al. [21] showed that long-term storage of 50% glycerol cryopreserved AM for durations up to 24 months at −80°C did not significantly impair the histology of AM. Wagner et al. [14] used 85% glycerol for cryopreserved AM, and their histological examinations had no significant alterations following cryopreservation, either for straight cryopreservation or with glycerol. They also demonstrated that neither tensile strength nor Young's modulus was significantly influenced by the storage method. In addition, they also detected a significant increase in tensile strength over storage time, independent of the stor-

Some groups have found that storage of AM in 50% glycerol at −80°C decellularizes the AM and results in low viability [17–20]. Interestingly, the results from Wagner et al. [14] research showed that epithelial cells were not significantly reduced during freezing in comparison to stromal cells, possibly indicating a higher sensitivity of stromal AM cells to freezing damage than epithelial cells (**Figure 2**). Through repeated measurement analysis, storage time showed a significant effect on cell viability. Prabhasawat et al. [22, 23] reported that the use of a highly concentrated glycerol solution abolishes AM cell viability. The possible toxic effect of

To summarize, glycerol-cryopreserved AM retains the histological characteristics of fresh AM independent of an increase in glycerol concentration. Tensile strength and elasticity can also be better preserved, with tensile strength increasing with storage time. However, the cell viability of cryopreserved AM was significantly affected by storage time and glycerol concentration. In particular, the stromal cells were more sensitive. A previous study [24] showed that this method had little effect on the growth factors of AM. More research is needed to confirm the effect of

## *Cryopreservation in Ophthalmology DOI: http://dx.doi.org/10.5772/intechopen.91312*

*Cryopreservation - Current Advances and Evaluations*

and minimizing infection risk [13, 14].

**Figure 1.**

*Amniotic membrane (AM).*

AM combined with specific clinical trials.

**1.2 General cryopreservation techniques**

**1.3 Cryopreservation methods on AM**

*1.3.1 Glycerol-cryopreservation*

can be in their "window period" and escape detection, further limiting the use of fresh AM. To overcome these concerns, different preservation methods have emerged, such as freezing, lyophilization, and cryopreservation. However, most methods result in the destruction of endogenous cells and cause varying degrees of extracellular matrix (ECM) damage, which can affect the functionality of AM and its clinical benefits for wound treatment [11, 12]. Cryopreservation was first introduced by Lee and Tseng and has been proven to achieve high success rate in AM transplantation, which has been distinguished from many methods for its attractive advantages of prolonging use span, optimally retaining tissue structure,

In this part, we classify the cryopreservation methods applied to amnion by commonly used cryoprotectant and analyze the influence of cryopreservation on

The AM is normally washed using balanced saline solution containing antibiotics such as streptomycin, penicillin, neomycin, and amphotericin prior to storage. Pieces of AM resting on a carrier are placed in a vial containing cryoprotectant solution at a controlled cooling rate. Storage temperatures of −80°C are often utilized,

Glycerol storage was first introduced in the Netherlands in 1984 to preserve donor skin for transplantation [13]. Positive results over subsequent decades have led to its clinical acceptance, including in the preservation of AM. Glycerol

has led to higher cell viability and higher bFGF secretion for up to three months of AM storage [14]. After strict preservation and sterilization processes, pieces of AM resting on a carrier are placed in a vial containing storage solution. Tseng's laboratory introduced a methodology of glycerol (86%) in Dulbecco's Modified Eagle Medium at a ratio of 1:1 [15, 16]. The most common

with the maximum storage times ranging between 1 and 2 years [1, 11, 12]. The main disadvantage of cryopreservation is the requirement of a deepfreezing facility, which is expensive, cumbersome, and frequently unavailable, especially in underdeveloped countries. In addition, maintaining stable storage

temperatures during transportation is also relatively difficult.

**140**

cryopreservation protocol reported in the literature involves the use of 50% glycerol and storage at −80°C [17–21]. Undiluted and 98% glycerol have also been reported to be clinically effective [15].

In 2011, Thomasen et al. [21] showed that long-term storage of 50% glycerol cryopreserved AM for durations up to 24 months at −80°C did not significantly impair the histology of AM. Wagner et al. [14] used 85% glycerol for cryopreserved AM, and their histological examinations had no significant alterations following cryopreservation, either for straight cryopreservation or with glycerol. They also demonstrated that neither tensile strength nor Young's modulus was significantly influenced by the storage method. In addition, they also detected a significant increase in tensile strength over storage time, independent of the storage method.

Some groups have found that storage of AM in 50% glycerol at −80°C decellularizes the AM and results in low viability [17–20]. Interestingly, the results from Wagner et al. [14] research showed that epithelial cells were not significantly reduced during freezing in comparison to stromal cells, possibly indicating a higher sensitivity of stromal AM cells to freezing damage than epithelial cells (**Figure 2**). Through repeated measurement analysis, storage time showed a significant effect on cell viability. Prabhasawat et al. [22, 23] reported that the use of a highly concentrated glycerol solution abolishes AM cell viability. The possible toxic effect of glycerol is responsible for that.

To summarize, glycerol-cryopreserved AM retains the histological characteristics of fresh AM independent of an increase in glycerol concentration. Tensile strength and elasticity can also be better preserved, with tensile strength increasing with storage time. However, the cell viability of cryopreserved AM was significantly affected by storage time and glycerol concentration. In particular, the stromal cells were more sensitive. A previous study [24] showed that this method had little effect on the growth factors of AM. More research is needed to confirm the effect of glycerol cryopreservation on AMs.

**Figure 2.** *Pathways of cellular injury during freeing.*

## *1.3.2 DMSO-cryopreservation*

DMSO has been used as an alternative for AM in glycerol-cryopreservation. An increasing concentration of DMSO is used instead of washing the AM with an antibiotic-saline solution after placenta collection [12]. Azuara-Blanco et al. [25] used 4%, 8%, and 10% DMSO, while Kubo et al. [26] used 0.5 M, 0.1 M, and 0.15 M DMSO for washing. AMs can be stored in 10% or 0.15 M DMSO at −80°C for several months without significant damage. In general, solutions containing DMSO are used less often for AM cryopreservation compared to glycerol, due to high toxicity [12]. However, AM storage solutions containing DMSO have been studied a lot regarding its ability to increase cell viability in AM under experimental conditions [2].

A cryopreservation method with DMSO from Duan-Arnold's group [24] showed a retained cell viability of over 80%. Cryopreserved AM tested after three months of storage showed no changes in the tissue architecture and collagen IV, which exists in the basement membrane, compared with fresh AM. However, in 2015, Yazdanpanah et al. [8] showed that the viability of epithelial cells in fresh AMs was estimated at 97% after staining with trypan blue, decreasing to about 50% in DMSO cryopreserved tissues after six months. They evaluated the effects of cryopreservation on AM angiogenesis modulation activity compared to fresh tissue in an animal model, showing that cryopreserved AM has the same effect on angiogenesis as fresh AM. The epithelial surface of cryopreserved AM inhibited angiogenesis, and the mesenchymal surface augmented vessel sprouting and length. In 2013, Tehrani et al. [27] used 10% DMSO as a cryoprotectant to evaluate the antibacterial properties of AM after preservation in vitro. The results of this study showed that the antibacterial property of AM was maintained after cryopreservation, but was dependent on bacterial genus and strain.

To sum up, the literature we collected on DMSO-cryopreserved AM showed no significant differences in tissue integrity and biological properties (antibacterial and angiogenesis modulation) compared with fresh AM. However, although many research groups use DMSO as a cryoprotectant, the data related to cell viability vary. These conflicting results can be attributed to several factors, including differing cryopreservation procedures and storage times.

## **1.4 Controversy on cryopreserved AM**

## *1.4.1 Variable cell viabilities*

In 2000, Kruse et al. [18] believed that devitalized AM exhibited therapeutic effects, and their data showed that the preservation of viable cells in AM provided no additional benefits. This conclusion led to the development of cryopreservation methods including AM devitalization steps. One of them, known as the CRYOTEK® process, includes a freezing step before cryopreservation, resulting in devitalized tissue [28]. However, Yan et al. [29] demonstrated that the combination of exogenous cells and acellular AM resulted in faster wound closure compared with acellular AM alone. Duan-Arnold et al. [24] demonstrated that endogenous viable cells allow cryopreserved AM with higher angiogenic, anti-inflammatory, antioxidant, fibroblast, and keratinocyte chemo-attractive activities when compared with AM in devitalization. Before 2001, most studies reported that cell viability of 50% or less at cryopreserved post-thaw with cells failing to survive after 18 months of storage at −80°C [26, 30]. Since then, scientists have been attempting to improve the cryopreservation method, for improved cell viability retention. For example, the cryopreservation protocol invented by the group of Duan Arnold et al. [24]

**143**

*Cryopreservation in Ophthalmology*

*1.4.2 Storage temperature*

diameter of 15 or 16 mm.

effects.

scientists.

*DOI: http://dx.doi.org/10.5772/intechopen.91312*

can maintain 70% or greater cell viability after 24 months of storage at −80°C. AM storage solutions containing dimethyl sulfoxide (DMSO) have been studied, mostly under experimental conditions, and shown the ability to increase AM cell viability [2]. Although the survival of amniotic cells is related to storage time, different cryopreservation steps can also affect cell viability, thus exerting different clinical

The best storage temperature (−196°C or − 80°C) is also a controversial issue for cryopreservation. AMs stored at −196°C have showed morphology similar to fresh AM in both preservation media, and AM stored at −80°C showed disruption of the stromal matrix [2, 31]. However, −80°C is still widely used by international

To sum up, cryopreservation protocols are not standardized. Preparation and sterilization before cryopreservation, as well as the selection of cryoprotectant during cryopreservation, will lead to high variability in cell viability [2, 32]. Different storage temperature and storage time also affects the structure and function of amniotic membrane. It is important to establish adaptable protocols for the clinical banking of AM that include verification of graft quality and viability before its

PROKERA®Slim (PKS) (Bio-Tissue, Inc., Miami, FL, USA) is a Class II medical device approved by the Food and Drug Administration in 2003 to be used as a temporary AM patch for delivering the biological actions of AM to a corneal surface without using sutures. It contains a piece of cryopreserved AM clipped into a concave poly-carbonate dual-ring system, like a symblepharon ring, that conforms to the corneal and limbal surface like a contact lens. The ring system has an inner

It has become the most common commercially available cryopreserved AM product in ophthalmology and is applied to various ocular surface and orbital disorders. It is a safe and effective method that makes AM transplantation sutureless and adhesiveless, contributing to healing and reconstruction of the ocular surface and orbit with minimal side effects [33]. However, PROKERA is not recommended for eyes with functioning blebs or glaucoma drainage implants because of the opposi-

Corneal disease is one of the world's leading causes of blindness. Corneal scarring and haze due to various factors can affect vision, making corneal transplantation an important means of treatment for corneal diseases [35–37]. Advances in corneal preservation techniques have improved the survival rate of corneal grafts [38] and have largely contributed to the development of modern corneal transplant surgery [39]. With the flourishing of corneal preservation technology, breakthroughs have been made in preservation times and corneal activity. Nevertheless,

release for transplantation, whether in the trial or clinical stage.

**1.5 Commercially available cryopreserved AM**

*1.5.1 PROKERA®Slim (PKS)/PROKERA®(PK)*

tional positioning of the retaining ring [34].

**2. Cryopreservation of cornea**

**2.1 Introduction**

## *Cryopreservation in Ophthalmology DOI: http://dx.doi.org/10.5772/intechopen.91312*

can maintain 70% or greater cell viability after 24 months of storage at −80°C. AM storage solutions containing dimethyl sulfoxide (DMSO) have been studied, mostly under experimental conditions, and shown the ability to increase AM cell viability [2]. Although the survival of amniotic cells is related to storage time, different cryopreservation steps can also affect cell viability, thus exerting different clinical effects.

## *1.4.2 Storage temperature*

*Cryopreservation - Current Advances and Evaluations*

DMSO has been used as an alternative for AM in glycerol-cryopreservation. An increasing concentration of DMSO is used instead of washing the AM with an antibiotic-saline solution after placenta collection [12]. Azuara-Blanco et al. [25] used 4%, 8%, and 10% DMSO, while Kubo et al. [26] used 0.5 M, 0.1 M, and 0.15 M DMSO for washing. AMs can be stored in 10% or 0.15 M DMSO at −80°C for several months without significant damage. In general, solutions containing DMSO are used less often for AM cryopreservation compared to glycerol, due to high toxicity [12]. However, AM storage solutions containing DMSO have been studied a lot regarding its ability to increase cell viability in AM under experimental

A cryopreservation method with DMSO from Duan-Arnold's group [24] showed a retained cell viability of over 80%. Cryopreserved AM tested after three months of storage showed no changes in the tissue architecture and collagen IV, which exists in the basement membrane, compared with fresh AM. However, in 2015, Yazdanpanah et al. [8] showed that the viability of epithelial cells in fresh AMs was estimated at 97% after staining with trypan blue, decreasing to about 50% in DMSO cryopreserved tissues after six months. They evaluated the effects of cryopreservation on AM angiogenesis modulation activity compared to fresh tissue in an animal model, showing that cryopreserved AM has the same effect on angiogenesis as fresh AM. The epithelial surface of cryopreserved AM inhibited angiogenesis, and the mesenchymal surface augmented vessel sprouting and length. In 2013, Tehrani et al. [27] used 10% DMSO as a cryoprotectant to evaluate the antibacterial properties of AM after preservation in vitro. The results of this study showed that the antibacterial property of AM was maintained after cryopreservation, but was dependent on

To sum up, the literature we collected on DMSO-cryopreserved AM showed no significant differences in tissue integrity and biological properties (antibacterial and angiogenesis modulation) compared with fresh AM. However, although many research groups use DMSO as a cryoprotectant, the data related to cell viability vary. These conflicting results can be attributed to several factors, including differing

In 2000, Kruse et al. [18] believed that devitalized AM exhibited therapeutic effects, and their data showed that the preservation of viable cells in AM provided no additional benefits. This conclusion led to the development of cryopreservation methods including AM devitalization steps. One of them, known as the CRYOTEK® process, includes a freezing step before cryopreservation, resulting in devitalized tissue [28]. However, Yan et al. [29] demonstrated that the combination of exogenous cells and acellular AM resulted in faster wound closure compared with acellular AM alone. Duan-Arnold et al. [24] demonstrated that endogenous viable cells allow cryopreserved AM with higher angiogenic, anti-inflammatory, antioxidant, fibroblast, and keratinocyte chemo-attractive activities when compared with AM in devitalization. Before 2001, most studies reported that cell viability of 50% or less at cryopreserved post-thaw with cells failing to survive after 18 months of storage at −80°C [26, 30]. Since then, scientists have been attempting to improve the cryopreservation method, for improved cell viability retention. For example, the cryopreservation protocol invented by the group of Duan Arnold et al. [24]

*1.3.2 DMSO-cryopreservation*

conditions [2].

bacterial genus and strain.

*1.4.1 Variable cell viabilities*

cryopreservation procedures and storage times.

**1.4 Controversy on cryopreserved AM**

**142**

The best storage temperature (−196°C or − 80°C) is also a controversial issue for cryopreservation. AMs stored at −196°C have showed morphology similar to fresh AM in both preservation media, and AM stored at −80°C showed disruption of the stromal matrix [2, 31]. However, −80°C is still widely used by international scientists.

To sum up, cryopreservation protocols are not standardized. Preparation and sterilization before cryopreservation, as well as the selection of cryoprotectant during cryopreservation, will lead to high variability in cell viability [2, 32]. Different storage temperature and storage time also affects the structure and function of amniotic membrane. It is important to establish adaptable protocols for the clinical banking of AM that include verification of graft quality and viability before its release for transplantation, whether in the trial or clinical stage.

## **1.5 Commercially available cryopreserved AM**

## *1.5.1 PROKERA®Slim (PKS)/PROKERA®(PK)*

PROKERA®Slim (PKS) (Bio-Tissue, Inc., Miami, FL, USA) is a Class II medical device approved by the Food and Drug Administration in 2003 to be used as a temporary AM patch for delivering the biological actions of AM to a corneal surface without using sutures. It contains a piece of cryopreserved AM clipped into a concave poly-carbonate dual-ring system, like a symblepharon ring, that conforms to the corneal and limbal surface like a contact lens. The ring system has an inner diameter of 15 or 16 mm.

It has become the most common commercially available cryopreserved AM product in ophthalmology and is applied to various ocular surface and orbital disorders. It is a safe and effective method that makes AM transplantation sutureless and adhesiveless, contributing to healing and reconstruction of the ocular surface and orbit with minimal side effects [33]. However, PROKERA is not recommended for eyes with functioning blebs or glaucoma drainage implants because of the oppositional positioning of the retaining ring [34].

## **2. Cryopreservation of cornea**

## **2.1 Introduction**

Corneal disease is one of the world's leading causes of blindness. Corneal scarring and haze due to various factors can affect vision, making corneal transplantation an important means of treatment for corneal diseases [35–37]. Advances in corneal preservation techniques have improved the survival rate of corneal grafts [38] and have largely contributed to the development of modern corneal transplant surgery [39]. With the flourishing of corneal preservation technology, breakthroughs have been made in preservation times and corneal activity. Nevertheless,

cryopreservation is the only current method that can virtually preserve tissue structure for a long time.

Meanwhile, the development of modern eye banks have been accompanied by the advancement of corneal preservation technology. The establishment of an eye bank provides favorable conditions for corneal transplantation [40, 41].

## **2.2 Corneal transplantation and preservation**

The idea of replacing the turbid cornea with transparent tissue was first proposed by Pellier de Quengsy in 1789 [42, 43]. In 1824, Reisinger exploited animal corneas in surgery [44], which was named keratoplasty. Later in the nineteenth century, a large number of animal experiments helped doctors realize that inter-species transplantation was a necessary condition to avoid corneal opacity after transplantation [45–47]. In view of this, researchers began to experiment with human corneal transplantation. Early corneal transplantation relied on living donor tissues due to fears relating to transplanting dead tissue. The first successful full-thickness corneal transplantation (including all corneal layers) was completed in 1905 [48]. It was not until the 1930s that the cornea of the deceased donor was used and the entire eye was kept in a glass jug (wet room) for several days [49].

In 1912, Magitot reported that excised corneal grafts could be preserved in red blood cells at 5°C for eight days [50–52]. The grafts were successfully used for corneal lamellar transplantation [53]. At first, the freshness of the cornea was considered key to corneal transplantation [54, 55]. However, Ukrainian doctor Filatov systematically reported the application of corpus corneal tissue to clinical practice [56], which possessed an inter-generational meaning. It opened a new era of corneal preservation and transplantation [57–59]. These developments led to the establishment of the world's first ophthalmology bank in New York in the 1940s. The preservation technique of the original eye bank was very simple [60, 61]: eyeballs were kept in a small glass bottle in a humid and cool environment [62]. Immediate removal of the eyeball after donor death was the only way to ensure the quality of the corneal grafts [63–65]. Eye banks were established in major cities, such as London, to guarantee that eyeballs were promptly forwarded [66, 67]. In the early 1950s, the activity of CECs was first considered as an important factor affecting transplantation [68–70]. The emphasis on preservation techniques was transferred to maintain the activity and integrity of CECs [71, 72]. Since then, corneal preservation techniques have been increasingly successful, resulting in approximately 40,000 corneal transplants per year in the United States, 20,000 per year in Europe, and thousands per year in other countries, such as India.

## **2.3 Corneal preservation methods**

Corneal preservation is divided into two categories according to the survival of CECs: inactive and active preservation [73–75]. The former method includes dry preservation and cryopreservation [76–78] and operates under the principle of removing corneal moisture while inhibiting enzyme activity and autolysis in cells for long-term preservation [79, 80]. Common preservatives are glycerin, molecular sieves, and silica gel [81–83], which can preserve intact lamellar collagen structure [84]. Active preservation comprises short-term (hours to two days), medium-long term (7 to 30 days) and long-term (months to years) preservation. In terms of storage conditions, it utilizes normal (34~37°C), low (usually 4°C) and deep low (subzero) temperatures [85–88].

Short-term corneal storage mainly refers to the preservation of wet rooms, the simplest and most convenient of all corneal storage technologies. For this reason,

**145**

edema.

**2.4 Cryopreservation**

endothelial cells.

tion and toxicity in these situations.

**2.5 Corneal cryopreservation technology**

*Cryopreservation in Ophthalmology*

*DOI: http://dx.doi.org/10.5772/intechopen.91312*

is stored at 4°C for 4 to 14 days [89].

it is still the basic technology for preserving cornea in the eye banks of developing countries. As for medium-term corneal preservation, corneal preservation solution

The prolongation of corneal preservation allows more preparation for patients

At present, there are several corneal preservation methods applied in global eye banks, but none of those is perfect. Each preservation method has its own advantages and disadvantages, which differ from the preservation temperature, the composition of the preservation solution, and the penetrant preventing matrix

After donor death, the sudden stop of the aqueous humor causes nutrient and oxygen shortages, leading to final depletion at room temperature, which can, in turn, lead to autolysis of the corneal cells and initial damage to the cornea [92]. During the period from donor death to corneal removal and storage, the donor's corpse is exposed to room temperatures, necessitating minimal time delays to ensure that the initial donor cornea is healthy and intact along with functional

The acceptable short storage time, as well as organ damage, poses a logistical challenge to organ storage and ultimately affects grafts and patient survival. Prolonged storage times can cause many transplantable organs, further exacerbating the growing imbalance between organ supply and demand. Organ cryopreservation is used to preserve long-lived cells and tissues. Theoretically, the storage of biological materials, including cells, tissues, and organs for transplantation at a low temperature (i.e., in liquid nitrogen at −160°C) is uncertain [93, 94]. Such a technique would have the potential to alter the way in which organs are recovered, distributed, and utilized for transplantation. However, ice is the biggest enemy in the cryopreservation of organs and tissues. Ice crystals, especially intracellular ice, can cause significant cellular damage and destroy the complex macroscopic tissues of intact organs. In this field, current developments are used to avoid the formation of ice, or mitigate it, during cryogenic storage. Any successful organ cryopreservation strategy requires a delicate balance between the relative needs of cryopreserva-

In 1954, Eastcott first adopted a cryopreserved human cornea for transplantation successfully [95, 96], pretreating the keratin tissue in glycerol before freezing it in a mixture of alcohol and carbon dioxide for cryopreservation of the full-thickness cornea [97]. This method generally removes the cornea under the protection of a cryogen to −80°C, and stores it in liquid nitrogen at −196°C. Therefore, the CECs are in a dormant state. The state can completely inhibit the metabolism

and flexible adjusting of operation times, while also satisfying blood test and corneal transportation times. With the improvement of preservation techniques, the composition of the corneal preservation solution has been constantly changing. A certain concentration of chondroitin sulfate is added to modify M-K solution, which can alleviate the swelling state during preservation. Optisol corneal medium preservation solution was proposed by Lindstrom and has become the most common preservation solution in US eye banks, which is mainly a mixture of K-liquid and Dexsol solution [90]. Long-term corneal preservation refers to organ culture storage and cryopreservation. Organ culture is to simulate the presence of a normal

human cornea environment with medium at 30–37°C [91].

## *Cryopreservation in Ophthalmology DOI: http://dx.doi.org/10.5772/intechopen.91312*

*Cryopreservation - Current Advances and Evaluations*

**2.2 Corneal transplantation and preservation**

was kept in a glass jug (wet room) for several days [49].

structure for a long time.

cryopreservation is the only current method that can virtually preserve tissue

bank provides favorable conditions for corneal transplantation [40, 41].

Meanwhile, the development of modern eye banks have been accompanied by the advancement of corneal preservation technology. The establishment of an eye

The idea of replacing the turbid cornea with transparent tissue was first proposed by Pellier de Quengsy in 1789 [42, 43]. In 1824, Reisinger exploited animal corneas in surgery [44], which was named keratoplasty. Later in the nineteenth century, a large number of animal experiments helped doctors realize that inter-species transplantation was a necessary condition to avoid corneal opacity after transplantation [45–47]. In view of this, researchers began to experiment with human corneal transplantation. Early corneal transplantation relied on living donor tissues due to fears relating to transplanting dead tissue. The first successful full-thickness corneal transplantation (including all corneal layers) was completed in 1905 [48]. It was not until the 1930s that the cornea of the deceased donor was used and the entire eye

In 1912, Magitot reported that excised corneal grafts could be preserved in red blood cells at 5°C for eight days [50–52]. The grafts were successfully used for corneal lamellar transplantation [53]. At first, the freshness of the cornea was considered key to corneal transplantation [54, 55]. However, Ukrainian doctor Filatov systematically reported the application of corpus corneal tissue to clinical practice [56], which possessed an inter-generational meaning. It opened a new era of corneal preservation and transplantation [57–59]. These developments led to the establishment of the world's first ophthalmology bank in New York in the 1940s. The preservation technique of the original eye bank was very simple [60, 61]: eyeballs were kept in a small glass bottle in a humid and cool environment [62]. Immediate removal of the eyeball after donor death was the only way to ensure the quality of the corneal grafts [63–65]. Eye banks were established in major cities, such as London, to guarantee that eyeballs were promptly forwarded [66, 67]. In the early 1950s, the activity of CECs was first considered as an important factor affecting transplantation [68–70]. The emphasis on preservation techniques was transferred to maintain the activity and integrity of CECs [71, 72]. Since then, corneal preservation techniques have been increasingly successful, resulting in approximately 40,000 corneal transplants per year in the United States, 20,000 per

year in Europe, and thousands per year in other countries, such as India.

Corneal preservation is divided into two categories according to the survival of CECs: inactive and active preservation [73–75]. The former method includes dry preservation and cryopreservation [76–78] and operates under the principle of removing corneal moisture while inhibiting enzyme activity and autolysis in cells for long-term preservation [79, 80]. Common preservatives are glycerin, molecular sieves, and silica gel [81–83], which can preserve intact lamellar collagen structure [84]. Active preservation comprises short-term (hours to two days), medium-long term (7 to 30 days) and long-term (months to years) preservation. In terms of storage conditions, it utilizes normal (34~37°C), low (usually 4°C) and deep low

Short-term corneal storage mainly refers to the preservation of wet rooms, the simplest and most convenient of all corneal storage technologies. For this reason,

**2.3 Corneal preservation methods**

(subzero) temperatures [85–88].

**144**

it is still the basic technology for preserving cornea in the eye banks of developing countries. As for medium-term corneal preservation, corneal preservation solution is stored at 4°C for 4 to 14 days [89].

The prolongation of corneal preservation allows more preparation for patients and flexible adjusting of operation times, while also satisfying blood test and corneal transportation times. With the improvement of preservation techniques, the composition of the corneal preservation solution has been constantly changing. A certain concentration of chondroitin sulfate is added to modify M-K solution, which can alleviate the swelling state during preservation. Optisol corneal medium preservation solution was proposed by Lindstrom and has become the most common preservation solution in US eye banks, which is mainly a mixture of K-liquid and Dexsol solution [90]. Long-term corneal preservation refers to organ culture storage and cryopreservation. Organ culture is to simulate the presence of a normal human cornea environment with medium at 30–37°C [91].

At present, there are several corneal preservation methods applied in global eye banks, but none of those is perfect. Each preservation method has its own advantages and disadvantages, which differ from the preservation temperature, the composition of the preservation solution, and the penetrant preventing matrix edema.

## **2.4 Cryopreservation**

After donor death, the sudden stop of the aqueous humor causes nutrient and oxygen shortages, leading to final depletion at room temperature, which can, in turn, lead to autolysis of the corneal cells and initial damage to the cornea [92]. During the period from donor death to corneal removal and storage, the donor's corpse is exposed to room temperatures, necessitating minimal time delays to ensure that the initial donor cornea is healthy and intact along with functional endothelial cells.

The acceptable short storage time, as well as organ damage, poses a logistical challenge to organ storage and ultimately affects grafts and patient survival. Prolonged storage times can cause many transplantable organs, further exacerbating the growing imbalance between organ supply and demand. Organ cryopreservation is used to preserve long-lived cells and tissues. Theoretically, the storage of biological materials, including cells, tissues, and organs for transplantation at a low temperature (i.e., in liquid nitrogen at −160°C) is uncertain [93, 94]. Such a technique would have the potential to alter the way in which organs are recovered, distributed, and utilized for transplantation. However, ice is the biggest enemy in the cryopreservation of organs and tissues. Ice crystals, especially intracellular ice, can cause significant cellular damage and destroy the complex macroscopic tissues of intact organs. In this field, current developments are used to avoid the formation of ice, or mitigate it, during cryogenic storage. Any successful organ cryopreservation strategy requires a delicate balance between the relative needs of cryopreservation and toxicity in these situations.

## **2.5 Corneal cryopreservation technology**

In 1954, Eastcott first adopted a cryopreserved human cornea for transplantation successfully [95, 96], pretreating the keratin tissue in glycerol before freezing it in a mixture of alcohol and carbon dioxide for cryopreservation of the full-thickness cornea [97]. This method generally removes the cornea under the protection of a cryogen to −80°C, and stores it in liquid nitrogen at −196°C. Therefore, the CECs are in a dormant state. The state can completely inhibit the metabolism

of cells, eliminate the toxic effects caused by the accumulation of metabolites, and avoid the need to change the liquid during organ culture. In addition, it also restrains microorganisms during cryopreservation, protecting the cornea from microbial invasion.

The components currently contained in corneal cryoprotectants include DMSO, propylene glycol, ChS, and sucrose. DMSO is a relatively stable protective agent to maintain the integrity of corneal cells, while sucrose molecules act as buffers in corneal protection, and ChS improves CEC activity in cryopreservation [98]. DMSO began to be treated as a tissue preservative to preserve cultured rabbit CECs by Smith [99]. Shortly thereafter, Mueller injected a preservation solution containing DMSO into the anterior chamber of an eyeball, placing the eyeball in a preservation solution containing glycerol. The cornea was removed before surgery for full-thickness transplantation [100, 101]. In 1965, Capella [102] used DMSO as an antifreeze to improve a cryopreservation method, ensuring corneal graft activity. According to another report [103], the clinical application of cryopreservation techniques has little differences in techniques. The corneal tissue must be preserved eight hours after death. By increasing the level of DMSO, it eventually reaches a concentration of 7.5%. The classic four-step cooling is to initiate a cooling rate at 1.5~2°C/min, drop the temperature to −30°C, change to 5–7°C/min, and ultimately maintain −80°C [104, 105].

It is still essential to further explore the rate of cooling to keep CEC activity and reduce cell loss [106, 107]. Temperature-controlled thawing before transplantation is a key step in protecting the corneal endothelium. At present, the prevalent view is that rapid rewarming could decrease the contact of cells with high concentrations of electrolytes and reduce cell damage [108]. The thawing process of the cryopreserved cornea must be strictly controlled, as the solute containing DMSO has endotoxicity once the temperature exceeds 37°C [109]. Cryopreservation would impair the morphology and function of the corneal endothelium. During the thawing process, an ascending solute concentration, the formation of crystals, changes in pH, and osmotic pressure will reduce the survival rate of CECs [110]. Glycerol, polyvinylpyrrolidone, and DMSO can all be used as cryopreservatives, but DMSO is currently the most widely used [111, 112].

## **2.6 Effect of corneal cryopreservation**

The ultra-low temperature preservation method overcomes the drawbacks of most other corneal preservation methods, significantly prolonging corneal preservation time, reducing pollution, and avoiding the toxic effects of its own metabolic substances. Electron microscopy can observe changes in the subcellular morphology of CECs caused by cryopreservation, some of which are considered irreversible [113]. Studies have shown that, after cryopreservation, the barrier function of endothelial cells is impaired. Compared with wet room preservation and MK solution preservation, cryopreserved corneal grafts have been completely transparent for a long time after surgery. For one-year cryopreservation, 55% of endothelial cells were deactivated, while the rate of CECs preserved by MK solution was only 21–22% [114]. There are barely significant structural differences in microbiological, histological, and ultrastructural features when comparing long-term cryopreservation of tissue (>7 years) and short-term cryopreserved cat corneal sclera (<1 year) [115]. As such, tissues cryopreserved for up to 10 years could be used for tectonic support without structural or microbial barriers.

Under suitable conditions, no crystal solidification occurs during the freezing process, called vitrification [116]. Vitrification requires a high concentration of

**147**

continue.

**Conflict of interest**

We declare that we have no conflicts of interest.

*Cryopreservation in Ophthalmology*

corneal vitrification [118, 119].

12 years [121].

corneas are difficult to get.

**3. Conclusion**

*DOI: http://dx.doi.org/10.5772/intechopen.91312*

cryoprotectant, yet theoretically, tissue could be stored in a very low temperature environment without forming intracellular or extracellular crystals, and corneal endothelium damage could be avoided significantly [117]. Glycerol, 1,2-propanetriol, and 2,3-butanediol are all considered as eligible cryopreservation agents for

Studies have found that an effective concentration of a single cryopreservative is toxic to CECs, yet the mixture of preservatives or the addition of preservatives at low temperatures seems to reduce toxicity [120]. As a means of corneal preservation, further study is warranted to investigate whether vitrification would achieve good results. In 1981, Sperling used corneal grafts in a corneal preservation solution at the early stage and carried out a cryopreservation operation later. After rewarming, the cornea was transferred to a preservation solution, identifying corneal activity. The following studies indicated that the corneal grafts maintained transparency 71% of the time after 1 year and 58% of the time after

In our previous study, we performed lamellar keratoplasty combined with keratopigmentation in 22 corneal leukoma eyes using glycerol-cryopreserved corneal tissues, and no graft-rejection occurred during the 3 years of follow-up. Moreover, the outcome of a low graft rejection rate in glycerol-cryopreserved corneal tissues was also confirmed by our preceding study in treating Terrien marginal degeneration. In the subsequent study, for patients with refractive herpes simplex keratitis, 3 eyes of 27 eyes (11.1%) suffered allograft stromal rejection, all eyes reversed after prompt medication. Meanwhile, only 2 eyes (7.41%) exhibited refractive herpes simplex keratitis recurrence and the main site was located at the margin of the graft and the recipient bed. This result is consistent with the theory that grafts survive better when compared with reports clarifying that up to 33% of patients have suffered recurrence using fresh grafts. The recurrence rate in fresh grafts may be partially related to the long-term usage of topical steroid eye drops; however, it may be much more closely correlated with fewer keratocytes in the cryopreserved donor tissue to reactivate immune-inflammatory responses [122–124]. Based on the above information, glycerol-cryopreserved corneal tissues can be effectively and biosafely used with a low rejection and recurrence rate in corneal transplantation, especially in developing countries where good donor

The cryopreservation method can preserve the activity of the AM and cornea for extended periods up to several years, solving the problem of preservation time and activity deterioration. However, equipment complications, expensive technical support, and transport difficulties have become impediments to widespread use. The functional status of AM, endothelial cells, and corneal transparency have been of vital importance in the development of cryopreservation. As researchers become more aware of the function and properties of CECs, attempts to find a more conducive method and media for the preservation of AMs and corneas will

## *Cryopreservation in Ophthalmology DOI: http://dx.doi.org/10.5772/intechopen.91312*

*Cryopreservation - Current Advances and Evaluations*

microbial invasion.

maintain −80°C [104, 105].

currently the most widely used [111, 112].

**2.6 Effect of corneal cryopreservation**

support without structural or microbial barriers.

of cells, eliminate the toxic effects caused by the accumulation of metabolites, and avoid the need to change the liquid during organ culture. In addition, it also restrains microorganisms during cryopreservation, protecting the cornea from

The components currently contained in corneal cryoprotectants include DMSO, propylene glycol, ChS, and sucrose. DMSO is a relatively stable protective agent to maintain the integrity of corneal cells, while sucrose molecules act as buffers in corneal protection, and ChS improves CEC activity in cryopreservation [98]. DMSO began to be treated as a tissue preservative to preserve cultured rabbit CECs by Smith [99]. Shortly thereafter, Mueller injected a preservation solution containing DMSO into the anterior chamber of an eyeball, placing the eyeball in a preservation solution containing glycerol. The cornea was removed before surgery for full-thickness transplantation [100, 101]. In 1965, Capella [102] used DMSO as an antifreeze to improve a cryopreservation method, ensuring corneal graft activity. According to another report [103], the clinical application of cryopreservation techniques has little differences in techniques. The corneal tissue must be preserved eight hours after death. By increasing the level of DMSO, it eventually reaches a concentration of 7.5%. The classic four-step cooling is to initiate a cooling rate at 1.5~2°C/min, drop the temperature to −30°C, change to 5–7°C/min, and ultimately

It is still essential to further explore the rate of cooling to keep CEC activity and reduce cell loss [106, 107]. Temperature-controlled thawing before transplantation is a key step in protecting the corneal endothelium. At present, the prevalent view is that rapid rewarming could decrease the contact of cells with high concentrations of electrolytes and reduce cell damage [108]. The thawing process of the cryopreserved cornea must be strictly controlled, as the solute containing DMSO has endotoxicity once the temperature exceeds 37°C [109]. Cryopreservation would impair the morphology and function of the corneal endothelium. During the thawing process, an ascending solute concentration, the formation of crystals, changes in pH, and osmotic pressure will reduce the survival rate of CECs [110]. Glycerol, polyvinylpyrrolidone, and DMSO can all be used as cryopreservatives, but DMSO is

The ultra-low temperature preservation method overcomes the drawbacks of most other corneal preservation methods, significantly prolonging corneal preservation time, reducing pollution, and avoiding the toxic effects of its own metabolic substances. Electron microscopy can observe changes in the subcellular morphology of CECs caused by cryopreservation, some of which are considered irreversible [113]. Studies have shown that, after cryopreservation, the barrier function of endothelial cells is impaired. Compared with wet room preservation and MK solution preservation, cryopreserved corneal grafts have been completely transparent for a long time after surgery. For one-year cryopreservation, 55% of endothelial cells were deactivated, while the rate of CECs preserved by MK solution was only 21–22% [114]. There are barely significant structural differences in microbiological, histological, and ultrastructural features when comparing long-term cryopreservation of tissue (>7 years) and short-term cryopreserved cat corneal sclera (<1 year) [115]. As such, tissues cryopreserved for up to 10 years could be used for tectonic

Under suitable conditions, no crystal solidification occurs during the freezing process, called vitrification [116]. Vitrification requires a high concentration of

**146**

cryoprotectant, yet theoretically, tissue could be stored in a very low temperature environment without forming intracellular or extracellular crystals, and corneal endothelium damage could be avoided significantly [117]. Glycerol, 1,2-propanetriol, and 2,3-butanediol are all considered as eligible cryopreservation agents for corneal vitrification [118, 119].

Studies have found that an effective concentration of a single cryopreservative is toxic to CECs, yet the mixture of preservatives or the addition of preservatives at low temperatures seems to reduce toxicity [120]. As a means of corneal preservation, further study is warranted to investigate whether vitrification would achieve good results. In 1981, Sperling used corneal grafts in a corneal preservation solution at the early stage and carried out a cryopreservation operation later. After rewarming, the cornea was transferred to a preservation solution, identifying corneal activity. The following studies indicated that the corneal grafts maintained transparency 71% of the time after 1 year and 58% of the time after 12 years [121].

In our previous study, we performed lamellar keratoplasty combined with keratopigmentation in 22 corneal leukoma eyes using glycerol-cryopreserved corneal tissues, and no graft-rejection occurred during the 3 years of follow-up. Moreover, the outcome of a low graft rejection rate in glycerol-cryopreserved corneal tissues was also confirmed by our preceding study in treating Terrien marginal degeneration. In the subsequent study, for patients with refractive herpes simplex keratitis, 3 eyes of 27 eyes (11.1%) suffered allograft stromal rejection, all eyes reversed after prompt medication. Meanwhile, only 2 eyes (7.41%) exhibited refractive herpes simplex keratitis recurrence and the main site was located at the margin of the graft and the recipient bed. This result is consistent with the theory that grafts survive better when compared with reports clarifying that up to 33% of patients have suffered recurrence using fresh grafts. The recurrence rate in fresh grafts may be partially related to the long-term usage of topical steroid eye drops; however, it may be much more closely correlated with fewer keratocytes in the cryopreserved donor tissue to reactivate immune-inflammatory responses [122–124]. Based on the above information, glycerol-cryopreserved corneal tissues can be effectively and biosafely used with a low rejection and recurrence rate in corneal transplantation, especially in developing countries where good donor corneas are difficult to get.

## **3. Conclusion**

The cryopreservation method can preserve the activity of the AM and cornea for extended periods up to several years, solving the problem of preservation time and activity deterioration. However, equipment complications, expensive technical support, and transport difficulties have become impediments to widespread use. The functional status of AM, endothelial cells, and corneal transparency have been of vital importance in the development of cryopreservation. As researchers become more aware of the function and properties of CECs, attempts to find a more conducive method and media for the preservation of AMs and corneas will continue.

## **Conflict of interest**

We declare that we have no conflicts of interest.

## **Funding**


## **Author details**

Yuting Shao† , Chao Chen† , Qi Zhou, Jun Yang, Xiao Lv, Mingyue Lin and Yanlong Bi\* Department of Ophthalmology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China

\*Address all correspondence to: biyanlong@tongji.edu.cn

† Co-first author.

© 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.

**149**

*Cryopreservation in Ophthalmology*

[1] Dua HS, Gomes JA, King AJ,

in ophthalmology. Survey of

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[10] Seo JH, Kim YH, Kim JS. Properties of the amniotic membrane may be applicable in cancer therapy. Medical Hypotheses. 2008;**70**(4):812-814. DOI:

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biomaterials.2009.09.034

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[13] Hermans MHE. Clinical-experience

[9] Talmi YP, Sigler L, Inge E, Finkelstein Y, Zohar Y. Antibacterial properties of human amniotic

Ophthalmology. 2004;**49**(1):51-77. DOI: 10.1016/j.survophthal.2003.10.004

[2] Hettiarachchi D, Dissanayake VHW, Goonasekera HWW. Optimizing amniotic membrane tissue banking protocols for ophthalmic use. Cell and Tissue Banking. 2016;**17**(3):387-397. DOI: 10.1007/s10561-016-9568-3

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[7] Hao YX, Ma DHK, Hwang DG, Kim WS, Zhang F. Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea. 2000;**19**(3):348-352. DOI: 10.1097/00003226-200005000-00018

cell.2013.0090

*Cryopreservation in Ophthalmology DOI: http://dx.doi.org/10.5772/intechopen.91312*

## **References**

*Cryopreservation - Current Advances and Evaluations*

1.The National Nature Science Foundation of China (81470028).

Leaders in Medical Disciplines in Shanghai (2017BR060).

2.The Municipal Human Resources Development Program for Outstanding

3.Shanghai Shenkang Hospital Development Project: A Three- Year Action Plan

**Funding**

(16CR3027A).

**148**

**Author details**

† Co-first author.

, Chao Chen†

provided the original work is properly cited.

Medicine, Shanghai, China

, Qi Zhou, Jun Yang, Xiao Lv, Mingyue Lin and

Department of Ophthalmology, Tongji Hospital, Tongji University School of

© 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,

\*Address all correspondence to: biyanlong@tongji.edu.cn

Yuting Shao†

Yanlong Bi\*

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[8] Yazdanpanah G, Paeini-Vayghan G, Asadi S, Niknejad H. The effects of cryopreservation on angiogenesis modulation activity of human amniotic membrane. Cryobiology. 2015;**71**(3):413-418. DOI: 10.1016/j. cryobiol.2015.09.008

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[10] Seo JH, Kim YH, Kim JS. Properties of the amniotic membrane may be applicable in cancer therapy. Medical Hypotheses. 2008;**70**(4):812-814. DOI: 10.1016/j.mehy.2007.08.008

[11] Jirsova K, Jones GLA. Amniotic membrane in ophthalmology: Properties, preparation, storage and indications for grafting—A review. Cell and Tissue Banking. 2017;**18**(2):193-204. DOI: 10.1007/s10561-017-9618-5

[12] Riau AK, Beuerman RW, Lim LS, Mehta JS. Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction. Biomaterials. 2010;**31**(2):216-225. DOI: 10.1016/j. biomaterials.2009.09.034

[13] Hermans MHE. Clinical-experience with glycerol-preserved donor skin treatment in partial thickness burns. Burns. 1989;**15**(1):57-59. DOI: 10.1016/0305-4179(89)90074-0

[14] Wagner M, Walter P, Salla S, Johnen S, Plange N, Rutten S, et al. Cryopreservation of amniotic membrane with and without glycerol additive. Graefes Archive for Clinical and Experimental Ophthalmology. 2018;**256**(6):1117-1126. DOI: 10.1007/ s00417-018-3973-1

[15] Lee SH, Tseng SCG. Amniotic membrane transplantation for persistent epithelial defects with ulceration. American Journal of Ophthalmology. 1997;**123**(3):303-312. DOI: 10.1016/ S0002-9394(14)70125-4

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[18] Kruse FE, Joussen AM, Rohrschneider K, You LT, Sinn B, Baumann J, et al. Cryopreserved human amniotic membrane for ocular surface reconstruction. Graefe's Archive for Clinical and Experimental Ophthalmology. 2000;**238**(1):68-75. DOI: 10.1007/s004170050012

[19] Shortt AJ, Secker GA, Lomas RJ, Wilshaw SP, Kearney JN, Tuft SJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells. Biomaterials. 2009;**30**(6):1056-1065. DOI: 10.1016/j.biomaterials.2008.10.048

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[21] Thomasen H, Pauklin M, Noelle B, Geerling G, Vetter J, Steven P, et al. The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane. Current Eye Research. 2011;**36**(3):247- 255. DOI: 10.3109/02713683.2010.542267

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[23] Prabhasawat P, Tseng SCG. Impression cytology study of epithelial phenotype of ocular surface reconstructed by preserved human amniotic membrane. Archives of Ophthalmology-Chic. 1997;**115**(11):1360-1367. DOI: 10.1001/ archopht.1997.01100160530001

[24] Duan-Arnold Y, Gyurdieva A, Johnson A, Uveges TE, Jacobstein DA, Danilkovitch A. Retention of endogenous viable cells enhances the antiinflammatory activity of cryopreserved amnion. Advances in Wound Care. 2015;**4**(9):523-533. DOI: 10.1089/ wound.2015.0636

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Current Eye Research. 2011;**36**(3):247- 255. DOI: 10.3109/02713683.2010.542267

[22] Prabhasawat P, Kosrirukvongs P, Booranapong W, Vajaradul Y. Application of preserved human amniotic membrane for corneal surface reconstruction. Cell and Tissue Banking. 2000;**1**(3):213-222. DOI:

10.1023/A:1026542702099

[23] Prabhasawat P, Tseng SCG.

[24] Duan-Arnold Y, Gyurdieva A, Johnson A, Uveges TE, Jacobstein DA, Danilkovitch A. Retention of endogenous

inflammatory activity of cryopreserved amnion. Advances in Wound Care. 2015;**4**(9):523-533. DOI: 10.1089/

[25] Azuara-Blanco A, Pillai CT, Dua HS. Amniotic membrane transplantation for ocular surface reconstruction. British Journal of Ophthalmology. 1999;**83**(4):399-402. DOI: 10.1136/

[26] Kubo M, Sonoda Y, Muramatsu R, Usui M. Immunogenicity of human amniotic membrane in experimental xenotransplantation. Investigative Ophthalmology & Visual Science.

viable cells enhances the anti-

wound.2015.0636

bjo.83.4.399

2001;**42**(7):1539-1546

cryobiol.2013.08.010

process.html

[27] Tehrani FA, Ahmadiani A,

Niknejad H. The effects of preservation procedures on antibacterial property of amniotic membrane. Cryobiology. 2013;**67**(3):293-298. DOI: 10.1016/j.

[28] Amniox Medical, Inc. CRYOTEK process. Available from: www. amnioxmedical.com/CRYOTEK-

phenotype of ocular surface reconstructed by preserved human amniotic membrane. Archives of Ophthalmology-Chic. 1997;**115**(11):1360-1367. DOI: 10.1001/ archopht.1997.01100160530001

Impression cytology study of epithelial

[15] Lee SH, Tseng SCG. Amniotic membrane transplantation for persistent epithelial defects with ulceration. American Journal of Ophthalmology. 1997;**123**(3):303-312. DOI: 10.1016/

[16] Malhotra C, Jain AK. Human amniotic membrane transplantation: Different modalities of its use in ophthalmology. World Journal of Transplantation. 2014;**4**(2):111-121.

Mandrycky C, He H, O'Connell J, Tseng SCG. Comparison of cryopreserved amniotic membrane and umbilical cord tissue with dehydrated amniotic membrane/chorion tissue. Journal of Wound Care. 2014;**23**(10):465. DOI: 10.12968/jowc.2014.23.10.465

S0002-9394(14)70125-4

DOI: 10.5500/wjt.v4.i2.111

[18] Kruse FE, Joussen AM, Rohrschneider K, You LT, Sinn B, Baumann J, et al. Cryopreserved human

amniotic membrane for ocular surface reconstruction. Graefe's Archive for Clinical and Experimental Ophthalmology. 2000;**238**(1):68-75. DOI: 10.1007/s004170050012

[19] Shortt AJ, Secker GA, Lomas RJ, Wilshaw SP, Kearney JN, Tuft SJ, et al. The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells. Biomaterials. 2009;**30**(6):1056-1065. DOI: 10.1016/j.biomaterials.2008.10.048

[20] Niknejad H, Deihim T, Solati-Hashjin M, Peirovi H. The effects of preservation procedures on amniotic membrane's ability to serve as a substrate for cultivation of endothelial cells. Cryobiology. 2011;**63**(3):145-151. DOI: 10.1016/j.cryobiol.2011.08.003

[21] Thomasen H, Pauklin M, Noelle B, Geerling G, Vetter J, Steven P, et al. The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane.

[17] Cooke M, Tan EK,

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ICL.0b013e31818c25bf

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iovs.17-22218

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**156**

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**159**

Section 4

Dynamics of Water

Content in Plant Tissues

During Cooling and

Heating

## Section 4
