Methodologies for Cryopreservation of Mammalian Germline Cells and Tissues

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* This work was supported by Next-Generation BioGreen 21 Program (PJ011347), Republic of Korea.

†Corresponding author : Phone: +82-31-670-4687, E-mail: byryu@cau.ac.kr

Methodologies for Cryopreservation of Mammalian Germline Cells and Tissues

Polash Chandra Karmakar, Sang-Eun Jung and Buom-Yong Ryu^†

Department of Animal Science & Technology, Chung-Ang University, Anseong 456-756, Republic of Korea

ABSTRACT

Until today, success in germline cells and tissue cryopreservation is limited mainly due to the poor understanding of the complex physiological processes can lead to cell damage during cryopreservation. Germline cells, from both male and female, have unique ability to differentiate into one or more cell lines and thus it becomes a crucial point to store them in subzero temperature with the minimal damage of their functional properties and maximum recovery of unchanged and viable cells when thawed. In the past three decades, a vast research has been performed using various different animal models which in fact have led to development of new methodologies and optimization of older one. However, successful use of animal model has provided the opportunity in research with human germline cells and tissues preservation, but not in all the cases. Therefore, the use of new cryo-protective chemicals and modified protocols have been often found in different groups of researchers based on the types, physical structures, utility and animal species of the specimens to be cryopreserved. This review discusses about the basics of different types of cryopreservation methodologies and commonly used optimized protocols and cryoprotectants for germline cells and tissues preservation.

(Key words : Germline cells and tissues, Cryopreservation, Cryoprotectant, Fertility preservation)

INTRODUCTION

Cryopreservation or cryoconservation is defined as a process where biological substances such as cells, tissues, organelles, extracellular matrix, and even organs are preserved by cooling to very low temperatures, ty- pically at −80℃ by using dry ice (solid CO2) or into liquid nitrogen at −196℃. This preservation phenome- non is special because it gives the maximum pro- tection to the biologically susceptible substances from extremely low temperature shock and as well as from damages caused by ice formation during freezing and unregulated chemical kinetics (Pegg, 2007). It is need- less to say that the history of cryopreservation began with the demand of preserving tissue, organelles and organs in terms of medical purpose such as grafting, transplantation etc. In the early period cryopreservation theory was proposed and developed for preserving human blood sample (Mazur, 1970) and even- tually researchers got success in preserving different tissues (Karlsson and Toner, 1996), and mammalian li- ver (Rubinsky et al., 1994) type organs with the de-

velopment of preservation methodologies and cryoprotectant chemicals. In the similar way, methodologies of long term preservation of male or female germ cells and testis or ovary tissues were developed in the next era for the demand of fertility preservation and medications for infertile people.

Spermatogonial stem cells (SSCs) are the foundation of sperm production in adult through spermatogenesis because they have the ability to undergo both self-re- newal and differentiation by several mitotic and meiotic division processes throughout the lifetime of a male (De Rooij et al., 1992). On the other hand, the ovum or egg cell is the female reproductive cell in oogamous organisms which are produced from oogenesis process with the similar mitotic and meiotic procedures (Bukovsky et al., 2005). So, when it is the question about preserving the fertility in both male and female, researchers found the importance of uninterrupted functional and stemness properties of the germline cells with minimal loss of cells while thawing and this strategy was followed for the cryopreservation of SSCs, ovum, and testis and ovary tissues. Therefore many of the preservation techniques and methodologies

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Fig. 1. Basic methodologies used in slow freezing, vitrification and freeze drying for germplasm preservation. In this figure, CPAs represents cryoprotective agents or cryoprotectants and LN2 represents liquid nitrogen.

had been developed based on the cellular states of germ cells and the animal species.

The current review aims to light on the major cryopreservation methodologies along with efficient cryoprotectant commonly used for the preservation of germline cells and tissue of both male and female, which is the other means of sequential researches on fertility preservation and reproductive medications.

CRYOPRESERVATION METHODS

Increased understanding about the cryo-injury cases has continually helped to improve cryopreservation techniques and thus, several methods have been developed for various types of cells, tissues and organs. The two most commonly used cryopreservation methods for preserving animal germplasm are slow-freezing and vitrification in which they relate to the same physico- chemical relationships. Therefore the choice of selecting cryopreservation methods depends on the type and the time period of biological substances to be preserved.

The generalized procedures for cryopreservation of germplasm are described in Fig. 1.

Slow Freezing

Slow freezing, also known as slow programmable freezing (SPF), (Vutyavanich et al., 2010) is a set of well established techniques developed during the early 1970s which enabled the first birth from frozen human embryo during 1984. Since then, sample freezing using programmable sequences have been practiced world- wide for freezing oocytes, skin, blood products, embryo, sperm, stem cells and general tissue, and with little modifications in slow-freezing methods where required. Generally in this method, cells or tissues in a medium are cooled to below freezing point where ice masses formed with pure crystalline water. The parts between the growing ice masses are known as unfrozen fraction in where all cells or tissues and all sol- utes are confined (Rapatz et al., 1960). A comparatively higher and sustainable concentration of sugar, salt and cryoprotectants are used due to increase in osmotic strength that cause sufficient efflux of water from the cells and minimize the chance of intercellular ice for-

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mation (Deller et al., 2014). Thus, as cooling continues slowly, the viscousity of the unfrozen fraction increases too high for any further crystallization and turns into amorphous solid having no ice crystals. Although the cooling rate differs between cells of differing size, tissue types and water permeability, about 1℃/minute rate is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulphoxide (DMSO). The 1℃ / minute rate can be achieved by using devices such as a rate-controlled freezer or a benchtop portable freezing container.

Vitrification

The term “vitrification” also refers as “glass formation” is the transformation from a liquid to a solid in the absence of crystallization. In mid-1980s, researchers Greg Fahy and William F. Rall practically introduced this method to the reproductive cryopreservation techniques and in the next decades researchers working on cryobiology claimed that vitrification provides benefits to cryopreservation methods by without damaging substances due to ice crystal formation. As the result of successful cryopreservation by vitrification, Lilia Kule- shova first reported to achieve vitrification of woman’s eggs (oocytes), which finally resulted in live birth (Kuleshova et al., 1999), and also vitrification of tissue engineered constructs (Kuleshova et al., 2004). Gener- ally, vitrification method begins with using a medium having a very high solute concentration so that ice can- not form in any part of the sample. As there is no ice forms, cooling does not have to be slow. However to reduce the damage by quick freezing, cells or tissues are rapidly cooled from a temperature at which chill- ing injury and cold shock play no role (e.g. room temperature). The samples to be preserved are brought in- to a medium that has a very high concentration of cryoprotectants, and solutions will solidify to a glass without any risk of intracellular or extracellular ice formation during cooling if this concentration is high en- ough. A series of different cryoprotectant concentration is used for reducing damage due to abrupt osmotic changes, extremely low water potential or chemical toxicity of high cryoprotectant concentrations. Rall WF worked on vitrification of mouse embryos where he first equilibrated with 25 percent vitrification solution at room temperature, increasing it to 50 percent at 4℃

and finally to 100 percent vitrification solution (Rall WF, 1987). They are then rapidly packed and transferred into liquid nitrogen. The stepwise increase of cryoprotective agent concentration reduces osmotic effects, while the low temperature and rapid transfer help to prevent damage by chemical toxicity. In addition to this, it is possible to reduce chemical toxicity by

using mixtures of various permeant CPAs, or addition of non-permeant CPAs (60 g/litre polyethylene glycol) (Rall, 1987) or 60 g/litre bovine serum albumin (BSA) (van Wagtendonk-de Leeuw et al., 1997).

Freeze Drying

Freeze drying with directional method is usually the alternative technique to slow freezing where the sample is advanced at a constant velocity through a linear temperature gradient due to the directional solidifica- tion (Arav and Natan, 2017). This temperature gradient facilitated very homogenous and accurate cooling of the samples. The samples are propagated constantly also results in continuous seeding and thus it could control inter-cellular (biological substances) ice crystal growth in a direction opposite to the direction in which the sample is moved down the temperature gradient.

Highly heat-conductive metal block is used surround- ing the sample which efficiently removes heat away as it freezes. Due to the directional movement of the samples heat also continuously moves away from the ice front into the unfrozen fraction of the sample but controlled heat dissipation protects the ice front from melting. In this way the morphology of cell-friendly ice crystal contributes to reducing physical damages of frozen samples (Arav and Natan, 2009; Arav et al., 2010).

Generally, the strategy for freeze drying is to bring the substances to a vitrified glass state at the primary step. Then the water portion and melted ice is re- moved by applying a vacuum environment to the ma- terials. The glass transition temperature increases in this stage and ultimately reaches the level higher than ambient temperature. Thus, at the end of the procedure, the dried substances can be stored at ambient temperature.

Storage of freeze-dried biological material is extremely cost efficient as it could be kept at ambient temperature and no expensive cryogenic storage equip- ments or liquid nitrogen are required. The negative side of this procedure is that it generally reduces cell viability. So, this could not be used for standard in- semination procedures or preserving sensitive tissues.

However, freeze-dried sperm have been successfully used by intracytoplasmic sperm injection (ICSI) method to produce live offspring in mice and rabbits (Liu et al., 2004). In addition, Loi et al. successfully produce apparently healthy embryos using somatic cell nuclear transfer (SCNT) by using freeze-dried somatic cells (Loi et al., 2008(Jan), 2008(Aug)). Thus, freeze drying is use- ful for of genetic resource banking with the objective of regenerating live animals and recovering lost breeds.

Therefore, freeze-dried gametes and somatic cells can

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already be used for conservation of germplasm in- tended for genetic research and could be milestone for future medication.

METHODS APPLICABLE FOR THE CRYOPRESERVATION OF GAMETES,

GERMLINE CELLS AND TISSUES

Cryopreservation of Spermatozoa

The male gamete or sperm cryopreservation (sperm banking), also known as semen cryopreservation, is a vital field in reproductive biology and infertility research area for last few decades. For centuries, scientist has been fascinated with the functions of spermatozoa at low temperatures. With the discovery and development of cryoprotective agents, successful cryopreservation of spermatozoa from different species has been established regarding cost effectiveness and maintenance of animal lines, and has insight into reproduction technologies to become partial realities in the human in- fertility treatment (Benson et al., 2012). Importantly, spe- rm or semen cryopreservation is linked to the challenges of retaining the functional properties of spermatozoa without damaging their aspherical shape, complex cytoskeleton, and the molecular mechanisms of activation and capacitation. Therefore, before the discovery of cryoprotectants, a successful semen freezing protocol was proposed as suppressing the extracellular ice formation and was aided by adding chemical agents or by cooling rapidly which was actually similar to vitrification of sperm cells. Luyet (1938) and others men- tioned that physical damages of sperm during cryopreservation was occurred due to intracellular ice formation but Lovelock made the realization about per- meating cryoprotective agents allow the reduction of water level during the cryo-process (Lovelock, 1953 (Mar) and 1953(May)). In the next decades, several experiments have been performed for seeking of successful semen preservation methods. As a result, the first attempt of sperm cryopreservation was followed as freeze-dry (FD) method in 1949 by Polge et al. who mixed fowl semen with Ringer’s solution containing glycerol and FD (removing 90% of water) but after storing at room temperature for 2 hours, he found 50%

of spermatozoa regained motility (Polge et al., 1949).

Then freeze-drying attempt was taken for human [Sherman, 1954] and bull (Sacke et al., 1961) sperm preservation but those experiments was not successful for regaining viable state of sperms. This scenario has been change with next several years and the first re-

ports of successful cryopreservation of mouse spermatozoa were published in 1990 by three independent groups of Japanese investigators (Tada et al., 1990). In Tada’s method, spermatozoa were rapidly frozen in two steps (37 to ―70℃ for solid CO2 and ―70 to ―196

℃ for liquid nitrogen) as pellets and 18% raffinose was used to protect from cold-shock whereas Yokoyama et al. in the same year reported of using Dulbecco's PBS containing raffinose in combination with glycerol, DM- SO or skim milk as freezing protective agents. Nakaga- ta and Takeshima subsequently improved the methods (Nakagata and Takeshima, 1992) and demonstrate for the first time that cryopreserved spermatozoa can fertil- ize cryopreserved oocytes in vitro and developed nor- mal live offspring after embryo transfer (Nakagata, 1993). Later on, many researcher were interested on the rate of survivability and functions of cryo-freezed sperm of mouse, rat and comparatively larger animals (monkey, bull, porcine, even human), by analyzing re- lation of water permeability and spermatozoa plasma membrane (Noiles et al., 1997), and by reducing os- motic injury by using appropriate CPA (Gao et al., 1995; Agca et al., 2005; Si et al., 2006). Therefore, the detail effect of sperm cytoskeleton, plasma membrane potential and proteome of cryopreserved sperms have also been studied well and cryoprotectant like glycerol, ethylene glycol, DMSO, propylene glycol has been wi- dely used for preserving mammalian sperm with some modifications where required.

Cryopreservation of Oocyte

Oocyte or female egg cryopreservation is procedure for preserving a female egg, stored at ultra-low temperature, fertilized, and transferred to the uterus as embryos to facilitate a pregnancy. Oocyte cryopreservation can increase the chance of future pregnancy of those women that (1) are patients of cancer but have not yet start chemotherapy, (2) undergoing treatment with assisted reproductive technologies and (3) would like to take children in future.

The first successful freezing of a mammalian oocyte was in the mouse, by Whittingham DG at 1977. In this study researcher reported live offspring obtained after IVF of mouse frozen oocytes where DMSO was used as a storage medium and stored under liquid nitrogen at ―196℃. Despite this success, scientist discovered several problems with oocyte freezing. The sperm attach- ing point in the egg, zona pellucid became harden due to freeze-thaw cycle and resulted decreased rates of fertilization in frozen thawed mouse oocytes (Johnson, 1989; Carroll et al., 1990). Oocyte also has temperature- sensitive meiotic spindle inside it which seemed to be found as a target of freezing damage (Pickering and

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Johnson, 1987; Van der Elst et al., 1988; Aigner et al., 1992). Thus, for these genetic anomalies, production of mouse embryos from frozen-thawed oocytes was a major problem. For a while, ultrarapid freezing and vitrification were found as solutions for this (Surrey and Quinn, 1990) but soon fetal defective development or problems in genetic nature also appeared here (Van der Elst et al., 1998). Therefore the protocol and type of using cryoprotectant has been changed by the period of time.

Oocytes can be cryopreserved successfully using slow freezing, vitrification or ultrarapid freezing methods. Some scientists have done extensive research on the methodologies of oocyte cryopreservation. Vitrifica- tion is found as more favorable method than slow cooling for several animal species (Kuleshova and Lopata, 2002; Paffoni et al., 2011; Levi et al., 2014). It is need- less to say that the protocols for vitrification have been developed with the time course and the invention of suitable cryopretectants. DMSO, ethylene glycol (EG), 1,2-propanediol (PROH) are the main permeable cryoprotectant used as single or combination (i.e. EG + DMSO or EG + PROH) according to the species. For mouse oocyte cryopreservation using slow freezing, PROH is most commonly used (Gook et al., 1993); al- though potential genotoxicity has been found in using PROH (Aye et al., 2010). When vitrification method is used, EG becomes efficient universal cryoprotectant for preserving mouse (Hotamisligil et al., 1996) and human spermatozoa [Kuwayama et al., 2005; Yoon et al., 2003].

In the same method, bovine oocyte has been reported to be preserved with mixture of EG and DMSO (Chian et al., 2004; Vajta et al., 1998). Later on, inspired from the bovine model some researchers also got success on preserving human oocyte with cryoprotectants mixtures (i.e. EG + DMSO or EG + PROH) in vitrification me- thod (Chang et al., 2008; Chian et al., 2009; Lucena et al., 2006; Cobo et al., 2008; Kuwayama et al., 2005). In the subsequent studies on vitrification, some other chemicals have been also used as a cryoprectant or addi- tives. Effective production of live mouse pup has been reported by Watanabe et al. who used carboxylated Ɛ-poly-L-lysine as a cryoprotectant (Watanabe et al., 2013). Similarly, Lee et al. reported three antifreeze pro- teins such as Flavobacterium frigoris ice-binding protein (FfIBP, from bacteria), Glaciozyma sp. ice-binding protein (LeIBP, from yeast) and Type III AFP (from fish) could improve murine oocyte quality during cryo and reduce reactive oxygen production during freeze-thaw (Lee et al., 2015) and thus got improved embryo de- velopment.

Cryopreservation of Spermatogonia

The various cryopreservation methods had been optimized to increase survival rates of spermatogonia and uninterrupted functional properties of spermatogonial stem cells (SSCs) after thawing. The preservation methods were similar to that of somatic cells and no study was found related to efficiency evaluation of cryopreservation of SSCs with highest survival rate. The recovery rate and stemness properties of cryopreserved mouse SSCs have been confirmed by germ cell trans- plantation into sterilized mice (Avarbock et al., 1996).

Some other studies have reported that SSCs from other mammal species (livestock and human) possess a capacity to survive after long-term cryopreservation (Ka- natsu et al., 2003; Schlatt et al., 2002; Wu et al., 2012).

Izadyar et al. (2002) reported a detailed slow freezing method where spermatogonia A cell suspensions of calf were cryopreserved in medium with glycerol or DMSO (Izadyar et al., 2002). Then, using the same cryopreser- vation method, the comparison between cryoprotectant DMSO and EG was reported (Frederickx et al., 2004) and colony formation ability of frozen thawed mouse spermatogonia was also studied (Koruji et al., 2007).

Hermann et al. (2007) described cryopreservation of rhesus macaque SSCs with DMSO where SSCs retained their engraftment potential after cryopreservation (Her- mann et al., 2007). Lee et al. demonstrated that slow- freezing method of cryopreservation of murine SSCs with media containing DMSO and 200 mM trehalose serves as more effective than DMSO alone (Lee et al., 2013), confirmed by germ cell transplantation. This research group also reported that 2.5% PEG (molecular weight 1000) can also be an effective sole agent for ef- ficient SSC cryopreservation (Lee et al., 2013). More- over, Kim et al. also reported slow freezing cryopreser- vation of putative pre-pubertal bovine SSCs and in- dicated that the presence of 200 mM trehalose in media can be an efficient cryopreservation method for bo- vine SSCs (Kim et al., 2015).

Cryopreservation of Testis Tissue

Preservation of testis tissue mainly conducted due to the fertility preservation of prepubertal cancer patients before chemotherapy because sperm banking is impos- sible here (Keros et al., 2007; Kvist et al., 2006). There- fore, with ongoing researches on cryopreservation methods of testis tissue, it becomes important of knowing about cell type, age and state of sample to be pre- served (Milazzo et al., 2008).

For cryopreserving testicular biopsies, slow-freezing is one of most popular method (Goossens et al., 2008;

Jahnukainen et al., 2007; Milazzo et al., 2008) and fer- tility with live birth have been achieved after micro- injection with spermatozoa (Ohta and Wakayama, 2005;

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Shinohara et al., 2002). However, in the early studies, researchers faced several problems in cryopreserved tissues like inactive spermatocytes, loss of seminiferous tubules etc (Jahnukainen et al., 2007), and thus the pre- servation protocol was needed to be optimized (Jahnu- kainen et al., 2007; Ohta and Wakayama, 2005). Testicu- lar tissue cryopreservation might be a better option than spermatogonial freezing, because frozen tissue preserves the cell–cell contacts and requires more perme- able cryoprotectants. Keros et al. reported that more Leydig cells survived when DMSO was used in slow freezing method (80% compared with 50% with pro- panediol) (Keros et al., 2005). He also successfully cryo- preserved testis fragments from prepubertal boys with higher tissue integrity (Keros et al., 2007). Besides slow freezing, vitrification was also tried for male gonads cryopreservation and comparison of these two protocols was conducted (Abrishami et al., 2010; Zeng et al., 2009) but germ cell stemness function was not tested after tissue grafting. Curaba et al. designed a vitri- fication method for prepubertal mouse testicular tissue and compared it with a slow-freezing (Curaba et al., 2011) which resulted similar efficiency. Gouk et al. con- ducted several aspects of the vitrification protocols (Gouk et al., 2011) and Unni et al. tried to optimize three cryoprotectants (GLY, DMSO, and EG) in adult and prepubertal testis tissue (Unni et al., 2012). Con- sidering their findings, DMSO can be used as effective CP for immature and ethylene glycol for adult tes- ticular tissue (Unni et al., 2012).

Cryopreservation of Ovarian Tissue

Ovarian tissue (OT) cryopreservation has been ap- plied in clinical practice for ovarian function recovery in human (Aubard et al., 2001), domestic animals (Wa- llin et al., 2009; Arav et al., 2010) and endangered spe- cies (Shaw et al., 2000). Successful OT cryopreserva- tions, which resulted live birth have been reported in different species like mice (Takahashi et al., 2001), rab- bit (Almodin et al., 2004), birds (Liu et al., 2010), sheep (Salle et al., 2003) and human (Donnez et al., 2012;

Revelli et al., 2013). Therefore methods for preserving OT have been developed to keep the tissue adequately functional after thawing. Among the recent protocols, Bagis et al. evaluated antifreeze proteins for better pres- ervation of transgenic mouse overies by vitrification where DMSO, EG and media supplemented with su- crose were used (Bagis et al., 2008). Another research group compared between vitrification and controlled rate freezing of human ovarian tissue and proposed better preservation by vitrification with combination of PROH, EG, DMSO and polyvinylpyrrolidone (PVP) (Ke- ros et al., 2009). Sheikhi et al also reported their vitri-

fication protocol with PROH, EG, DMSO and PVP could keep the ultrastructure of ovarian follicle and stroma similar like non-vitrified control (Sheikhi et al., 2011). Study also showed the similar morphological integrity and follicular proliferation of cryopreserved ovarian tissue by slow freezing and vitrification methods (Klocke et al., 2014). However, vitrification protocols with different combinations of cryoprotectants have been generally accepted by most of the researchers work with OT cryopreservation (Sanfilippo et al., 2015; Lee et al., 2015).

CONCLUSION

The cryopreservation protocols and the use of suitable cryoprotectant regarding their concentration, combination or permeability is a growing science in the reproductive research field and fertility restoration techniques. The importance of preservation has been increased with the time period. The involvements of effective cryopreservation techniques enlarge the production; enhance the research on life survivability in agricultural and veterinarian field. On the other hand, germline cell or tissue cryopreservation increases the chance of fertility restoration of cancer patients, iatro- genic infertile personnel and endanger species. There- fore, the successful development of protocols in germline cell cryopreservation will advance not only in human fertility related technologies, but will be of enor- mous benefit to shape the future of its applications in the biomedical field.

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(Received: May 12 2017/

Revised: May 26 2017/

Accepted: May 29 2017)