cryopreservation-cbe658-blog

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Introduction to Cryopreservation

Cryopreservation can be defined as the storage of living biological material at ultralow temperature which is from -80 to – 190 oC in order to inhibit to zero of the microbial activities. In another words, the cryopreservation is able to retain the biological and chemical reactions from happened in the living cells a phenomenon that can lead to the possible long-term preservation of cells and tissues (Jang et al., 2017). Cell injury at fast cooling rates is caused by intracellular ice formation in the living cells whereas slow cooling causes osmotic changes due to the effects of exposure to highly concentrated intra- and extracellular solutions or to mechanical interactions between cells and the extracellular ice (Jang et al., 2017).However, this technique is one of advanced method in preservation and the most importantly it is considered safe and economically viable for long term conservation of many plant (Pinto et al., 2016). The temperature of the ultralow could be provided by liquid nitrogen which at this state, the theory says it could dramatically disrupted, allowing material storage without any alteration for, in theory, an unlimited period. Other advantages of this technique are the small space needed for storage, the absence of possible contaminants and low maintenance requirements (Pinto et al., 2016).

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Following video shows brief technique on cryo preservation technique.

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Mechanism of Cryopreservation

During the state of ultralow temperature, it is undeniable freezing the cells or tissues will cause fatal or cell injury to most living organisms. Thus, the main thing need to make sure to avoid any lethal damage to living material is the dehydration and water crystallization during freezing (Pinto et al., 2016). This is because the low temperature will cause the living material froze inside out which possibly lethal to the materials. The dehydration process is the prior to cryopreservation which can be carried out by physical methods such as exposing the sample to a laminar flow cabinet. However, the dehydration is more uniform and efficient when using a closed system containing silica gel (Pinto et al., 2016).

           The important step after cryopreservation process is the thawing of the living materials which is important to avoid any lethal damage to the living materials because of formation of larger ice crystals of lethal dimensions during warming (Vanderzwalmen et al, 2013). In another words, the rate of thawing plays as important rules as the nature of the medium in which explants are warmed and then rehydrated is critical to the recovery of the explant after cryopreservation (Pinto et al., 2016).

           To be more specified, the process of cryopreservation can be divided into three major process which is the mixing of CPAs with cells or tissues before cooling, cooling of the cells or tissues to a low temperature and its storage, warming of the cells or tissues and removal of CPAs from the cells or tissues after thawing (Jang et al., 2017). For the first step of cryopreservation is introduced of CPA which is usually a fluid, reduces the freezing injury from the cryopreservation process. The CPA must capable to penetrate the living cells or tissues without high level of toxicity (Jang et al., 2017) used to reduce the amount of ice formed at any given temperature, depending on the cell type, cooling rate, and warming rate. CPA liquid can be divided into two types which is cell membrane permeating such as dimethyl sulfoxide and glycerol. Another type is non-membrane permeating such as 2-methyl-2,4-pentanediol and polymers such as polyvinyl pyrrolidone, hydroxyethyl starch, and various sugars (Jang et al., 2017).

           During the freezing process, the process also known as vitrification process which manipulated by freezing rate and concentration of CPA liquid. According to (Jang et al., 2017) if cooling is sufficiently slow, cells could efflux intracellular water rapidly enough to eliminate super-cooling and thus prevent intracellular ice formation (Vanderzwalmen et al, 2013). There is also slow freezing process instead of rapid freezing process which use typical cooling rate at 1oC/min at 1m of CPA concentration. However, slow freezing has a high risk of freeze injury due to the formation of extracellular ice (Jang et al., 2017). On the other side, the advantages of slow freezing are that it has a low risk of contamination during the procedures and does not demand high manipulation skills (Jang et al., 2017).

           Eventhough the slow freezing has its disadvantages, it could be fixed by immediate transformation directly from the aqueous phase state after direct exposure to liquid nitrogen. The process requires cooling of the cells or tissues to deep cryogenic temperatures with liquid nitrogen) after their exposure to high concentrations of CPA in the ratio of 40–60%, weight/volume, with subsequent rapid cooling to avoid ice nucleation (Jang et al., 2017). This alteration of slow freezing process is called vitrification which depended on three major factors which is viscosity of the sample, second is cooling and warming rates and third is sample volume. Thus, a delicate balance must be maintained among all the relevant factors to ensure successful vitrification (Jang et al., 2017).

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Following video discuss on What is Semen/Sperm Cryopreservation:

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Applications and Previous Study of Cryopreservation in Fisheries Industry

In 1949, Polge et al. (1949) successfully cryopreserved the avian spermatozoa using glycerol as a cryoprotectant. Thereafter, cryopreservation of male gamete became possible. Blaxter (1953) applied a similar approach for fish gametes and reported success with Atlantic herring spermatozoa, achieving approximately 80% cellular motility after thawing. Since then, cryopreservation of fish sperm has been studied and has been successful in more than 200 species (Kopeika et al. 2007; Tiersch et al. 2007; Tsai et al., 2010) and techniques of sperm management have been established for freshwater and marine fish species, including carp, salmonids, catfish, cichlids, medakas, white-fish, pike, milkfish, grouper, cod, and zebrafish (Scott and Baynes 1980; Harvey and Ashwood-Smith 1982; Stoss and Donaldson 1983; Babiak et al. 1995; Suquet et al. 2000). Many studies on cryopreservation of fish sperm have been carried out on economically important freshwater species and attempts to cryopreserve sperm from the marine fish species tended to be more successful when compared with those obtained from the freshwater fish (Tsvetkova et al. 1996). Although freshwater fish sperm are generally more difficult to cryopreserve, the fertilization rates obtained from the cryopreserved marine fish sperm are similar to those obtained with mammalian species (Tsvetkova et al. 1996). Controlled-rate slow cooling in cryopreservation has been mainly used for fish sperm. Common carp has been studied using frozen-thawed sperm with 95% fertilization and hatching rate.

Salmonid species spermatozoa have been successfully cryopreserved (Lahnsteiner 2000). Another well studied cryopreserved group is cyprinids and some of these cyprinid fishes are widely farmed throughout Asia and Europe. A fertilization and hatching rate of 95% using the frozen-thawed sperm has been reported for the common carp and these results are not significantly different from fresh sperm (Magyary et al. 1996). Tilapias are among the exotic freshwater fishes that have been successfully established for fish farming in Taiwan; they have been cryopreserved successfully and produced 40-80% motility with cryoprotectant DMSO (Chao et al. 1987). The sperm of more than 30 marine fish species have been cryopreserved successfully (Suquet et al. 2000). Generally, high survival and fertilization capacity has been obtained in frozen-thawed spermatozoa when compared to freshwater species (Drokin 1993; Gwo 2000).

Successful cryopreservation of the sperm of aquatic invertebrate has been carried out for sea urchin, oyster, starfish, abalone and coral (Adams et al. 2004). Dimethyl sulfoxide has also been reported as a successful cryoprotectant for sperm cryopreservation; the concentration range used was 5 to 30% for these species. Various levels of motility, ranging from <5% to 95%, have been reported for the cryopreserved aquatic invertebrate sperm (Dunn and McLachlan 1973).

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Example of application in cryopreservation: Salmon Sperm

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References

Adams SL, Hessian PA, Mladenov PV. Cryopreservation of sea urchin (Evechinus chloroticus) sperm. CryoLetters, 2004; 25(4): 287-289.

Babiak I, Glogowsky, Brzuska JE, Szumiec J, Adamek J, Cryopreservation of sperm of common carp Cyprinus carpio. Aquaculture Res. 1995; 28: 567-571. 

Blaxter JHS. Sperm storage and cross-fertilization of spring and autumn spawning herring. Nature. 1953; 172: 1189-1190.

Chao NH, Chao WC, Liu KC, Liao IC. The properties of tilapia sperm and its cryopreservation. J. Fish. Biol. 1987; 30: 107-118. 

Drokin SI. Phospholipid distribution and fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in sperm of some freshwater and marine species of fish. Aquat. Living Resour. 1993; 6: 49-56.

Dunn RS, McLachlan J. Cryopreservation of echinoderm sperm. Can. J. Zool. 1973; 51: 666-669.

Harvey B, Ashwood-Smith MJ. Cryopretectant penetration and supercooling in the eggs of samonid fishes.Cryobiology. 1982; 19: 29-40. 

Janga, T. H., Park, S. C., Yanga, J. H., Kima, J. Y., Seoka, J. H., & Parka, U. S. (2017). Cryopreservation and its clinical applications. Integrative Medicine Research, 12-18.

Kopeika E, Kopeika J, Zhang T. Cryopreservation of fish sperm. Methods Mol. Biol. 2007; 368: 203-17. 

Lahnsteiner F. Semen cryopreservation in the Salmonidae and in the northern pike; in special issue: cryopreservation of gametes in aquatic species. Aquacult. Res. 2000; 31: 245-258.  

Magyary I, Urbányi B, Horváth L. Cryopreservation of common carp (Cyprinus carpio L.) sperm II Optimal conditions for fertilization. J. Appl. Ichthyol. 1996; 12: 117-119.  

Pint, M. d., Paiva, R., Silva, D. P., Santos, P. A., & Freitas, R. T. (2014). Cryopreservation of coffee zygotic embryos dehydration and osmotic rehydration. Ciência e Agrotecnologia, 380-389.

Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949; 164: 666.

Scott AP, Baynes SM. A review of the biology, handing and storage of salmonid spermatozoa. J. Fish Biol. 1980; 17: 707-739.

Stoss J, Donaldson EM. Studies on cryopreservation of eggs from rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus Kisutch). Aquaculture. 1983; 31: 51-65. 

Suquet M, Dreanno C, Fauvel C, Cosson J, Billard R. Cryopreservation of sperm in marine fish. Aquaculture Res.2000; 31(3): 231-243. 

Tiersch TR, Yang H, Jenkins JA, Dong Q. Sperm cryopreservation in fish and shellfish. Soc. Reprod. Fertil. Suppl.2007; 65: 493-508.

Tsai S, Lin C. Effects of cryoprotectant on the embryos of banded coral shrimp (Stenopus hispidus), preliminary studies to establish freezing protocols. CryoLetters. 2009; 30(5): 373-381.

Tsvetkova LI, Cosson J, Linhart O, Billard R. Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser baeri and A. ruthenus. J. Appl. Ichthyol. 1996; 12: 107-112.

Vanderzwalmen, P., & Papatheodorou, P. (2013). Cryopreservation of Human Unfertilized and Fertilized Oocytes. Journal of Reproductive Medicine and Endocrinology, 45-54.