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Journal articles on the topic 'Tissues Cryopreservation'

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Author: Grafiati

Published: 10 December 2022

Last updated: 28 January 2023

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1

Crowley, Conor A., William P. W. Smith, K. T. Matthew Seah, Soo-Keat Lim, and Wasim S. Khan. "Cryopreservation of Human Adipose Tissues and Adipose-Derived Stem Cells with DMSO and/or Trehalose: A Systematic Review." Cells 10, no. 7 (July 20, 2021): 1837. http://dx.doi.org/10.3390/cells10071837.

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Adipose tissue senescence is implicated as a major player in obesity- and ageing-related disorders. There is a growing body of research studying relevant mechanisms in age-related diseases, as well as the use of adipose-derived stem cells in regenerative medicine. The cell banking of tissue by utilising cryopreservation would allow for much greater flexibility of use. Dimethyl sulfoxide (DMSO) is the most commonly used cryopreservative agent but is toxic to cells. Trehalose is a sugar synthesised by lower organisms to withstand extreme cold and drought that has been trialled as a cryopreservative agent. To examine the efficacy of trehalose in the cryopreservation of human adipose tissue, we conducted a systematic review of studies that used trehalose for the cryopreservation of human adipose tissues and adipose-derived stem cells. Thirteen articles, including fourteen studies, were included in the final review. All seven studies that examined DMSO and trehalose showed that they could be combined effectively to cryopreserve adipocytes. Although studies that compared nonpermeable trehalose with DMSO found trehalose to be inferior, studies that devised methods to deliver nonpermeable trehalose into the cell found it comparable to DMSO. Trehalose is only comparable to DMSO when methods are devised to introduce it into the cell. There is some evidence to support using trehalose instead of using no cryopreservative agent.

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2

Arav, Amir. "Cryopreservation by Directional Freezing and Vitrification Focusing on Large Tissues and Organs." Cells 11, no. 7 (March 22, 2022): 1072. http://dx.doi.org/10.3390/cells11071072.

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The cryopreservation of cells has been in routine use for decades. However, despite the extensive research in the field, cryopreservation of large tissues and organs is still experimental. The present review highlights the major studies of directional freezing and vitrification of large tissues and whole organs and describes the different parameters that impact the success rate of large tissue and organ cryopreservation. Key factors, such as mass and heat transfer, cryoprotectant toxicity, nucleation, crystal growth, and chilling injury, which all have a significant influence on whole-organ cryopreservation outcomes, are reviewed. In addition, an overview of the principles of directional freezing and vitrification is given and the manners in which cryopreservation impacts large tissues and organs are described in detail.

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3

Müller-Schweinitzer, Else. "Cryopreservation of vascular tissues." Organogenesis 5, no. 3 (July 2009): 97–104. http://dx.doi.org/10.4161/org.5.3.9495.

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4

Xu, Feng, Sangjun Moon, Xiaohui Zhang, Lei Shao, Young Seok Song, and Utkan Demirci. "Multi-scale heat and mass transfer modelling of cell and tissue cryopreservation." Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 368, no. 1912 (February 13, 2010): 561–83. http://dx.doi.org/10.1098/rsta.2009.0248.

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Cells and tissues undergo complex physical processes during cryopreservation. Understanding the underlying physical phenomena is critical to improve current cryopreservation methods and to develop new techniques. Here, we describe multi-scale approaches for modelling cell and tissue cryopreservation including heat transfer at macroscale level, crystallization, cell volume change and mass transport across cell membranes at microscale level. These multi-scale approaches allow us to study cell and tissue cryopreservation.

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5

Lee, Sanghoon, Ki-Jin Ryu, Boram Kim, Dahyeon Kang, Yoon Young Kim, and Tak Kim. "Comparison between Slow Freezing and Vitrification for Human Ovarian Tissue Cryopreservation and Xenotransplantation." International Journal of Molecular Sciences 20, no. 13 (July 8, 2019): 3346. http://dx.doi.org/10.3390/ijms20133346.

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Two methods for the cryopreservation of human ovarian tissue were compared using a xenotransplantation model to establish a safe and effective cryopreservation method. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital. The tissues were transplanted into 112 ovariectomized female severe combined immunodeficient mice 4 weeks after slow freezing or vitrification cryopreservation. Tissues were retrieved 4 weeks later. Primordial follicular counts decreased after cryopreservation and xenotransplantation, and were significantly higher in the slow freezing group than in the vitrification group (p < 0.001). Immunohistochemistry and TUNEL assay showed that the Ki-67 and CD31 markers of follicular proliferation and angiogenesis were higher in the slow freezing group (p < 0.001 and p = 0.006, respectively) and DNA damage was greater in the vitrification group (p < 0.001). Western blotting showed that vitrification increased cellular apoptosis. Anti-Müllerian hormone expression was low in transplanted samples subjected to both cryopreservation techniques. Electron microscopy revealed primordial follicle deformation in the vitrification group. Slow freezing for ovarian tissue cryopreservation is superior to vitrification in terms of follicle survival and growth after xenotransplantation. These results will be useful for fertility preservation in female cancer patients.

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6

Bakhach, Joseph. "The cryopreservation of composite tissues." Organogenesis 5, no. 3 (July 2009): 119–26. http://dx.doi.org/10.4161/org.5.3.9583.

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7

Cui, X. D., D. Y. Gao, B. F. Fink, H. C. Vasconez, and L. L. Q. Pu. "Cryopreservation of human adipose tissues." Cryobiology 55, no. 3 (December 2007): 269–78. http://dx.doi.org/10.1016/j.cryobiol.2007.08.012.

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8

Hughes, Sean M., April L. Ferre, Sarah E. Yandura, Cory Shetler, Chris A. R. Baker, Fernanda Calienes, Claire N. Levy, et al. "Cryopreservation of human mucosal tissues." PLOS ONE 13, no. 7 (July 30, 2018): e0200653. http://dx.doi.org/10.1371/journal.pone.0200653.

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9

Islam, Nadia, Ugwoke Sunday Paul, Rana Alhamdan, Juan Hernandez-Medrano, Bruce K. Campbell, Peter Marsters, and Walid E. Maalouf. "Steroids and miRNAs in assessment of ovarian tissue damage following cryopreservation." Journal of Molecular Endocrinology 62, no. 4 (May 2019): 207–16. http://dx.doi.org/10.1530/jme-18-0237.

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Ovarian cortical tissue cryopreservation is a relatively novel approach to preserving fertility in women diagnosed with cancer. However, the effects of freezing-thawing are not fully understood, mainly due to the lack of suitable methods to assess tissue’s survival after thawing. Disparities in steroid production have been associated with ovarian failure by disrupting folliculogenesis, ovulation and oocyte apoptosis. Moreover, specific miRNAs, identified in human ovarian follicles, are thought to play a fundamental role in folliculogenesis. In this study, we investigated the possible interplay between the ovarian steroidal production and miRNA expression patterns in spent culture media, as potential non-invasive markers for ovarian tissue damage after cryopreservation. Cryopreservation of ovarian cortical tissue decreased (P < 0.05) both steroid production (oestradiol and progesterone) and expression of miRNA-193b and 320A in spent culture media over 5 days; however, expression of miRNA-24 increased (P < 0.05). The number of primordial follicles was also reduced (P < 0.05) in fresh-cultured and cryopreserved-cultured cortical tissues when compared with fresh tissues. Downregulation of miRNA-193b and miRNA-320A together with upregulation of miRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production. Thus, there appears to be an interplay between these miRNAs, ovarian steroid production and cell damage, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.

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10

Matsuura, Yoshitaka, Michiharu Sakamoto, Shuichi Ogino, Jun Arata, and Naoki Morimoto. "Inactivated Nevus Tissue with High Hydrostatic Pressure Treatment Used as a Dermal Substitute after a 28-Day Cryopreservation Period." BioMed Research International 2021 (February 24, 2021): 1–9. http://dx.doi.org/10.1155/2021/3485189.

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Background. Giant congenital melanocytic nevi (GCMN) treatment remains controversial. While surgical resection is the best option for complete removal, skin shortage to reconstruct the skin defect remains an issue. We report a novel treatment using a high hydrostatic pressurization (HHP) technique and a cryopreservation procedure. However, cryopreservation may inhibit revascularization of implanted nevus tissue and cultured epidermal autograft (CEA) take. We aimed to investigate the influence of the cryopreservation procedure on the HHP-treated dermis specimen and CEA take on cryopreserved tissue. Methods. Nevus tissue harvested from a patient with GCMN was inactivated with HHP of 200 MPa and then cryopreserved at -30°C for 28 days. The cryopreserved specimen was compared with fresh (HHP-treated without cryopreservation) tissue and with untreated (without HHP treatment) tissue to evaluate the extracellular matrix, basal membranes, and capillaries. Cultured epidermis (CE) take on the cryopreserved tissue was evaluated following implantation of the cryopreserved nevus tissue with CE into the subcutis of nude mice. Results. No difference was observed between cryopreserved and fresh tissue in terms of collagen or elastic fibers, dermal capillaries, or basement membranes at the epidermal-dermal junction. In 4 of 6 samples (67%), applied CE took on the nevus tissues and regenerated the epidermis in the cryopreserved group compared with 5 of 6 samples (83%) in the fresh group. Conclusion. Cryopreservation at -30°C for 28 days did not result in significant damage to inactivated nevus tissue, and applied CE on the cryopreserved nevus tissues took and regenerated the epidermis. Inactivated nevus tissue with HHP can be used as a dermal substitute after 28-day cryopreservation.

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von Schönfeldt, V., R. Chandolia, L. Kiesel, E. Nieschlag, S. Schlatt, and B. Sonntag. "Assessment of follicular development in cryopreserved primate ovarian tissue by xenografting: prepubertal tissues are less sensitive to the choice of cryoprotectant." REPRODUCTION 141, no. 4 (April 2011): 481–90. http://dx.doi.org/10.1530/rep-10-0454.

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Improvements in cancer survival rates have renewed interest in the cryopreservation of ovarian tissue for fertility preservation. We used the marmoset as a non-human primate model to assess the effect of different cryoprotectives on follicular viability of prepubertal compared to adult ovarian tissue following xenografting. Cryopreservation was performed with dimethylsulfoxide (DMSO), 1,2-propanediol (PrOH), or ethylene glycol (EG) using a slow freezing protocol. Subsequently, nude mice received eight grafts per animal from the DMSO and the PrOH groups for a 4-week grafting period. Fresh, cryopreserved–thawed, and xenografted tissues were serially sectioned and evaluated for the number and morphology of follicles. In adult tissue, the percentage of morphologically normal primordial follicles significantly decreased from 41.2±4.5% (fresh) to 13.6±1.8 (DMSO), 9.5±1.7 (PrOH), or 6.8±1.0 (EG) following cryopreservation. After xenografting, the percentage of morphologically normal primordial (26.2±2.5%) and primary follicles (28.1±5.4%) in the DMSO group was significantly higher than that in the PrOH group (12.2±3 and 5.4±2.1% respectively). Proliferating cell nuclear antigen (PCNA) staining suggests the resumption of proliferative activity in all cellular compartments. In prepubertal tissues, primordial but not primary follicles display a similar sensitivity to cryopreservation, and no significant differences between DMSO and PrOH following xenografting were observed. In conclusion, DMSO shows a superior protective effect on follicular morphology compared with PrOH and EG in cryopreserved tissues. Xenografting has confirmed better efficacy of DMSO versus PrOH in adult but not in prepubertal tissues, probably owing to a greater capacity of younger animals to compensate for cryoinjury.

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12

Valli-Pulaski, H., M. Sukhwani, KA Peters, and KE Orwig. "Optimizing Cryopreservation of Human Testicular Tissues." Reproductive BioMedicine Online 37 (November 2018): e4. http://dx.doi.org/10.1016/j.rbmo.2018.06.008.

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13

Lightfoot, Fred, Michael Taylor, Kelvin G. M. Brockbank, and Cindy Hastings. "The Use of Ultrarapid Freezing and Freeze Substitution to Verify Vitrification and/or Ice Formation in Vascular Tissue." Microscopy Today 8, no. 9 (November 2000): 16–21. http://dx.doi.org/10.1017/s1551929500059381.

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The expanding field of cryobiology, in particular that of the study of vitrification for long term storage of tissues for transplantation, has demonstrated that ice is damaging to smooth muscle tissue. Consequently conventional methods such as ultrarapid freezing and freeze substitution are becoming routine protocols to determine the quality of cryopreservation. This article introduces the scientific community to the PS-1000 cryofixation unit (Delaware Diamond Knives, Wilmington, DE) which provides both ultrarapid freezing and a means of validation of freeze substitution methods. When any tissue is frozen or vitrif ed for clinical use it is imperative to know the structural and functional integrity of these tissues. Ice formation within the extracellular matrix and dehydration of multi-cellular tissues, using conventional cryopreservation, is the principal reason why these methods frequently prove to be ineffective.

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Bit, Arindam, Awanish Kumar, Abhishek Kumar Singh, Albert A. Rizvanov, Andrey P. Kiassov, Pradeep Kumar Patra, Munish Kumar, and Akalabya Bissoyi. "Crosstalk between Substrates and Rho-Associated Kinase Inhibitors in Cryopreservation of Tissue-Engineered Constructs." Stem Cells International 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/1380304.

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It is documented that human mesenchymal stem cells (hMSCs) can be differentiated into various types of cells to present a tool for tissue engineering and regenerative medicine. Thus, the preservation of stem cells is a crucial factor for their effective long-term storage that further facilitates their continuous supply and transportation for application in regenerative medicine. Cryopreservation is the most important, practicable, and the only established mechanism for long-term preservation of cells, tissues, and organs, and engineered tissues; thus, it is the key step for the improvement of tissue engineering. A significant portion of MSCs loses cellular viability while freeze-thawing, which represents an important technical limitation to achieving sufficient viable cell numbers for maximum efficacy. Several natural and synthetic materials are extensively used as substrates for tissue engineering constructs and cryopreservation because they promote cell attachment and proliferation. Rho-associated kinase (ROCK) inhibitors can improve the physiological function and postthaw viability of cryopreserved MSCs. This review proposes a crosstalk between substrate topology and interaction of cells with ROCK inhibitors. It is shown that incorporation of ionic nanoparticles in the presence of an external electrical field improves the generation of ROCK inhibitors to safeguard cellular viability for the enhanced cryopreservation of engineered tissues.

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15

Zhang, Chaocan, Youliang Zhou, Li Zhang, Lili Wu, Yanjun Chen, Dong Xie, and Wanyu Chen. "Hydrogel Cryopreservation System: An Effective Method for Cell Storage." International Journal of Molecular Sciences 19, no. 11 (October 25, 2018): 3330. http://dx.doi.org/10.3390/ijms19113330.

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At present, living cells are widely used in cell transplantation and tissue engineering. Many efforts have been made aiming towards the use of a large number of living cells with high activity and integrated functionality. Currently, cryopreservation has become well-established and is effective for the long-term storage of cells. However, it is still a major challenge to inhibit cell damage, such as from solution injury, ice injury, recrystallization and osmotic injury during the thawing process, and the cytotoxicity of cryoprotectants. Hence, this review focused on different novel gel cryopreservation systems. Natural polymer hydrogel cryopreservation, the synthetic polymer hydrogel cryopreservation system and the supramolecular hydrogel cryopreservation system were presented, respectively. Due to the unique three-dimensional network structure of the hydrogel, these hydrogel cryopreservation systems have the advantages of excellent biocompatibility for natural polymer hydrogel cryopreservation systems, designability for synthetic polymer hydrogel cryopreservation systems, and versatility for supramolecular hydrogel cryopreservation systems. To some extent, the different hydrogel cryopreservation methods can confine ice crystal growth and decrease the change rates of osmotic shock in cell encapsulation systems. It is notable that the cryopreservation of complex cells and tissues is demanded in future clinical research and therapy, and depends on the linkage of different methods.

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16

Fancsovits, Péter, János Urbancsek, László Fónyad, Anna Sebestyén, Gézáné †Csorba, Ádám Lehner, Zita Kaszás, János Rigó jr., and Attila Bokor. "Kezdeti tapasztalataink a petefészekszövet-fagyasztás bevezetésével." Orvosi Hetilap 157, no. 49 (December 2016): 1947–54. http://dx.doi.org/10.1556/650.2016.30582.

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Introduction: The oncological treatment may damage ovarian function. To prevent this, it is possible to cryopreserve the ovarian tissue, and to keep the samples for long-term storage. The frozen-thawed tissue could be retransplanted after chemo- or radiotherapy. Aim: The aim of our study was to examine the effect of cryopreservation on the viability of ovarian tissue. Method: We analyzed the survival of frozen-thawed donated ovarian tissues. The quality of the follicles and hormone production in fresh and frozen-thawed samples were compared. Results: Histological analysis showed that the number of viable follicles was reduced by 23% in the frozen-thawed samples. However, viable follicles still presented in post thawing ovarian tissues. Maximal estradiol production in frozen-thawed tissues was 908 pg/ml and hormone production was similar to the control tissues. The maximal progesterone production was 1.95 ng/ml post thawing, but these values were lower than the progesterone production of fresh tissues. Conclusions: The method of ovarian cryopreservation used in our laboratory was able preserve the viability of follicles in frozen-thawed ovarian tissues. Orv. Hetil., 2016, 157(49), 1947–1954.

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Pacchiarotti, Jason, Thomas Ramos, Kyle Howerton, Scott Greilach, Karina Zaragoza, Marnie Olmstead, and Fariborz Izadyar. "Developing a Clinical-Grade Cryopreservation Protocol for Human Testicular Tissue and Cells." BioMed Research International 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/930962.

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Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients.

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Iroshini Welewanni and Dharshani Bandupriya. "Coconut Cryopreservation: Present Status and Future Prospects." CORD 33, no. 1 (April 1, 2017): 21. http://dx.doi.org/10.37833/cord.v33i1.54.

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Coconut is one of the most important small holder crops worldwide. Conservation of coconut as seeds or field gene banks is not effective due to a range of limitations. Cryopreservation, which is the conservation of living propagules at very low temperature (-196ºC), is the only method available currently for the long-term conservation of germplasm for problem plant species such as recalcitrant and vegetatively propagated plant species. This review summarizes different cryopreservation techniques that have been published from 1984 until the present in relation to different coconut material; it includes a brief discussion about short and medium-term cryopreservation before describing long-term preservation. It discusses factors affecting the process and success of cryopreservation, such as selection of plant material, pre-culture of tissues, osmoprotection, dehydration, cryo-storage, thawing and post-culturing of tissues, and finally to plants. The review also describes histological and ultra-structural studies on and the use of molecular markers to assess genetic stability after cryopreservation. Limitations and future directions related to coconut cryopreservation are discussed. Additional experiments are identified that will need to be undertaken to improve our understanding of the different cryopreservation methods.

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Fujihara, Mayako, Jun-ichi Shiraishi, Manabu Onuma, Yoshiyuki Ohta, and Miho Inoue-Murayama. "Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival." Animals 12, no. 11 (June 2, 2022): 1434. http://dx.doi.org/10.3390/ani12111434.

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For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.

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Rabin, Yoed, and Jedediah Lewis. "Organ Banking." Mechanical Engineering 139, no. 05 (May 1, 2017): 44–49. http://dx.doi.org/10.1115/1.2017-may-3.

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This article focuses on various technological innovations in the field of cryopreservation of human tissues. In order to further explore how engineers from various disciplines can offer a broader and improved set of tools to tackle preservation challenges, ASME and the Organ Preservation Alliance are co-organizing the Summit on Organ Banking through Converging Technologies in Boston, Mass., in August 2017. Techniques for successful cryopreservation have been developed over the past five decades for several tissue types. To achieve vitrification, cryobiologists introduce glass-promoting solutions known as cryoprotective agents (CPAs) into the tissue. As researchers push the boundaries on the ability to cryopreserve bulky tissues and large organs, a new thermal challenge emerges called rapid cooling, which can potentially give rise to dangerous thermomechanical stress driven by the tendency of the material to contract with temperature.

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Thuwanut, Paweena, Pierre Comizzoli, Alongkorn Pimpin, Weerayut Srituravanich, Wisan Sereepapong, Kamthorn Pruksananonda, Charoen Taweepolcharoen, Punkavee Tuntiviriyapun, Chanakarn Suebthawinkul, and Porntip Sirayapiwat. "Influence of hydrogel encapsulation during cryopreservation of ovarian tissues and impact of post-thawing in vitro culture systems in a research animal model." Clinical and Experimental Reproductive Medicine 48, no. 2 (June 1, 2021): 111–23. http://dx.doi.org/10.5653/cerm.2020.04056.

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Objective: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality.Methods: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. Results: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2%±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls.Conclusion: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.

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Terry, Claire, Ragai R. Mitry, Sharon C. Lehec, Paulo Muiesan, Mohamed Rela, Nigel D. Heaton, Robin D. Hughes, and Anil Dhawan. "The Effects of Cryopreservation on Human Hepatocytes Obtained from Different Sources of Liver Tissue." Cell Transplantation 14, no. 8 (September 2005): 585–94. http://dx.doi.org/10.3727/000000005783982765.

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Successful cryopreservation of human hepatocytes is important to establish hepatocyte banks for clinical use or in vitro research. The availability of donor tissue from unused liver segments/lobes and non-heart-beating donors (NHBD) has provided newer sources of hepatocytes. The quality of hepatocytes at the time of cryopreservation is important as cells isolated from liver tissue of borderline quality may not withstand the stresses associated with cryopreservation and subsequent thawing. Human hepatocytes were cryopreserved after isolation from mainly donor tissues (n = 40). In vitro assessment of the viability and function of the fresh and thawed cryopreserved hepatocytes was performed. Viability, attachment efficiency, enzyme activity, and albumin production of hepatocytes were all significantly decreased, and LDH leakage significantly increased, on thawing after cryopreservation. The viability of cryopreserved hepatocytes isolated from tissue rejected for orthotopic liver transplantation (36 ± 15%) was significantly lower than those isolated from tissue where part was used for liver transplantation (47 ± 14%, p = 0.002), but there were no significant differences in functional parameters. The viability of cryopreserved hepatocytes isolated from NHBD tissue (29 ± 9%, p = 0.001) and from steatotic donor tissue (35 ± 11%, p = 0.019) was significantly lower than those isolated from normal donor tissue (49 ± 14%). There was no difference in functional parameters, except for albumin production of hepatocytes from NHBD tissue (2.9 ±1.0 μg/h/mg protein) being significantly lower than those from normal donor tissue (4.8 ± 2.8 μg/h/mg protein, p = 0.03). The viability and attachment efficiency of cryopreserved hepatocytes isolated from liver tissue from resections for tumors was significantly higher, and the LDH leakage significantly lower, than those isolated from all donor tissue. Hepatocytes isolated from NHBD and steatotic tissue were more vulnerable to the effects of cryopreservation. Further research is required to improve hepatocyte isolation and cryopreservation protocols for different types of liver tissue.

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Wang, Min-Rui, Wenlu Bi, Mukund R. Shukla, Li Ren, Zhibo Hamborg, Dag-Ragnar Blystad, Praveen K. Saxena, and Qiao-Chun Wang. "Epigenetic and Genetic Integrity, Metabolic Stability, and Field Performance of Cryopreserved Plants." Plants 10, no. 9 (September 13, 2021): 1889. http://dx.doi.org/10.3390/plants10091889.

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Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.

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Vásquez-Rivera, Andrés, Kim K. Sommer, Harriëtte Oldenhof, Adam Z. Higgins, Kelvin G. M. Brockbank, Andres Hilfiker, and Willem F. Wolkers. "Simultaneous monitoring of different vitrification solution components permeating into tissues." Analyst 143, no. 2 (2018): 420–28. http://dx.doi.org/10.1039/c7an01576c.

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Linkova, Daria D., Yulia P. Rubtsova, and Marfa N. Egorikhina. "Cryostorage of Mesenchymal Stem Cells and Biomedical Cell-Based Products." Cells 11, no. 17 (August 29, 2022): 2691. http://dx.doi.org/10.3390/cells11172691.

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Mesenchymal stem cells (MSCs) manifest vast opportunities for clinical use due both to their ability for self-renewal and for effecting paracrine therapeutic benefits. At the same time, difficulties with non-recurrent generation of large numbers of cells due to the necessity for long-term MSC expansion ex vivo, or the requirement for repeated sampling of biological material from a patient significantly limits the current use of MSCs in clinical practice. One solution to these problems entails the creation of a biobank using cell cryopreservation technology. This review is aimed at analyzing and classifying literature data related to the development of protocols for the cryopreservation of various types of MSCs and tissue-engineered structures. The materials in the review show that the existing techniques and protocols for MSC cryopreservation are very diverse, which significantly complicates standardization of the entire process. Here, the selection of cryoprotectors and of cryoprotective media shows the greatest variability. Currently, it is the cryopreservation of cell suspensions that has been studied most extensively, whereas there are very few studies in the literature on the freezing of intact tissues or of tissue-engineered structures. However, even now it is possible to develop general recommendations to optimize the cryopreservation process, making it less traumatic for cells.

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Ijpma, Gijs, Chu Qiao Liang, Linda Kachmar, Alice Panariti, Andrea Benedetti, Jean-Pierre Lavoie, and Anne-Marie Lauzon. "Maintenance of contractile function of isolated airway smooth muscle after cryopreservation." American Journal of Physiology-Lung Cellular and Molecular Physiology 315, no. 5 (November 1, 2018): L724—L733. http://dx.doi.org/10.1152/ajplung.00064.2018.

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Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

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Cui, Xiangdong, Dayong Y. Gao, Betsy F. Fink, Henry C. Vasconez, and Brian Rinker. "Cryopreservation of composite tissues and transplantation: Preliminary studies." Cryobiology 55, no. 3 (December 2007): 295–304. http://dx.doi.org/10.1016/j.cryobiol.2007.08.013.

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PU, L., X. CUI, B. FINK, M. CIBULL, and D. GAO. "Cryopreservation of adipose tissues: The role of trehalose." Aesthetic Surgery Journal 25, no. 2 (March 2005): 126–31. http://dx.doi.org/10.1016/j.asj.2005.01.003.

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Yokonishi, Tetsuhiro, and Takehiko Ogawa. "Cryopreservation of testis tissues and in vitro spermatogenesis." Reproductive Medicine and Biology 15, no. 1 (August 5, 2015): 21–28. http://dx.doi.org/10.1007/s12522-015-0218-4.

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Brockbank, K. G. M. "Ice-free cryopreservation of natural and engineered tissues." Cryobiology 73, no. 3 (December 2016): 403. http://dx.doi.org/10.1016/j.cryobiol.2016.09.021.

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SAKAI, Akira. "Cryopreservation of Cultured Plant Cells, Tissues, and Embryos." Kagaku To Seibutsu 30, no. 7 (1992): 441–48. http://dx.doi.org/10.1271/kagakutoseibutsu1962.30.441.

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Zheng, Huanqin, Ying Chen, Fangli Lu, Man Liu, Xiaoyan Yang, Xiaoyin Fu, Ying Zhao, Bo Huang, Shiguang Huang, and Lloyd H. Kasper. "Cryopreservation of Toxoplasma gondii in infected murine tissues." Parasitology Research 111, no. 6 (June 16, 2012): 2449–53. http://dx.doi.org/10.1007/s00436-012-2991-x.

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Choi, Jin-Sik, Byung-Joo Lee, Hee-Young Park, Ji-Sun Song, Sung-Chan Shin, Jin-Choon Lee, Soo-Geun Wang, and Jin Sup Jung. "Effects of Donor Age, Long-Term Passage Culture, and Cryopreservation on Tonsil-Derived Mesenchymal Stem Cells." Cellular Physiology and Biochemistry 36, no. 1 (2015): 85–99. http://dx.doi.org/10.1159/000374055.

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Objectives: Human mesenchymal stem cells (MSCs) are efficacious in various cellular therapeutic applications and have been isolated from several tissues. Recent studies have reported that human tonsil tissue contains a new source of progenitor cells, potentially applicable for cell-based therapies. Information about the effects of donor age, long-term passage and cryopreservation are essential for clinical applications and cell-based therapies. Therefore, the authors investigated how the morphology, cell-surface markers, proliferation potential and differentiation capacity of tonsil-derived MSCs (T-MSCs) were affected by donor age, long-term passage, and cryopreservation. Materials and Methods: T-MSCs were isolated from tonsillar tissue of 20 patients undergoing tonsillectomy. Authors evaluated the effects of donor-age, long-term passage, and cryopreservation on the morphology, surface markers, proliferation potential and differentiation capacities of T-MSCs. Results: T-MSCs exhibited a fibroblast-like, spindle-shaped appearance. There were no significant morphological differences according to donor age, long-term passage or cryopreservation. T-MSCs isolated from donors of various ages were positive for markers CD90, CD44, and CD73, but negative for CD45, CD31, and HLA-DR. There were no significant differences in the expression of positive and negative surface markers as a function of donor age, long-term passage and cryopreservation. T-MSCs from different donor age groups showed similar proliferation potentials after passage 2. After long-term passage and cryopreservation, there were no significant morphological differences. Cryopreservation did not affect the proliferation potential of T-MSCs, but there was a significant decrease in the proliferation potential in long-term passage T-MSCs (passage 15). The effect of donor age, long-term passage and cryopreservation on the in vitro adipogenic, osteogenic, and chondrogenic differentiation potential of T-MSCs was not significant. Conclusion: The effect of donor age, long-term passage culture, and cryopreservation on T-MSC properties are negligible, except for the proliferation capacity of long-term cultured T-MSCs. Therefore, T-MSCs are considered to be promising MSCs that can be used as future alternative sources for autologous or allogenic MSCs.

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Rabin, Y., and P. S. Steif. "Thermal Stresses in a Freezing Sphere and its Application to Cryobiology." Journal of Applied Mechanics 65, no. 2 (June 1, 1998): 328–33. http://dx.doi.org/10.1115/1.2789058.

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Thermal stresses in an inwardly solidifying sphere are studied analytically. A closed-form solution is given which accounts for thermal expansion associated with temperature gradients and volume changes associated with phase transition. Consistent with the target application of cryopreservation of biological solutions and tissues, the material is modeled as elastic-perfectly plastic. Parametric studies using appropriate material properties and typical cryopreservation protocols suggest that strains associated with phase transition lead to far higher stresses than those associated with thermal expansion, with important implications for cryopreservation procedures.

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Arutyunyan, I. V. Arutyunyan, and T. K. Dubovaya. "Cryopreservation of tissue-engineered constructs in regenerative medicine." CLINICAL AND EXPERIMENTAL MORPHOLOGY 10, no. 2 (2021): 6–12. http://dx.doi.org/10.31088/cem2021.10.2.6-12.

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The transplantation of artificial tissues and organs is gradually becoming a part of our reality. At the same time, researchers are facing a problem common to all transplantologists, i.e. the need for a long-term storage of a biomedical product (transplant) without losing its properties. The possibility to cryopreserve cells adhered to various scaffolds' surface was first presented about 20 years ago. However, the data on the technology as a whole remains unsystematized and controversial. This review aimed to analyze the literature on tissue-engineered constructs (TEC) cryopreservation of different scientific groups to create a unified approach in assessing the technique's efficacy necessary for further regenerative medicine development. The comparison of studies on TEC cryopreservation conducted by various research groups is hampered not only by the lack of standardized protocols but also by different approaches to assessing the result. As experimental data were accumulated, the cryopreservation efficacy was reassessed from meeting the basic requirements for the structure preservation (thawed TEC retains its integrity, cells are partially alive and attached to the matrix) to focusing on the final result (thawed TEC retains its functional properties and is ready to be transplanted). Many of the currently used in vitro research methods presented in the review allow one to look for new ways of increasing the TEC cryopreservation efficacy; however, in our opinion, the next step on the way to introducing the technology into clinical practice should be research on experimental animals. Keywords: tissue engineered construction, cryopreservation, efficacy estimation

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Bebbere, Daniela, and Sara Succu. "New Challenges in Cryopreservation: A Reproductive Perspective." Animals 12, no. 13 (June 21, 2022): 1598. http://dx.doi.org/10.3390/ani12131598.

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Conde, Marcus Cristian Muniz, Luiz Alexandre Chisini, Guillermo Grazioli, Alejandro Francia, Rodrigo Varella de Carvalho, Jose Carlos Bernedo Alcázar, Sandra Beatriz Chavez Tarquinio, and Flávio Fernando Demarco. "Does Cryopreservation Affect the Biological Properties of Stem Cells from Dental Tissues? A Systematic Review." Brazilian Dental Journal 27, no. 6 (December 2016): 633–40. http://dx.doi.org/10.1590/0103-6440201600980.

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Abstract This systematic review evaluated if different cryopreservation protocols could affect biological properties (Cell survival rate (CSR), proliferation, differentiation, maintenance of stem cell markers) of stem cells obtained from dental tissues (DSC) post-thaw. An electronic search was carried out within PubMed and ISI Web Science by using specific keyword. Two independent reviewers read the titles and abstracts of all reports respecting predetermined inclusion/exclusion criteria. Data were extracted considering the biological properties of previously cryopreserved DSCs and previously cryopreserved dental tissues. DSCs cryopreserved as soon as possible after their isolation presents a CSR quite similar to the non-cryopreserved DSC. Dimethyl sulfoxide (DMSO) [10%] showed good results related to cell recovery post-thaw to cryopreserve cells and tissues for periods of up to 2 years. The cryopreservation of DSC in a mechanical freezer (-80°C) allows the recovery of stem cells post-thaw. The facilities producing magnetic field (MF), demand a lower concentration of cryoprotectant, but their use is not dispensable. It is possible to isolate and cryopreserve dental pulp stem cell (DPSC) from healthy and diseased vital teeth. Cryopreservation of dental tissues for late DSC isolation, combined with MF dispensability, could be valuable to reduce costs and improve the logistics to develop teeth banks.

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Braye, Aude, Herman Tournaye, and Ellen Goossens. "Setting Up a Cryopreservation Programme for Immature Testicular Tissue: Lessons Learned After More Than 15 Years of Experience." Clinical Medicine Insights: Reproductive Health 13 (January 2019): 117955811988634. http://dx.doi.org/10.1177/1179558119886342.

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Young boys undergoing gonadotoxic treatments are at high risk of spermatogonial stem cell (SSC) loss and fertility problems later in life. Stem cell loss can also occur in specific genetic conditions, eg, Klinefelter syndrome (KS). Before puberty, these boys do not yet produce sperm. Hence, they cannot benefit from sperm banking. An emerging alternative is the freezing of testicular tissue aiming to preserve the SSCs for eventual autologous transplantation or in vitro maturation at adult age. Many fertility preservation programmes include cryopreservation of immature testicular tissue, although the restoration procedures are still under development. Until the end of 2018, the Universitair Ziekenhuis Brussel has frozen testicular tissues of 112 patients between 8 months and 18 years of age. Testicular tissue was removed in view of gonadotoxic cancer treatment (35%), gonadotoxic conditioning therapy for bone marrow transplantation (35%) or in boys diagnosed with KS (30%). So far, none of these boys had their testicular tissue transplanted back. This article summarizes our experience with cryopreservation of immature testicular tissue over the past 16 years (2002-2018) and describes the key issues for setting up a cryopreservation programme for immature testicular tissue as a means to safeguard the future fertility of boys at high risk of SSC loss.

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Jarošová, Rea, Petra Ondráčková, Zdeněk Patočka, and Zbyšek Sládek. "Comparison of cryoprotective methods for histological examination of rat and porcine lung tissue." Acta Veterinaria Brno 90, no. 2 (2021): 225–31. http://dx.doi.org/10.2754/avb202190020225.

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Proper histological evaluation of lung tissue and accurate antigen detection by immunodetection techniques require histological tissue processing to preserve tissue reactivity and open alveolar spaces. In this study, we focused on testing and comparing different procedures of tissue cryopreservation. Sucrose or Tissue Tek were used with several methods of freezing samples by supercooled liquids and liquid nitrogen. Changes in tissue caused during the freezing of samples and the effect of cryoprotectants on the tissue were recorded. Rat and porcine pulmonary tissues were used in this experiment. This study aimed to optimize the process of lung cryopreservation with emphasis on enabling proper anatomical evaluation and preserving a high tissue immunoreactivity. The best results were obtained by inflating pulmonary parenchyma with a 1 : 1 mixture of O.C.T. with phosphate buffered saline (PBS) frozen in supercooled n-heptane placed on dry ice. Pulmonary tissue prepared in this way enables to perform proper histological evaluation and to detect target molecules by immunohistochemical analysis.

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SHIOGAMA, TOSHIAKI, YOKO MULLEN, HILLAR KLANDORF, MASAZUMI TERADA, and WILLIAM R. CLARK. "AN IMPROVED CRYOPRESERVATION PROCEDURE FOR HUMAN FETAL PANCREAS TISSUES." Transplantation 44, no. 5 (November 1987): 602–6. http://dx.doi.org/10.1097/00007890-198711000-00003.

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Karlsson, Jens O. M., and Mehmet Toner. "Long-term storage of tissues by cryopreservation: critical issues." Biomaterials 17, no. 3 (January 1996): 243–56. http://dx.doi.org/10.1016/0142-9612(96)85562-1.

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Barra-Jiménez, Azahara, Tuija S. Aronen, Jesús Alegre, and Mariano Toribio. "Cryopreservation of embryogenic tissues from mature holm oak trees." Cryobiology 70, no. 3 (June 2015): 217–25. http://dx.doi.org/10.1016/j.cryobiol.2015.02.006.

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Jiang, Yu, Wen-Qian Zhu, Xian-Chun Zhu, Ning-Ning Cai, Rui Yang, Huan Cai, and Xue-Ming Zhang. "Cryopreservation of calf testicular tissues with knockout serum replacement." Cryobiology 92 (February 2020): 255–57. http://dx.doi.org/10.1016/j.cryobiol.2020.01.010.

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LI, Yu-bin, Can-quan ZHOU, Guo-fen YANG, Qiong WANG, and Yu DONG. "Modified vitrification method for cryopreservation of human ovarian tissues." Chinese Medical Journal 120, no. 2 (January 2007): 110–14. http://dx.doi.org/10.1097/00029330-200701020-00007.

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Taylor, Michael J., Bradley P. Weegman, Simona C. Baicu, and Sebastian E. Giwa. "New Approaches to Cryopreservation of Cells, Tissues, and Organs." Transfusion Medicine and Hemotherapy 46, no. 3 (2019): 197–215. http://dx.doi.org/10.1159/000499453.

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46

Hazubska-Przybył, Teresa, Paweł Chmielarz, Marcin Michalak, and Krystyna Bojarczuk. "Cryopreservation of embryogenic tissues of Picea omorika (Serbian spruce)." Plant Cell, Tissue and Organ Culture (PCTOC) 102, no. 1 (February 10, 2010): 35–44. http://dx.doi.org/10.1007/s11240-010-9701-0.

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Salaj, T., I. Matušíková, L. Fráterová, B. Piršelová, and J. Salaj. "Regrowth of embryogenic tissues of Pinus nigra following cryopreservation." Plant Cell, Tissue and Organ Culture (PCTOC) 106, no. 1 (December 3, 2010): 55–61. http://dx.doi.org/10.1007/s11240-010-9893-3.

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Rong-sheng, Cai, Xue De-lin, and Jiang Xian-hui. "Cryopreservation and culture of the human fetal brain tissues." Journal of Tongji Medical University 13, no. 3 (September 1993): 138–42. http://dx.doi.org/10.1007/bf02886504.

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49

Wooi, K. H., M. J. Mahony, J. M. Shaw, and J. Clulow. "402. Cryopreservation of oocytes and follicular cells of the cane toad Bufo Marinus." Reproduction, Fertility and Development 20, no. 9 (2008): 82. http://dx.doi.org/10.1071/srb08abs402.

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Amphibians are currently the most threatened of all vertebrate groups with more than 30% of all known species in decline, facing extinction or recently extinct. Cryobanking of amphibian germ cells and reproductive tissues could be used to manage threatened species and provide insurance against extinction. However, cryopreservation of fully developed amphibian oocytes and whole embryos has not been achieved due to technical problems freezing such large cellular structures. As an alternative approach, we investigated the feasibility of developing protocols for the slow-cool freezing, storage and retrieval of developmentally competent amphibian ovarian follicles containing Stage I and II oocytes which are much smaller in size than later developmental stages. Ovarian follicles from euthanased Cane Toads were incubated in cryodiluents containing either glycerol or DMSO to assess cryoprotectant toxicity and response to slow cooling freezing protocols. The fluorescent live cell stain SYBR 14 and its counter stain propidium iodide was used to score the proportion of viable follicle cells before and after cryopreservation. Cryoprotectant type, concentration and exposure time all had significant effects (P < 0.05) on the viability of follicle cells, with significant interactions between these variables. Overall, glycerol was less toxic to follicle cells than DMSO. At higher concentrations, glycerol exerted high osmotic stress on oocytes, and there was evidence that DMSO triggered apoptosis in oocytes. The most effective cryopreservation protocol for stage I and II oocyte follicles resulted in a post-thaw recovery of a mean 70% of viable follicular cells. This protocol involved cryopreservation in 15% v/v glycerol, inclusion of seeding and temperature holding periods during cryopreservation, coupled with rapid thawing in a 30°C water bath. The successful cryopreservation of intact follicles in this study indicates the potential to recover functional ovarian tissues post cryopreservation for continuation of amphibian oogenesis in vitro or in vivo.

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Devi, Lalitha, Himesh Makala, Lavanya Pothana, Khemlal Nirmalkar, and Sandeep Goel. "Comparative efficacies of six different media for cryopreservation of immature buffalo (Bubalus bubalis) calf testis." Reproduction, Fertility and Development 28, no. 7 (2016): 872. http://dx.doi.org/10.1071/rd14171.

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Buffalo calves have a high mortality rate (~80%) in commercial dairies and testis cryopreservation can provide a feasible option for the preservation of germplasm from immature males that die before attaining sexual maturity. The aim of the present study was to evaluate combinations of 10 or 20% dimethylsulfoxide (DMSO) with 0, 20 or 80% fetal bovine serum (FBS) for cryopreservation of immature buffalo testicular tissues, subjected to uncontrolled slow freezing. Tissues cryopreserved in 20% DMSO with 20% FBS (D20S20) showed total, tubular and interstitial cell viability, number of early apoptotic and DNA-damaged cells, surviving germ and proliferating cells and expression of testicular cell-specific proteins (POU class 5 homeobox (POU5F1), vimentin (VIM) and actin α2 (ACTA2)) similar to that of fresh cultured control (FCC; P > 0.05). Expression of cytochrome P450, family 11, subfamily A (CYP11A1) protein and testosterone assay showed that only tissues cryopreserved in D20S20 had Leydig cells and secretory functions identical to that of FCC (P > 0.05). High expression of superoxide dismutase2 (SOD2), cold-inducible RNA-binding protein (CIRBP) and RNA-binding motif protein3 (RBM3) proteins in cryopreserved tissues indicated involvement of cell signalling pathways regulating cellular protective mechanisms. Similarity in expression of pro-apoptosis proteins transcription factor tumour protein P53 (TP53) and BCL2-associated X protein (BAX) in D20S20 cryopreserved tissues to that of FCC (P > 0.05) suggested lower apoptosis and DNA damage as key reasons for superior cryopreservation.

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