Biopolym. Cell. 2020; 36(2):122-132.
Methods
Vitrification of rat testicular tissue using biopolymers
1Volkova N. O., 1Yukhta M. S., 1Goltsev A. M.
  1. Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine
    23, Pereyaslavskaya Str., Kharkiv, Ukraine, 61015

Abstract

Aim. Toassessof the effect of cryopreservation by vitrification on morphofunctionalparametersof immature rat testicular tissue in a cryoprotective media based on polymeric carriers. Methods. Samples of immature rat testicles were rapidly immersed in liquid nitrogen under protection of the cryomedium 1 (15 % DMSO+18 % glycerol+0.5M sucrose) or the cryomedium 2 (15 % DMSO+18 % glycerol+15 % PEO) based on biopolymers (bovine serum albumin (BSA), collagen (CG) or fibrin (FG) gels). The comparison groupsincluded theintact control and fragments vitrified in Hank’s solution. Results. In contrast to BSA and CG, FG had a more pronounced protective effect. Its combination with the cryomedium 1 led to a decrease in the degree of retraction and desquamation of spermatogenic epithelium, as well as to a three-fold increase incell density compared to the negative control. The metabolic activity of testis tissue vitrified under protection of FG and the criomedium 1 increased 2.75 times compared tothe negative control (BSA and CG inthis cryomedium led to 2.1- and 2.0-fold increase, respectively). A similar trendwas observed in the LDH activity. Biopolymers in thecryomedium 2 had a positive effect uponhistological examination, but did not contribute to the preservation of metabolic activity of spermatogenic epithelial cells. Conclusion. Cryomedium 1 based on fibrin gel had the optimal properties among all studied combinations of biopolymers and cryoprotectants. The obtained data can be used for development of vitrification methods for human seminiferous tubules.
Keywords: Immature testicular tissue, spermatogenic epitelium, cryoprotective medium, collagen gel, fibrin gel, metabolic activity

References

[1] Keros V, Hultenby K, Borgstrom B, Fridstrom M, Jahnukainen K, Hovatta O. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. Human Reproduction. 2007; 22 (5): 1384-95.
[2] Stukenborg JB, Jahnukainen K, Hutka M, Mitchell RT. Cancer treatment in childhood and testicular function: the importance of the somatic environment. Endocrine connections. 2018; 7(2): R69-87.
[3] Ginsburg ES, Yanushpolsky EH, Jackson KV. In vitro fertilization for cancer patients and survivors. Fertility and Sterility. 2001; 75(4): 705-10.
[4] Poels J, Langendonckt A, Many MC, Wese FX, Wyns C. Vitrification preserves proliferation capacity in human spermatogonia. Hum Reprod. 2013;28(3): 578-589.
[5] Dimasi L. Meeting increased demands on cell-based processes by using defined media supplements. BioProcess International. 2011; 9(8): 48-58.
[6] Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L, Hovatta O. Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants. Hum Reprod. 2005;20(6): 1676-1687.
[7] Nicodemus GD, Bryant SJ. Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng Part B Rewires. 2008; 4: 149-65.
[8] Jafari M, Paknejad Z, Rad MR, Motamedian SR, Eghbal MJ, Nadjmi N, Khojasteh A. Polymeric scaffolds in tissue engineering: a literature review. J Biomed Mater Res B. 2017; 105(2): 431-59.
[9] Guarino V, Ambrosio L. Properties of biomedical foams for tissue engineering applications. In: Netti PA (ed.) Biomedical foams for tissue engineering applications. - Woodhead Publishing, Cambridge, 2014; pp 40-70.
[10] Gelse K, Posch E, Aigner T. Collagens - structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003; 55(12):1531-46.
[11] Allenspach AL, Kraemer TG. Ice crystal patterns in artificialgels of extracellular matrix molecules after quick-freezing and freeze-substitution. Cryobiology. 1989; 26: 170-9.
[12] Volkova N, Yukhta M, Goltsev A. Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats. Cell and tissue banking. 2018; 19(4), 819-26.
[13] Volkova NO, Yukhta MS, Chernyshenko LG, Stepanyuk LV, Sokil LV, Goltsev AM. Cryopreservation of Rat Seminiferous Tubules Using Biopolymers and Slow Non-Controlled Rate Cooling. Problems of Cryobiology and Cryomedicine. 2018; 28(4): 278-92.
[14] European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientifi c Purposes. European Treaty Series No. 123 Strasbourg, 18.III.1986. Strasbourg: Council of Europe; 1986. 53p.
[15] Campion SN, Carvallo FR, Chapin RE, Now-land WS, Beauchamp D, Jamon R, Koitz R, Winton TR, Cappon GD, Hurtt ME. Comparative assessment of the timing of sexual maturation in male Wistar Han and Spra-gue-Dawley rats. Reprod Toxicol. 2013; 38(7): 16-24.
[16] Chandrakasan G, Torchia DA, Piez KA. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution. J Biol Chem. 1976; 251: 6062-7.
[17] Milazzo JP, Vaudreuil L, Cauliez B, Gruel E, Masse L, Mousset-Simeon N, Mace B, Rives N. Comparison of conditions for cryopreservation of testicular tissue fromimmature mice. Hum Reprod. 2008; 23: 17-28.
[18] Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2): 55-63.
[19] Chiti MC, Dolmans MM, Donnez J, Amorim CA. Fibrin inreproductive tissue engineering: a review on its application as a biomaterial for fertility preservation. Annals of biomedical engineering. 2017; 45(7): 1650-63.
[20] Miyamoto Y, Enosawa S, Takeuchi T, Takezawa T. Cryopreservation in situ of cell monolayers on collagen vitri-gel membrane culture substrata: ready-to-use preparation of primary hepatocytes and ES cells. Cell Transplant. 2009; 18(5): 619-26.