Biopolym. Cell. 2021; 37(1):14-22.
Molecular and Cell Biotechnologies
The impact of cryopreservation with polyvinyl alcohol on the survival and functional activity of rat testis interstitial cells
1, 2, 3Pakhomov O. V., 1, 4Sidorenko O. S.
  1. Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine
    23, Pereyaslavskaya Str., Kharkiv, Ukraine, 61015
  2. V. N. Karazin Kharkiv National University
    4, Svobody Ave., Kharkiv, Ukraine, 61022
  3. Kharkiv National Medical University
    4, Nauki Ave., Kharkiv, Ukraine, 61022
  4. Institute for Scintillation Materials, NAS of Ukraine
    60, Lenin Ave., Kharkiv, Ukraine, 61001


Aim. To investigate the impact of polyvinyl alcohol (PVA) in cryoprotective media on the survival and proliferation of rat testis interstitial cells (ICs) after cryopreservation. Methods. Rat ICs were obtained by enzymatic processing of testis with collagenase (type I) and DNase I. The obtained cells were cryopreserved by three methods. Methods 1 and 2 implied the use of cryoprotective medium (CM) based on 1.4 M dimethylsulfoxide (DMSO) and 10 % fetal cow serum (FCS) but utilized different cooling rates to –70°C. Method 1: ICs were cooled with 1°C/min. Method 2: after initiation of crystallization the cells were cooled with 15°C/min to –40°C and with 20°C/min from –40 to –70°C. Method 3 implied the cooling rate of 1°C/min but FCS was replaced by 20 mg/ml PVA. When the temperature –70°C was reached the samples were plunged into liquid nitrogen (–196°C). After warming and removal of CM the ICs were cultured in Ham’s/F12 with and without human chorionic gonadotropin (hCG). Results. The use of method 2 improved the survival of Leydig cells in samples to 79.5 (70.0; 90.0) % comparing with method 1 (42.0 (37.0; 47.0) %). The use of PVA in method 3 did not have an effect on ICs and Leydig cell survival comparing with method 1. Cell culturing showed that the number of Leydig cells in wells rose to 17 (0.09; 0.39) and 0.12 (0.08; 0.14) ×106 after cryopreservation by method 2 and 3, respectively, when the cells were stimulated with hCG. These values are several times higher than the initial number of cells in the wells (0.65 (0.57;0.71) ×104). Conclusion. PVA in combination with other components of cryoprotective medium promoted the survival of Leydig cells capable of further proliferation in culture, especially, in the presence of hCG.
Keywords: testis interstitial cells, Leydig cells, cryopreservation, polyvinyl alcohol, cell proliferation, human chorionic gonadotropin


[1] Schlatt S, Honaramooz A, Boiani M, Schöler HR, Dobrinski I. Progeny from sperm obtained after ectopic grafting of neonatal mouse testes. Biol Reprod. 2003;68(6):2331-5.
[2] Woelders H, Windig J, Hiemstra SJ. How developments in cryobiology, reproductive technologies and conservation genomics could shape gene banking strategies for (farm) animals. Reprod Domest Anim. 2012;47 Suppl 4:264-73.
[3] Kirpatovskii VI, Efremov GD, Frolova EV, Kudryavtseva LV. Stimulation of Spermatogenesis and Synthesis of Testosterone by Allotransplantation of Neonatal Testicular Tissue under Tunica Albuginea of Cryptorchid Testis. Bull Exp Biol Med. 2019;166(4):497-502.
[4] Gao X, Chang XB, Wu RY, Zhan BY. Allotransplantation of cryopreserved human Leydig cells. Transplant Proc. 1994;26(6):3490.
[5] Tai J, Tze WJ, Johnson HW. Cryopreservation of rat Leydig cells for in vitro and in vivo studies. Horm Metab Res. 1994;26(3):145-7.
[6] Chen GR, Ge RS, Lin H, Dong L, Sottas CM, Hardy MP. Development of a cryopreservation protocol for Leydig cells. Hum Reprod. 2007;22(8):2160-8.
[7] Hurina TM, Pakhomov OV, Bozhok HA, Bondarenko TP. Method for cryopreservation of testicular cell suspen-sion of mammals. Patent UA, no. 91787, 2010.
[8] Gurina TM, Pakhomov AV, Bozhok GA. Effect of Cooling Rate under Various Temperature Intervals on Survival of Steroidogenic Potential of Suspension of Interstitial Cells of Adult Rat’s Testes. Problems of Cryobiology and Cryomedicine. 2007; 17(4): 394–402.
[9] Gurina TM, Pakhomov AV, Kyryliuk AL, Bozhok GA. Development of a cryopreservation protocol for testicular interstitial cells with the account of temperature intervals for controlled cooling below -60°C. Cryobiology. 2011;62(2):107-14.
[10] Truyen U, Parrish CR, Harder TC, Kaaden OR. There is nothing permanent except change. The emergence of new virus diseases. Vet Microbiol. 1995;43(2-3):103-22.
[11] Hinckley GT, Johnson CJ, Jacobson KH, Bartholomay C, McMahon KD, McKenzie D, Aiken JM, Pedersen JA. Persistence of pathogenic prion protein during simulated wastewater treatment processes. Environ Sci Technol. 2008;42(14):5254-9.
[12] van Os HC, Drogendijk AC, Fetter WP, Heijtink RA, Zeilmaker GH. The influence of contamination of culture medium with hepatitis B virus on the outcome of in vitro fertilization pregnancies. Am J Obstet Gynecol. 1991;165(1):152-9.
[13] Snyman E, Van der Merwe JV. Endotoxin-polluted medium in a human in vitro fertilization program. Fertil Steril. 1986;46(2):273-6.
[14] Petrenko YuA. Cryopreservation of human embryonic liver cells using DMSO and high molecular weight polymers. Problems of Cryobiology. 2003; (3): 80–7.
[15] Liu Y, Xu X, Ma XH, Liu J, Cui ZF. Effect of various freezing solutions on cryopreservation of mesenchymal stem cells from different animal species. Cryo Letters. 2011;32(5):425-35.
[16] Knight CA, Wen D, Laursen RA. Nonequilibrium antifreeze peptides and the recrystallization of ice. Cryobiology. 1995;32(1):23-34.
[17] Tekin K, Daşkın A. Effect of polyvinyl alcohol on survival and function of angora buck spermatozoa following cryopreservation. Cryobiology. 2019;89:60-67.
[18] Klinefelter GR, Hall PF, Ewing LL. Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod. 1987;36(3):769-83.
[19] Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2015;1257:3-19.
[20] Zakharov B, Fisyuk A, Fitch A, Watier Y, Kostyuchenko A, Varshney D, Sztucki M, Boldyreva E, Shalaev E. Ice Recrystallization in a Solution of a Cryoprotector and Its Inhibition by a Protein: Synchrotron X-Ray Diffraction Study. J Pharm Sci. 2016;105(7):2129-38.
[21] Bruyère P, Baudot A, Joly T, Commin L, Pillet E, Guérin P, Louis G, Josson-Schramme A, Buff S. A chemically defined medium for rabbit embryo cryopreservation. PLoS One. 2013;8(8):e71547.
[22] Liu Y, Xu X, Ma X, Martin-Rendon E, Watt S, Cui Z. Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions. Biotechnol Prog. 2010;26(6):1635-43.
[23] Riccetti L, De Pascali F, Gilioli L, Potì F, Giva LB, Marino M, Tagliavini S, Trenti T, Fanelli F, Mezzullo M, Pagotto U, Simoni M, Casarini L. Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro. Reprod Biol Endocrinol. 2017;15(1):2.
[24] Nisula B, Bartocci A. Choriogonadotropin and immunity: a reevaluation. Ann Endocrinol (Paris). 1984;45(4-5):315-9.
[25] Zhao S, Zhu W, Xue S, Han D. Testicular defense systems: immune privilege and innate immunity. Cell Mol Immunol. 2014;11(5):428-37.