Use of polyethyleneoxide and hydroxyethylstarch as blood plasma substitutes in the cryopreservation of testis interstitium cells in mice




cryopreservation, Leydig cells, testes, polyethylene oxide, hydroxyethyl starch


Purpose of the study: to investigate the impact of hydroxyethyl starch (HES) and polyethylene oxide (PEO) on the indicators of preservation of murine testis interstitial cells (IC) under cryopreservation.

Materials and methods. To isolate IC the enzymes were used: 0.2 mg/ml collagenase and 0.1 mg/ml DNase. The obtained cell suspension was cryopreserved in the solutions that contained 0; 0,7; 1,4; 2,1; 2,8 M of dimethyl sulfoxide (DMSO) and/or 10%, 20% fetal cow serum, 10 mg/ml PEO or HES. The samples (1 ml) were cooled at a rate of 1 °C/min to -80 °C then stored in liquid nitrogen (-196 °C). They were warmed at 37 °C in the water bath. Cryopreservation solution was removed. The number of cells and their preservation were assessed before and after with the assistance of Goryaev’s camera. Viability of IC, Leydig cell preservation and preservation of metabolic activity were measured with trypan blue dye, histochemical staining for 3β-hydroxysteroid dehydrogenase activity.

Results. It was shown that 1,4 M DMSO without supplements favored IC preservation. Addition to the cryopreservation solution 10% and 20% of fetal cow serum or 10 mg/ml HES increased total preservation of IC by more than 10% and Leydig cell cryopreservation by an average 15%. HES 10 mg/ml may decrease DMSO concentration to 0,7 M. This combination had the best indicators of total preservation of IC, preservation of viable cells and Leydig cells: 75,8 (53,3; 93,3), 55,6 (45,1; 69,4), 57,1 (40,2;70,3) %, respectively. PEO was ineffective.

Conclusion. High-molecular weight synthetic polymers such as HES can substitute protective properties of blood serum under cryopreservation and allow decreasing effective concentration of permeable cryoprotective such as DMSO.

Author Biographies

O. V. Pakhomov, Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine; V.N. Karazin Kharkiv National University; Private Higher Education Institution “Kharkiv International Medical University”, Kharkiv

PhD, senior researcher, Cryoendocrinology Department;

Associate professor, Biochemistry Department;

Department of Fundamental General Scientific Disciplines

E. R. Grabovetskaya, V.N. Karazin Kharkiv National University, Kharkiv

PhD, associate professor, Biochemistry Department

N. I. Filimonova, National University of Pharmacy of the MoH of Ukraine, Kharkiv

MD, professor, Department of Microbiology, Virology and Immunology

N. V. Dubinina, National University of Pharmacy of the MoH of Ukraine, Kharkiv

PhD, associate professor, Department of Microbiology, Virology and Immunology

O. G. Geyderikh, National University of Pharmacy of the MoH of Ukraine, Kharkiv

PhD, associate professor, Department of Microbiology, Virology and Immunology


  1. Deng, B., Bondarenko, T., Pakhomov, O. “Changes in Sexual Behavior of Orchidectomized Rats Under Influence of Allotransplantation of Testicular Interstitial Cell Suspension.” Cell Transplantation 26.5 (2017): 795–803.
  2. Pallotti, F., Pelloni, M., Gianfrilli, D., et al. “Mechanisms of Testicular Disruption from Exposure to Bisphenol A and Phtalates.” J Clin Med 9.2 (2020).
  3. Kahn, B.E., Brannigan, R.E. “Obesity and male infertility.” Curr Opin Urol 27.5 (2017): 441–5.
  4. Snyder, P.J., Lawrence, D.A. “Treatment of male hypogonadism with testosterone enanthate.” J Clin Endocrinol Metab 51.6 (1980): 1335-9.
  5. Velázquez, E., Bellabarba Arata, G. “Testosterone replacement therapy.” Arch Androl 41.2 (1998): 79–90.
  6. Lo, K.C., Lei, Z., Rao, C.V., et al. “De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells.” Endocrinology 145.9 (2004): 4011–5.
  7. Jiang, M.H., Cai, B., Tuo, Y., et al. “Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction.” Cell Res 24.12 (2014): 1466–85.
  8. Li, X., Xu, A., Li, K., et al. “CXCR4-SF1 bifunctional adipose-derived stem cells benefit for the treatment of Leydig cell dysfunction-related diseases.” J Cell Mol Med (2020).
  9. Tai, J., Tze, W.J., Johnson, H.W. “Cryopreservation of rat Leydig-cells for in-vitro and in-vivo studies.” Hormone and Metabolic Research 26.3 (1994): 145–7.
  10. Chen, G.R., Ge, R.S., Lin, H., et al. “Development of a cryopreservation protocol for Leydig cells.” Human Reproduction 22.8 (2007): 2160–8.
  11. Dokras, A., Sargent, I.L., Redman, C.W., Barlow, D.H. “Sera from women with unexplained infertility inhibit both mouse and human embryo growth in vitro.” Fertil Steril 60.2 (1993): 285–92.
  12. Kemmann, E. “Creutzfeldt-Jakob disease (CJD) and assisted reproductive technology (ART). Quantification of risks as part of informed consent.” Hum Reprod 13.7 (1998): 1777.
  13. Leung, P.C., Gronow, M.J., Kellow, G.N., et al. “Serum supplement in human in vitro fertilization and embryo development.” Fertil Steril 41.1 (1984): 36–9.
  14. van Os, H.C., Drogendijk, A.C., Fetter, W.P., et al. “The influence of contamination of culture medium with hepatitis B virus on the outcome of in vitro fertilization pregnancies.” Am J Obstet Gynecol 165.1 (1991): 152–9.
  15. Snyman, E., Van der Merwe, J.V. “Endotoxin-polluted medium in a human in vitro fertilization program.” Fertil Steril 46.2 (1986): 273–6.
  16. Gurtovenko, A.A., Anwar, J. “Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide.” J Phys Chem B 111.35 (2007): 10453–60.
  17. Petrenko, Y. “Cryopreservation of Human Embryonic Liver Cells Using DMSO and High Molecular Weight Polymers.” Problems of Cryobiology and Cryomedicine 3 (2003): 80–7.
  18. Liu, Y., Xu, X., Ma, X.H., et al. “Effect of various freezing solutions on cryopreservation of mesenchymal stem cells from different animal species.” Cryo Letters 32.5 (2011): 425–35.
  19. Scheinkönig, C., Kappicht, S., Kolb, H.J., Schleuning, M. “Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells.” Bone Marrow Transplant 34.6 (2004): 531–6.
  20. Graham, J.E., Meola, D.M., Kini, N.R., Hoffman, A.M. “Comparison of the effects of glycerol, dimethyl sulfoxide, and hydroxyethyl starch solutions for cryopreservation of avian red blood cells.” Am J Vet Res 76.6 (2015): 487–93.
  21. Imaizumi, K., Nishishita, N., Muramatsu, M., et al. “A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol.” PLoS One 9.2 (2014): e88696.
  22. Lee, Y.A., Kim, Y.H., Kim, B.J., et al. “Cryopreservation of mouse spermatogonial stem cells in dimethylsulfoxide and polyethylene glycol.” Biol Reprod 89.5 (2013): 109.
  23. Klinefelter, G.R., Hall, P.F., Ewing, L.L. “Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure.” Biol Reprod 36.3 (1987): 769–83.
  24. Best, B.P. “Cryoprotectant Toxicity: Facts, Issues, and Questions.” Rejuvenation Res 18.5 (2015): 422–36.
  25. Liu, Y., Xu, X., Ma, X., et al. “Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions.” Biotechnol Prog 26.6 (2010): 1635–43.
  26. Zakharov, B., Fisyuk, A., Fitch, A., et al. “Ice Recrystallization in a Solution of a Cryoprotector and Its Inhibition by a Protein: Synchrotron X-Ray Diffraction Study.” J Pharm Sci 105.7 (2016): 2129–38.
  27. Park, S., Lee, D.R., Nam, J.S., et al. “Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells.” Cryobiology 81 (2018): 65–73.
  28. Lionetti, F.J., Hunt, S.M. “Cryopreservation of human red cells in liquid nitrogen with hydroxyethyl starch // Cryobiology.” 12.2 (1975): 110–8.
  29. Lionetti, F.J., Hunt, S.M., Mattaliano, R.J., Valeri, C.R. “In vitro studies of cryopreserved baboon granulocytes.” Transfusion 18.6 (1978): 685–92.
  30. Bruyère, P., Baudot, A., Joly, T., et al. “A chemically defined medium for rabbit embryo cryopreservation.” PLoS One 8.8 (2013): e71547.
  31. Ferrucci, P.F., Martinoni, A., Cocorocchio, E., et al. “Evaluation of acute toxicities associated with autologous peripheral blood progenitor cell reinfusion in patients undergoing high-dose chemotherapy.” Bone Marrow Transplant 25.2 (2000): 173–7.
  32. Chen-Plotkin, A.S., Vossel, K.A., Samuels, M.A., Chen, M.H. “Encephalopathy, stroke, and myocardial infarction with DMSO use in stem cell transplantation.” Neurology 68.11 (2007): 859–61.



How to Cite

Pakhomov, O. V., Grabovetskaya, E. R., Filimonova, N. I., Dubinina, N. V., & Geyderikh, O. G. (2020). Use of polyethyleneoxide and hydroxyethylstarch as blood plasma substitutes in the cryopreservation of testis interstitium cells in mice. REPRODUCTIVE ENDOCRINOLOGY, (55), 67–71.



Experimental studies