ANDROGEN SYNTHESIZING ACTIVITY OF CRYOPRESERVED TESTICULAR INTERSTITIAL CELLS UPON TRANSPLANTATION

Abstract

Background. Cells isolated from the testes of mammals and humans can be used for scientific purposes, maintaining certain animal lines and breeds, preserving biological material from endangered species, as well as in reproductive technologies. Most approaches for cryopreserving such cells utilize blood serum (or its derivatives) and dimethyl sulfoxide (DMSO). This can lead to unstable results, the spread of infections, altered expression of certain cell genes, and the manifestation of DMSO’s toxic effects. In our previous studies, serum-free media for testicular interstitial cells (ICs) were develo­ped; the aim of this work was to investigate their ability to synthesize testo­sterone after cryopreservation.
Materials and Methods. ICs were obtained from mature rats via enzymatic digestion and cryopreserved in solutions containing 0.7 M DMSO and 100 mg/mL of one of the following polymers: dextran 40, hydroxyethyl starch, polyethylene oxide, or 1.4 M DMSO and 10% fetal bovine serum (FBS). The cooling rate was 1 °C/min. After cryopreservation, the cells were thawed in a water bath, the DMSO was removed, and their ability for basal and stimulated testosterone synthesis in vitro was assessed. Additionally, ICs were transplanted into castrated animals, and changes in free testosterone blood levels, seminal vesicle weight, and sexual behavior were examined.
Results. The capacity for stimulated testosterone synthesis was preserved only in cells cryopreserved in the solution containing dextran 40 (0.7DMSO + D40) and FBS (1.4DMSO + FBS). Cryopreserved ICs enhanced sexual behavior parameters in castrated rats upon transplantation without removing the cryoprotective medium (0.7DMSO + D40), including mount and intromission latency, the mount and intromission frequency, ejaculation ability, and copulatory efficiency. Moreover, they helped maintain free testosterone blood levels and seminal vesicle weight in castrated animals after transplantation.
Conclusions. It was demonstrated that ICs cryopreserved in the serum-free medium (0.7DMSO + D40) retained their ability to synthesize and secrete testosterone. Furthermore, the use of 0.7DMSO + D40 allows the immediate use of cells after thawing, bypassing the step of cryoprotectant removal, which could facilitate the translation of experimental protocols into practice.