Application of photocrosslinked gelatin, alginate and dextran hydrogels in the in vitro culture of testicular tissue

Testicular tissue culture in vitro is considered an important tool for the study of spermatogenesis and the treatment of male infertility. Although agarose hydrogel is commonly used in testicular tissue culture, the efficiency of spermatogenesis in vitro is limited. In this study, testicular tissues from adult mice were cultured using a gas-liquid interphase method based on agarose (Agarose), gelatin methacryloyl (GelMA), alginate methacryloyl (AlgMA), dextran methacryloyl (DexMA), and mixture GelMA-Agarose, AlgMA-Agarose, and DexMA-Agarose hydrogels, respectively, for 32 days in vitro. The integrity of the seminiferous tubules, the density and proportions of spermatogonia, spermatocytes, Sertoli cells, and testosterone concentrations were quantified and compared between groups. Properties of different hydrogels including compression modulus, Fourier Infrared Spectroscopy (FITR) spectra, pore size, water absorption, and water retention were tested to investigate how biochemical and physical properties of hydrogels affect the results of testicular tissue culture. The results indicate that testicular tissues cultured on AlgMA exhibited the highest seminiferous tubule integrity rate (0.835 ± 0.021), the presence of a high density of spermatocytes (2107.627 ± 232.082/mm2), and a high proportion of SOX9-positive well-preserved seminiferous tubules (0.473 ± 0.047) compared to all remaining experimental groups on day 32. This may be due to the high water content of AlgMA reducing the toxic effect of oxygen on testicular tissue. In the later period of culture, testicular tissues cultured on DexMA, not DexMA-Agarose, produced significantly more testosterone (18.093 ± 3.302 ng/mL) than the other groups, suggesting that DexMA is friendly to Leydig cells. Our study provides a new idea for the optimization of the gas-liquid interphase method for achieving in vitro spermatogenesis, facilitating the future achievement of efficient in vitro spermatogenesis in more species, including humans.