Document Type : Original Article
Authors
1
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
2
Department of Anatomical Sciences, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran. 3. Reproductive Health Research Center, Clinical Research Institute, Urmia University of Medical Sciences, Urmia, Iran.
3
Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
4
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
5
2. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. 6. Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
10.22074/ijfs.2024.2018868.1599
Abstract
Objective: The utilization of decellularized extracellular matrix (dECM) derived from animal testis tissue has demonstrated potential as a component of tissue-specific scaffolds. Current research is mostly centered around dECM as a natural resource for culturing testicular cells. This study aimed to assess firstly the comparison of Voytik-Harbin (VH) and Frytes protocol in creating Ram’s dECM testis hydrogel and secondly the evaluation of the best protocol effect on in vitro spermatogenesis.
Method: In this experimental study, the six testes of mature rams were decellularized and the hydrogel production was performed by 1) The Frytes protocol utilized a concentration of 1 mg/mL of pepsin, dissolved in either 0.1 or 0.01M HCl, and 2) the Voytik-Harbin protocol was involved 10 mg of pepsin per 100 mg of ECM in 0.5 M of acetic acid. Subsequently, mouse testicular cells were cultivated on collagen hydrogel as the control and the more effective testicular-derived hydrogel (TDH) to evaluate the early stages of in vitro spermatogenesis.
Results: While the Freytes protocol produced a homogeneous pre-gel solution with both HCl concentrations; elevating the pH to 7.4 loosened the hydrogel and made gelation problematic. In contrast, the VH protocol solidified the hydrogel and produced a strong hydrogel due to its gelation consistency. Furthermore, the prepared hydrogel by VH with 25 mg of dECM had a significantly higher priority in terms of rheology and structure (p<0.05). Following mouse testicular cell culture, TDH and collagen hydrogel did not differ significantly in terms of cell survival rates and the mRNA expression of early spermatogenesis genes.
Conclusion: Using the Voytik-Harbin protocol for producing ram TDH resulted in a firm hydrogel with a high frequency of repeat, which may be suited for testicular cell growth.
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