Department of Reproductive Biology, Faculty of Basic Sciences and Advanced Medical Technologies, Royan Institute, ACECR, Tehran, Iran.
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Biol Cell. 2024 Oct;116(10):e2400049. doi: 10.1111/boc.202400049. Epub 2024 Aug 23.
Extracellular matrix (ECM)-derived hydrogels are frequently used in three-dimensional (3D) cell culture and organoid formation in several tissues. However, in the 3D cultivation of testicular cells, the hyaluronic acid (HA) hydrogel has not received as much attention. This study examined the effects of three distinct composites, including HA-alginate (HA-Alg), HA-alginate-collagen (HA-Alg-Col), and HA-alginate-decellularized ECM (HA-Alg-dECM), on mouse testicular cell culture and in vitro spermatogenesis.
For the creation of composites, the concentration of biomaterials used was 0.5% HA, 1% alginate, 2.5 mg/mL collagen, and 25 mg/mL dECM derived from the testicles of Rams. After 3D culture of 5 days post-partum (dpp) mouse testicular cells for 14 days, HA-Alg was selected as a superior composite due to the greater number and size of the produced organoids. Then, cell culture was rerun by HA-Alg for 14 days, which was later extended for an additional 28 days. In addition, the 3D culture of 10 dpp mouse testicular cells was used to compare with 5 dpp mice on day 14. The morphology and gene expression were analyzed using appropriate techniques.
On day 14, the HA-Alg hydrogel showed significantly more organoids in terms of size and number than the other two groups (p < 0.05); nevertheless, none of the groups showed the expected signs of testis organoids. Remarkably, on day 14, the histology and immunostaining tests revealed features of hepatocyte-like cells (HLCs) and albumin production as a marker of HLC functionality. Furthermore, the analysis of gene expression verified the significant expression of angiogenesis markers (p < 0.01). After the extended culture to 28 days, 5 dpp testicular cells once more differentiated into erythrocytes and HLCs, while a small number of organoids showed the characteristic of renal cells. Cell culture of 10 dpp mice for 14 days showed a wide range of cell lineages, including renal, glandular, chondrocyte, and hepatocyte-like cells in comparison to the 5 dpp mice.
While the HA-Alg composite did not support spermatogenesis in the 3D culture of mouse testicular cells, it demonstrated an unpredicted potential for promoting the differentiation of neonate mouse testicular cells into HLC, erythrocytes, and other cell lineages.
细胞外基质（ECM）衍生的水凝胶常用于几种组织的三维（3D）细胞培养和类器官形成。然而，在睾丸细胞的 3D 培养中，透明质酸（HA）水凝胶并没有受到太多关注。本研究探讨了三种不同复合材料，包括透明质酸-藻酸盐（HA-Alg）、透明质酸-藻酸盐-胶原蛋白（HA-Alg-Col）和透明质酸-脱细胞 ECM（HA-Alg-dECM），对小鼠睾丸细胞培养和体外精子发生的影响。
为了制备复合材料，使用的生物材料浓度为 0.5%HA、1%藻酸盐、2.5mg/mL 胶原蛋白和 25mg/mL 源自公羊睾丸的脱细胞 ECM。在产后 5 天（5 dpp）的小鼠睾丸细胞进行 3D 培养 14 天后，由于产生的类器官数量更多且更大，因此选择 HA-Alg 作为更好的复合材料。然后，HA-Alg 再进行 14 天的细胞培养，随后又延长了 28 天。此外，还比较了 10 dpp 小鼠睾丸细胞的 3D 培养与 14 天的 5 dpp 小鼠。使用适当的技术分析形态和基因表达。
第 14 天，HA-Alg 水凝胶在大小和数量方面表现出明显更多的类器官，与其他两组相比具有统计学意义（p<0.05）；然而，没有一组显示出预期的睾丸类器官特征。值得注意的是，第 14 天，组织学和免疫染色测试显示出肝细胞样细胞（HLC）和白蛋白产生的特征，白蛋白是 HLC 功能的标志物。此外，基因表达分析证实了血管生成标志物的显著表达（p<0.01）。延长培养至 28 天后，5 dpp 睾丸细胞再次分化为红细胞和 HLC，而一小部分类器官表现出肾细胞的特征。与 5 dpp 小鼠相比，10 dpp 小鼠的细胞培养 14 天显示出广泛的细胞谱系，包括肾、腺、软骨细胞和肝细胞样细胞。
虽然 HA-Alg 复合材料在小鼠睾丸细胞的 3D 培养中不支持精子发生，但它表现出促进新生小鼠睾丸细胞分化为 HLC、红细胞和其他细胞谱系的意外潜力。
