细胞外基质 (ECM) 衍生的水凝胶经常用于三维 (3D) 细胞培养和多种组织中的类器官形成。然而，在睾丸细胞的3D培养中，透明质酸（HA）水凝胶并没有受到那么多的关注。本研究考察了三种不同复合材料对小鼠的影响，包括 HA-海藻酸盐 (HA-Alg)、HA-海藻酸盐-胶原蛋白 (HA-Alg-Col) 和 HA-海藻酸盐-脱细胞 ECM (HA-Alg-dECM)睾丸细胞培养和体外精子发生。为了创建复合材料，所用生物材料的浓度为 0.5% HA、1% 海藻酸盐、2.5 mg/mL 胶原蛋白和 25 mg/mL dECM（源自公羊睾丸）。对产后 5 天 (dpp) 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、尺寸更大，HA-Alg 被选为优异的复合材料。然后，用 HA-Alg 重新进行细胞培养 14 天，随后又延长了 28 天。此外，在第 14 天，将 10 dpp 小鼠睾丸细胞的 3D 培养物与 5 dpp 小鼠睾丸细胞进行比较。使用适当的技术分析形态和基因表达。在第 14 天，HA-Alg 水凝胶显示出明显更多的类器官。大小和数量均高于其他两组 (p < 0.05)；然而，没有一个组显示出预期的睾丸类器官迹象。值得注意的是，在第 14 天，组织学和免疫染色测试揭示了肝细胞样细胞 (HLC) 的特征以及作为 HLC 功能标志的白蛋白产生。此外，基因表达分析验证了血管生成标记物的显着表达（p < 0.01）。延长培养至28天后，5 dpp的睾丸细胞再次分化为红细胞和HLC，而少数类器官则表现出肾细胞的特征。与 5 dpp 小鼠相比，10 dpp 小鼠细胞培养 14 天显示出广泛的细胞谱系，包括肾脏、腺体、软骨细胞和肝细胞样细胞。虽然 HA-Alg 复合材料不支持 3D 精子发生通过对小鼠睾丸细胞进行培养，发现它具有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不可预测的潜力。© 2024 作者。 《细胞生物学》由 Wiley‐VCH GmbH 代表法国显微镜学会和法国细胞生物学学会出版。

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.© 2024 The Author(s). Biology of the Cell published by Wiley‐VCH GmbH on behalf of Société Française des Microscopies and Société Biologie Cellulaire de France.