Abstract：
Mimicking the microenvironment of seminiferous tubules plays an indispensable role in directing differentiation of stem cells toward germ cells in vitro. In this work, we fabricated electrospun gelatin (EG) mats (i.e., with diameter <500 nm) conditioned with Sertoli cells\' extracellular matrix (ECM) to simulate both 3D structures and composition of normal testis tissue. Sertoli cells were isolated from mice testis and represented through immunocytochemistry (ICC) staining for expression of vimentin, a specific marker of Sertoli cells. The morphological characteristics of ECM-coated scaffold were investigated under scanning electron microscope (SEM). The efficient elimination of cells was confirmed by MTT assay. Furthermore, the cyto/biocompatibility of ECM-conditioned EG scaffold was determined for Sertoli cells and embryonic stem cells (ESCs), alone and as in co-culture. According to the results, the designed scaffold provided a mat for cell proliferation with negligible toxicity (almost 100% cell viability). SEM micrographs displayed cells with elongated shape and complete stretching morphology when compared with those cultured on scaffold without ECM. Moreover, an enhanced differentiation of ESCs toward sperm-generating cells was obtained through co-culturing of Sertoli cells and ESCs, where cell viability was found almost 100%. Our findings introduce the ECM-conditioned EG scaffold as a potentially influential engineered substrate for in vitro guidance of stem cells differentiation by mimicking the native microenvironment.
摘要：
模拟生精小管的微环境在体外指导干细胞向生殖细胞分化中起着不可或缺的作用。在这项工作中,我们制造了电纺明胶(EG)垫(即，直径<500nm）用支持细胞细胞外基质（ECM）调节，以模拟正常睾丸组织的3D结构和组成。从小鼠睾丸中分离睾丸支持细胞，并通过免疫细胞化学（ICC）染色表达波形蛋白，支持细胞的特异性标记。在扫描电子显微镜下研究了ECM涂层支架的形态特征。通过MTT实验证实了细胞的有效消除。此外，对支持细胞和胚胎干细胞（ESCs）的ECM条件EG支架的细胞/生物相容性进行了测定，单独和共同文化。根据结果,设计的支架提供了细胞增殖的垫，毒性可忽略不计（几乎100％的细胞活力）。与在没有ECM的支架上培养的细胞相比，SEM显微照片显示细胞具有细长的形状和完全拉伸的形态。此外，通过共培养支持细胞和ESCs获得ESCs向精子生成细胞的增强分化，其中发现细胞活力几乎100%。我们的发现引入了ECM调节的EG支架，作为一种潜在有影响力的工程基质，用于通过模拟天然微环境来指导干细胞分化。
