Chen Yuexi, Gu Jianing, Cui Zekai, Sun Xihao, Liang Yuqin, Duan Chunwen, Li Xiaoxue, Su Zhanyu, Zhang Bo, Chen Jiansu, Wang Zheng
The First Clinical Medical College, Jinan University, Guangzhou 510632, China.
Guangzhou Aier Eye Institute, Guangzhou 510071, China.
Biomolecules. 2025 Mar 19;15(3):438. doi: 10.3390/biom15030438.
Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs). Here, we introduce a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities to generate human corneal stromal cell spheroids and promote cell stemness. We isolated hCSCs from SMILE-derived lenticules and maintained their physiological phenotype by culturing them in a medium supplemented with human corneal stromal extract (hCSE). Utilizing a PDMS microwell platform fabricated through 3D printing technology, we successfully generated 3D corneal stromal cell spheroids (3D-CSC) with uniform size and stable structure, exhibiting increased expression of pluripotency factors, including OCT4, NANOG, SOX2, KLF4, and PAX6. Furthermore, the iPS supernatant of E8-conditioned medium (E8-CM) significantly enhanced the stemness properties of these cells. RNA sequencing and proteomics analyses revealed that 3D-CSCs exhibited superior proliferation, differentiation, cell adhesion, migration, and neurogenesis compared to traditional monolayer cultures, underscoring the role of biophysical cues in promoting hCSCs stemness. In summary, this study presents an effective 3D cell culture platform that mimics the in vivo microenvironment, facilitating the enhancement of stemness properties and providing valuable insights into corneal tissue engineering and regenerative medicine, particularly for treating corneal opacities and diseases.
球形培养可促进人角膜基质细胞（hCSCs）的可塑性和干性。在此，我们介绍一种基于聚二甲基硅氧烷（PDMS）微孔平台的新型三维（3D）细胞培养系统，该平台由许多V型底部微腔组成，用于生成人角膜基质细胞球体并促进细胞干性。我们从飞秒激光小切口基质透镜切除术（SMILE）获得的透镜中分离出hCSCs，并通过在补充有人角膜基质提取物（hCSE）的培养基中培养来维持其生理表型。利用通过3D打印技术制造的PDMS微孔平台，我们成功生成了尺寸均匀、结构稳定的3D角膜基质细胞球体（3D-CSC），其多能性因子OCT4、NANOG、SOX2、KLF4和PAX6的表达增加。此外，E8条件培养基（E8-CM）的诱导多能干细胞（iPS）上清液显著增强了这些细胞的干性特性。RNA测序和蛋白质组学分析表明，与传统单层培养相比，3D-CSCs在增殖、分化、细胞黏附、迁移和神经发生方面表现更优，突出了生物物理线索在促进hCSCs干性中的作用。总之，本研究提出了一种有效的3D细胞培养平台，该平台模拟体内微环境，有助于增强干性特性，并为角膜组织工程和再生医学，特别是治疗角膜混浊和疾病提供了有价值的见解。
