Three D-Printed Pdms Microwell Platform Promotes The Stemness Of Human Keratocyte Spheroids From Smile-Derived Lenticules
Published 2024 - 42nd Congress of the ESCRS
Reference: FP19.08 | Type: Free paper | DOI: 10.82333/p7v8-wb67
Authors: Yuexi Chen* 1 , Jianing Gu 2 , Zekai Cui 2 , Xihao Sun 2 , Yuqin Liang 2 , Chunwen Duan 2 , Xiaoxue Li 2 , Jiansu Chen 3 , Zheng Wang 4
1Aier Eye Institute,Changsha,China;Refractive,Guangzhou Aier Eye hospital,GUANZHOU,China, 2Aier Eye Institute,Changsha,China, 3Refractive,Aier Eye Institute,Changsha,China, 4Refractive,Guangzhou Aier Eye Hospital,GUANZHOU,China
Purpose
To develop an effective approach for the physical reprogramming of human corneal stromal cells (hCSCs, also called keratocytes) derived from SMILE lenticules into stem-like cells without using transcription factors. Then, further, prove that these cells can be good seed cells for tissue engineering corneal construction and cell therapy.
Setting
Human keratocytes could be reprogrammed into induced pluripotent stem cells based on transcription factors. Besides, we found that adherent 3D spheroid culture keratocytes on PDMS displayed increased expression of several stemness genes, and the 3D-printed derived PDMS microwell platform improves the robustness of in vitro retinal organoid cultures. In this study, we first apply this platform to obtain Human healthy corneal stromal cells with high stem cell characteristic expression.
Methods
Primary hCSCs were isolated from SMILE-derived lenticules and cultured in a low-serum RIFA medium with soluble human corneal stromal extract (hCSE). A novel platform with 62 V-bottom micro-cavities and essential 8 (E8) medium was used to generate adherent 3D keratocyte spheroids in the polydimethylsiloxane (PDMS) microwell. The reprogramming results were compared between 3D spheroids with 2D cultures via qPCR and Immunofluorescence (IF) staining.
Results
The hCSCs were successfully isolated from SMILE-derived lenticules, exhibiting dendritic or stellate shapes. IF analysis showed that these cells strongly expressed keratocyte markers and were weakly stained with (myo)fibroblast markers after subculturing in RIFA + hCSE. We successfully generated efficient and homogeneous 3D spheroids in the PDMS microwell. The average diameter of 3D spheroids is 433.40 ± 68.66 µm. qPCR analysis showed that the expression of stem cell marker genes (SOX2, PAX6, OCT4, NANOG, NGFR, and NESTIN) was significantly greater in 3D spheroids than in 2D cells. Furthermore, IF staining revealed that KLF4, NANOG, SOX2, and PAX6 were positively expressed in adherent 3D spheroids but negatively in 2D cells.
Conclusions
Our results indicated that the physiological cells and culture conditions enhance reprogramming. Therefore, the adherent 3D spheroid culture of keratocytes using the PDMS microwell platform is a promising and safe strategy to promote reprogramming and enhance stemness, suggesting its potential application for developing implants in tissue engineering and regenerative medicine.