Here, we introduce a novel three-dimensional (3D) cell culture system based on a pol-ydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities for the generation of human corneal stromal spheroids. 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 prolif-eration, differentiation, cell adhesion, migration, and neurogenesis compared to tradi-tional monolayer cultures, underscoring the role of biophysical cues in promoting hCSCs stemness.