Efficient Fabrication Of Human Corneal Stromal Cell Spheroids And Promoting Cell Stemness Based On 3D-Printed Derived Polydimethylsiloxaned Microwell Platform

To create a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities for generating human corneal stromal cell spheroids and further promote the plasticity and stemness of human corneal stromal cells (hCSCs).

Cell therapy was an effective treatment for cornea diseases. However, the main challenge is finding seed cells with good viability and regenerative ability. Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs), which is crucial in regenerative medicine, offering a new source of cells for treating corneal diseases. However, the efficient fabrication of human corneal stromal cell spheroids and the promotion of cell stemness remains a challenge.

Using a PDMS microwell platform through 3D printing technology, we successfully generated 3D corneal stromal cell spheroids (3D-CSC) with uniform size and stable structure, achieving a mean diameter of 230.38 ± 22.12 µm. These spheroids exhibited significantly increased expression of key pluripotency factors, including OCT4, NESTIN, NANOG, SOX2, KLF4, and PAX6. Furthermore, the iPS cell-conditioned medium significantly enhanced the stemness  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.