Generation of primary pluripotent spheroid culture from human corneas using human platelet lysates

Abstract

Purpose : Corneal stromal cells are important for corneal wound healing, angiogenesis, and nerve regeneration. One common method to generate pure keratocytes is via spheroid culturing requiring either scraping corneal cells or digestion of collagen. We developed a simple and effective method to obtain spheroid cells from human corneas using human platete lysate (hPL).

Methods : Human corneas (n=5) not suitable for grafting were obtained with ethics approval (HREC 14/275). Each cornea was dissected and cultured in a 35mm petri dish with growth medium (10% hPL in DMEM supplemented with penicillin-streptomycin). Spheroid cells generated were subject to continuous sub-culturing. The cells was subjected to CK3, CK12, keratocan and lumican immunostaining and confocal imaging. qPCR was performed to detect stem cell markers.

Results : Spheroid cells were observed after 1 week culturing from each donor. The medium gelatinized and formed a matrix for multidimensional growth. The spheroid cells increased over time, and a border line formed around the explants between week 2 to week 3 (Fig 1A). Spheroid cells were harvested and re-cultured in a new dish, continuously generating adhering cells and spheroid cells (p5 and still ongoing, Fig 1B & C). Immunostaining results on adhered cells generated from spheroid cells, showed expression of keratocan and lumican but not CK3 and CK12. qPCR results detected the expression of Nanog and OCT4 (Cq=35±0.8 and Cq=33±0.02 respectively). The continuous culturing of spheroid cells without passage resulted in generation of sheet like structure (Fig 1D). No spheroid cells observed when substituting hPL with FBS.

Conclusions : We demonstrated a method to generate spheroid cells from human cornea explants with 100% successful rate using hPL. The 3D matrix environment formed by 10% hPL is likely the key for spheroid cell generation. Spheroid cells showed pluripotent potential, produced keratocytes and possible stromal sheet, which could be important to study keratocyte regeneration, wound healing and possible stromal reconstruction.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Fig 1. Spheroid cells tissue culture. A: spheroid cells observed in explant culture. B: sub-culturing of spheroid cells. C: Generation of keratocytes. D: Formation of stromal sheet like structure.

 

Fig 2: Detection of keratocan and lumican in cell culture.