Abstract
Purpose :
The limited availability of donor tissues for transplantation requires novel approaches for ex vivo cornea production. We utilized ‘smart’ cell culture carriers for the cultivation, gentle harvest and transplantation of human corneal endothelial cell (HCEC) layers to create in vitro produced HCEC sheets. These sheets can then be used to bring us one step closer to overcoming the shortage of donor corneas.
Methods :
In this study, we combine the advantages of a biohybrid material with an enzymatically degradable peptide material concept in order to create a new biorthogonal approach for ex vivo tissue regeneration. The results presented in this study were gained with the established cell line HCEC-B4G12, which exhibits a uniform morphology similar to primary cells. Briefly, peptide functionalized, fully synthetic culture carriers were seeded with HCECs and cultured until the formation of a functional tissue. The culture carrier was subsequently enzymatically degraded to release free-floating cell sheets, which were then transferred onto a de-endothelialized porcine cornea.
Results :
Release of the cultured HCEC layers show the suitability of enzymatically-degradable hydrogels for HCEC sheet generation. The cells cultured on the hydrogels exhibit a regular morphology and appropriate metabolic activity. Cells were positive for expression of function-associated marker proteins ZO-1, Na+/K+-ATPase, and the extracellular matrix proteins fibronectin, laminin and collagen IV. Upon application of the enzyme for the specific cleavage of the culture carrier, the HCEC layers gently detached as complete cell sheets with a diameter of approximately 1 cm. Neither the cell to cell contacts, the cell-matrix contacts nor the cell-surface proteins were impaired. The detached, floating HCEC sheets were stabilized by macroporous biohybrid hydrogels and transplanted onto porcine corneas in vitro. This illustrates the effective stabilization of the released, fragile tissue as well as meeting the geometrical challenges of the concave target tissue.
Conclusions :
This proposed tissue engineering technique has allowed, for the first time, the formation of physiologically relevant size human cornea endothelial lamella tissue suitable for surgical implantation in descemet membrane endothelial keratoplasty (DMEK). We envision, that with slight changes, this technique could be applied as a regenerative therapy for other ophthalmologic tissues.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.