Abstract
Purpose :
Created human corneal endothelial cell (HCEC) layers or sheets aimed for the revaluation of a degenerated corneal endothelium, but mostly failed in the clinical validation due to poor mechanical stability. The immature Descemet membrane in bioengineered grafts is the main obstacle to their clinical transition. Human corneal endothelial cells have poor extracellular deposition properties in vitro. The creation of an artificial Descemet membrane could resolve this issue, but a high collagen and laminin content, as well as 16±3 microns thickness, require advanced bioengineering techniques.
Methods :
We utilized peptide-functionalized, fully synthetic “smart” cell culture carriers to culture primary human MSC, which have high ECM deposition properties. Macromolecular crowding with Ficoll was used to enhance ECM secretion. Cells were cultured for ten days and removed by the diluted ammonium hydroxide solution. The formed membrane was reseeded with various cell types to validate cell adhesion and proliferation. After the confluent cell layer formation, the culture carrier was subsequently enzymatically degraded to release free-floating bioengineered Descemet membrane with attached cell layer, which was validated with various DMEK cannulas by mimicking in lab transplantation experiments
Results :
The microscopic and cell staining analyses revealed that cell to cell and cell-to-matrix contacts were impaired. We utilized peptide-functionalized, fully synthetic “smart” cell culture carriers to culture primary human MSCs, which have high ECM deposition properties additionally enhanced with Macromolecular crowding. Cells were cultured for 10 days and removed by ammonium hydroxide solution. The formed bioengineered Descemet membrane was reseeded with cell types to validate cell adhesion and proliferation. After the confluent cell layer formation, the culture carrier was subsequently enzymatically degraded to release free-floating bioengineered Descemet membrane with attached cell layer, which was validated with various cannulas by mimicking in lab transplantation experiments has not impacted the implant integrity and cell viability
Conclusions :
This proposed bioengineering technique has allowed, for the first time, the formation of endothelial lamella tissue with a bioengineered Descemet membrane which is shown to stabilize surgical implantation in model Descemet membrane endothelial keratoplasty DMEK experiments.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.