In vitro expansion of corneal endothelial cells on biomimetic substrates

Abstract

Corneal endothelial (CE) cells do not divide in vivo, leading to edema, corneal clouding and vision loss when the density drops below a critical level. The endothelium can be replaced by transplanting allogeneic tissue; however, access to donated tissue is limited worldwide resulting in critical need for new sources of corneal grafts. In vitro expansion of CE cells is a potential solution, but is challenging due to limited proliferation and loss of phenotype in vitro via endothelial to mesenchymal transformation (EMT) and senescence. We hypothesized that a bioengineered substrate recapitulating chemo-mechanical properties of Descemet's membrane would improve the in vitro expansion of CE cells while maintaining phenotype. Results show that bovine CE cells cultured on a polydimethylsiloxane surface with elastic modulus of 50 kPa and collagen IV coating achieved >3000-fold expansion. Cells grew in higher-density monolayers with polygonal morphology and ZO-1 localization at cell-cell junctions in contrast to control cells on polystyrene that lost these phenotypic markers coupled with increased α-smooth muscle actin expression and fibronectin fibril assembly. In total, these results demonstrate that a biomimetic substrate presenting native basement membrane ECM proteins and mechanical environment may be a key element in bioengineering functional CE layers for potential therapeutic applications.

Figure 1. CE cells lose differentiated morphology when cultured on glass or tissue culture polystyrene.

(a) Example of ex vivo, intact endothelium from the bovine cornea where cells exhibit a polygonal, mostly hexagonal shape and small size with the tight junction protein ZO-1 (red) present at cell borders and F-actin (green) located cortically. (b) CE cells cultured for one passage on a rigid glass substrate have reduced ZO-1 localization at the cell-cell borders and F-actin fibers present cortically as well as throughout the cell body. (c) CE cells cultured on TCPS for one passage appear similar to CE cells on glass, showing a loss of phenotypic shape and ZO-1 and F-actin localization relative to the ex vivo tissue. Cells are stained for nuclei (blue), tight junction protein ZO-1 (red) and F-actin (green) and scale bars are 50 μm.

Figure 2. Screening the response of CE cells to variable substrate elastic modulus and ECM protein coating.

CE cells were cultured on one of 36 different PDMS substrates with elastic modulus of 5, 50, 130, 830, 1340 or 1720 kPa and uncoated or coated with the ECM proteins fibronectin (FN), collagen type I (COLI), laminin (LAM), collagen type IV (COL4) or LAM and COL4. Representative fluorescent images show CE cells stained for nuclei (blue), ZO-1 (red) and F-actin (green), demonstrating distinct differences in morphology based on the substrates properties. In general, CE cells appeared to have a more polygonal morphology, continuous ZO-1 at the cell-cell border and cortical F-actin on substrates with an elastic modulus of 50 kPa for most of the ECM proteins. Similar results were observed for the COL4 coating for most of the elastic modulus formulations. Thus, the combination of elastic modulus and ECM protein that gave the best results in terms of the CE cell morphology most closely resembling that observed in vivo was an elastic modulus of 50 kPa and COL4 coating, which was selected for further cell expansion studies.

Figure 3. CE cells cultured on the biomimetic PDMS_50+COL4 substrate maintained a polygonal morphology, higher cell density, increased proliferation rate and smaller cell size.

(a) Representative phase contrast images showing the morphology of CE cells cultured on the three different substrates at four different passages. At P5, CE cells cultured on TCPS_COL4 and TCPS exhibited elongated, irregular cell morphology. By P8 the cells were enlarged and polarized with no resemblance to a hexagonal morphology. In contrast, CE cells cultured on PDMS_50+COL4 maintained a hexagonal like morphology up to P8. (b) Normalized cell density for CE cells cultured on TCPS (n = 4), TCPS_COL4 (n = 5) and PDMS_50+COL4 (n = 5) (mean ± s.d.). At all passages the density of CE cells on the PDMS_50+COL4 was significantly greater than on the TCPS and than on the TCPS_COL4 from P4 to P10. (c) Number of CE cells on the different substrates as a function of time, where each data point represents cell number prior to passaging (up to P5 for TCPS and TCPS_COL4 and P8 for PDMS_50+COL4, when CE morphology became non-polygonal). There was a >3000-fold increase in the total cell number on PDMS₅₀₊COL4 compared to approximately a 140-fold increase on TCPS and TCPS_COL4 (dashed lines are to guide the eye). (d) Cell area in the ex vivo endothelium and on TCPS, TCPS_COL4 and PDMS_50+COL4 at P1, P5 and P8 (mean ± s.e.m.). (*) indicating a statistically significant difference (P < 0.05) between PDMS_50+COL4 and TCPS and (#) indicating a statistically significant difference (P < 0.05) between PDMS_50+COL4 and both TCPS and TCPS_COL4.

Figure 4. CE cells cultured on PDMS_50+COL4 maintained phenotypic ZO-1 staining and hexagonal shape while inhibiting fibronectin fibril formation and α-SMA expression.

(a) Representative images of CE cells on TCPS, TCPS_COL4 and PDMS_50+COL4 stained for nuclei (blue) and ZO-1 (red) to show cell morphology and separate images stained for fibronectin (green) to show fibril assembly at the cell-substrate interface at P1 and P5. CE cells on PDMS_50+COL4 had more continuous ZO-1 staining at the cell-cell border and decreased fibronectin fibril formation relative to cell on TCPS and TCPS_COL4. (b) Representative images of CE cells on TCPS, TCPS_COL4 and PDMS_50+COL4 stained for nuclei (blue), F-actin (red) and α-SMA (green) to show cells that have undergone EMT to a fibroblast-like phenotype at P1 and P5. (c) Percentage of α-SMA positive cells at P1 and P5 showing the increase in EMT on TCPS and TCPS_COL4 relative to PDMS_50+COL4 (mean ± s.d., * indicates statistically significant difference relative to PDMS_50+COL4, P < 0.05). (d) Hexagon shape factor (HSF) of CE cells in ex vivo, intact endothelium and at P1and P5 TCPS, TCPS_COL4 and PDMS_50+COL4 (mean ± s.e.m., * indicates TCPS_COL4 had a statistically significant difference to TCPS and PDMS_50+COL4, P < 0.05, and # indicates PDMS_50+COL4 had a statistically significant difference to TCPS and TCPS_COL4, P < 0.05).

Figure 5. CE cells expanded on PDMS_50+COL4 formed a higher density, tissue engineered endothelium.

(a) Representative phase contrast images and fluorescent confocal images of CE cells expanded on TCPS, TCPS_COL4 and PDMS_50+COL4 until P5 and then seeded onto collagen type I gels to form a tissue engineered endothelium. CE cells expanded on PDMS_50+COL4 formed a complete monolayer more rapidly and had a polygonal morphology by 24 hours. At 48 hours CE cells stained for nuclei (blue), ZO-1 (red) and F-actin (green) appeared to have more continuous ZO-1 staining at the cell-cell border when expanded first on PDMS_50+COL4. (b) Quantification of cell density in the tissue engineered endothelium showed that CE cells expanded on PDMS_50+COL4 formed higher density monolayers (mean ± s.d., * indicates PDMS_50+COL4 had a statistically significant difference compared to TCPS and TCPS_COL4, P < 0.05).