Amar K. Singh

Elastin-like recombinamers as substrates for retinal pigment epithelial cell growth

The aim of this study is to investigate the use of elastin-like recombinamers (ELRs) as a substrate that can maintain the growth, phenotype, and functional characteristics of retinal pigment epithelial (RPE) cells efficiently and as a suitable carrier for the transplantation of autologous RPE cells for treatment of age-related macular degeneration (AMD). ELR films containing a bioactive sequence, RGD (ELR-RGD), and one with no specific sequence (ELR-IK) as control, were obtained by solvent-casting onto glass and subsequent cross-linking. ARPE19 cells were seeded on sterilized ELR films as well as on the control surfaces. Cells were analysed after 4, 24, 72, and 120 h to study cell adhesion, proliferation, cell viability, morphology, and specificity by staining with Trypan blue, DAPI, Rhodamin-Phalloidin and RPE65, ZO-1 antibodies and observing under fluorescence as well as electron microscope. ARPE19 cells seeded on both ELR films and controls were 100% viable and maintained their morphology and set of characteristics at the different time points studied. Cell proliferation on ELR-RGD was significantly higher than that found on ELR-IK at all time points, although it was less than the growth rate on polystyrene. ARPE19 cells grow well on ELR-RGD maintaining their phenotype. These results should be extended to further studies with fresh human RPE cells and in vivo studies to determine whether this ELR-RGD matrix could be used as a Bruchs membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

Bioactive substrates for human retinal pigment epithelial cell growth from elastin‐like recombinamers

The aim of this study was to investigate the use of bioactive RGD-containing elastin-like recombinamers (ELR-RGDs) as a substrate that can maintain human retinal pigment epithelial cell (hRPE) phenotype and growth pattern. Results obtained are compared with previously published behavior of ARPE19 cells. The extension of these results to hRPE is required because ARPE19 cells cannot be used clinically to treat age-related macular degeneration. hRPE cells were isolated, cultured, seeded, and grown on surface of glass, treated polystyrene (TCP), and solvent-cast ELR-RGD and ELR-IK film with no specific sequence. Cells were analyzed to study cell adhesion, proliferation, morphology, and RPE65 protein expression by staining with diamidino-2-phenylindole, Rhodamine-Phalloidin, and anti-RPE65 antibody at 12, 24, 72, 120, 168, and 360 h. hRPE cells always grew better on ELR-RGD than on glass and ELR-IK but not on TCP. The kinetic hRPE growth curves confirmed that growth differences started to appear at 24 h for these surfaces in ascending order of cell growths, namely glass, ELR-IK, ELR-RGD, and TCP. There was a clear difference at 360 h. ELR-RGD maintained hRPE cells stable morphology and RPE65 protein expression. ELR-RGD seems to be a good substrate for growing hRPE cells with stable morphology and RPE65 protein expression. As such, this work confirms our hypothesis regarding ELR-RGD substrates viability, which can be used as a Bruchs membrane prosthesis for further studies in animals. However, these results must subsequently be extrapolated to use of hRPE cells in animals to evaluate them as a transplantation vehicle in human.

Flow cytometry assessment of the purity of human retinal pigment epithelial primary cell cultures

Culturing of human retinal pigment epithelial cells (hRPE) is the initial step in cell therapy of some retinal diseases. To transfer these cells into clinical use, it is necessary to guarantee that they are well differentiated and contamination free. Fluorescence microscopy is the easiest method to do this, but it is associated with operator subjectivity, and the results are highly variable. The aim of this study was to demonstrate the practicality of implementing flow cytometry (FC) analysis to determine the purity of human RPE primary cell cultures. An ARPE19 cell line, human skin fibroblasts, hRPE, and human corneal epithelial cells were analysed by FC to determine the percentage of the hRPE population expressing RPE65 and epithelial and fibroblast proteins. The cell viability and DNA content also were determined. FC analysis showed that the hRPE cells were healthy, stable, and expressed RPE65 protein in the study working conditions. The density of RPE65 protein expression decreased during passages 2 to 10, which was confirmed using a Western blot technique. However, the hRPE cells did not express the 112-kDa epithelial and fibroblast proteins in the current working conditions. These findings suggested that FC facilitates a detailed analysis of human RPE primary cell cultures, a necessary step in developing new cell therapies for retinal diseases.

Chitosan feasibility to retain retinal stem cell phenotype and slow proliferation for retinal transplantation

Retinal stem cells (RSCs) are promising in cell replacement strategies for retinal diseases. RSCs can migrate, differentiate, and integrate into retina. However, RSCs transplantation needs an adequate support; chitosan membrane (ChM) could be one, which can carry RSCs with high feasibility to support their integration into retina. RSCs were isolated, evaluated for phenotype, and subsequently grown on sterilized ChM and polystyrene surface for 8 hours, 1, 4, and 11 days for analysing cell adhesion, proliferation, viability, and phenotype. Isolated RSCs expressed GFAP, PKC, isolectin, recoverin, RPE65, PAX-6, cytokeratin 8/18, and nestin proteins. They adhered (28 ± 16%, 8 hours) and proliferated (40 ± 20 cells/field, day 1 and 244 ± 100 cells/field, day 4) significantly low (P < 0.05) on ChM. However, they maintained similar viability (>95%) and phenotype (cytokeratin 8/18, PAX6, and nestin proteins expression, day 11) on both surfaces (ChM and polystyrene). RSCs did not express alpha-SMA protein on both surfaces. RSCs express proteins belonging to epithelial, glial, and neural cells, confirming that they need further stimulus to reach a final destination of differentiation that could be provided in in vivo condition. ChM does not alternate RSCs behaviour and therefore can be used as a cell carrier so that slow proliferating RSCs can migrate and integrate into retina.