Retinal pigment epithelium cell compositions
a technology of pigment epithelium and composition, which is applied in the field of red pigment epithelium cell composition, can solve the problems of limiting the widespread use of red pigment donor sources, affecting the viability and quality of red pigment cells, and affecting the practicality of clinical us
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example 1
[0259]Used herein are cell suspensions of RPE cells, derived from human embryonic stem cells (hESCs) through a process of directed differentiation under xeno-free, GMP manufacturing conditions. These cells were expanded on irradiated human umbilical cord fibroblast feeders (hUCFs). The expanded hESCs were then differentiated into retinal pigment epithelium (RPE) cells using Nicotinamide and Activin A. The RPE cells were then expanded and cryopreserved in cryopreservation medium.
[0260]Xeno-free GMP grade HAD-C 102 hESCs were expanded as colonies on irradiated xeno-free GMP-grade CRD008 hUCFs that were seeded on recombinant human vitronectin (rhVTN) or on recombinant human Gelatin (rhGelatin). hESC expansion was carried out in the presence of NUTRISTEM® medium that contains human serum albumin in addition to the growth factors basic FGF and TGF beta (Biological Industries). Expanded hESCs were then transferred to a suspension culture to initiate differentiation in a directed manner un...
example 2
[0262]Post thaw vitality and viability were assessed for therapeutic RPE cells cryopreserved in cryopreservation media with 5% dimethyl sulfoxide (DMSO) (Media 2, CS5) at cell densities of 1.5×106 and 5×106. Results were compared to the results of cells that were cryopreserved in 90% human serum (HS) with 10% DMSO using a controlled freezing machine (e.g., an isopropanol containing slow cooling apparatus). After thawing of 3 vials of each composition frozen in each cryopreservation media, viability was tested using a cell counter. Cells of each vial were then seeded in a 12-well plate, at a density of 0.5×106 viable cells / well in a final volume of 2 mL DMEM containing 20% human serum per well, for 24 hours at 37° C. and 5% CO2. At the end of the incubation period, cultures were washed with PBS. Following TrypLE Select treatment, cells were enumerated using a cell counter. Percent vitality was then calculated by dividing the average number of viable adhered cells with the total numbe...
example 3
[0264]Therapeutic RPE cell compositions were formulated using xeno-free GMP-grade reagents, xeno-free GMP-grade cells (HAD-C 102-hESCs grown on irradiated CRD008), as described in Example 1.
[0265]Assessment of CRALBP+PMEL17+ cells for measurement of RPE purity was performed at the end of the differentiation phase. As shown in Table 1a and Table 1 b, purity of RPE cells was at least 98.76% or greater for all RTA RPE cell therapy compositions formulated with CS2 (Media 1) or CS5 (Media 2).
[0266]Tight junctions generated between RPE cells enable the generation of the blood-retinal barrier and a polarized PEDF and VEGF secretion. PEDF is secreted to the apical side where it acts as an anti-angiogenic and neurotropic growth factor. VEGF is mainly secreted to the basal side, where it acts as a proangiogenic growth factor on the choroidal endothelium. RPE polarization (barrier function and polarized PEDF and VEGF secretion) was measured in a transwell system in cells at the end of the prod...
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