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Brittni Scruggs, Budd A Tucker, Chunhua Jiao, Janet Riley, Edwin M Stone, Robert F Mullins, Elliott H Sohn; Dissociation protocol and optimal conditions for clinical-grade induced pluripotent stem cell-derived retinal precursor cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3736.
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© ARVO (1962-2015); The Authors (2016-present)
There is no defined dissociation protocol for GMP-grade induced pluripotent stem cell-derived (iPSC) retinal precursor cells (RPCs), and there are few data defining the effects of retinal cell transplantation conditions on cell viability. We tested the hypotheses that human iPSC-derived RPCs could be isolated after dissociation from neurospheres and that cell viability could be improved by optimizing the temperature, storage conditions, and injection conditions.
Skin fibroblasts were obtained from patients with known retinal disease to generate iPSCs, which were then grown to produce eyecup-like structures. These neurospheres at early (60 days), intermediate (90 days), and later (>150 days) stages were dissociated in the presence of either dispase or papain solutions. The dissociation protocol was optimized for cell yield, and isolated cells were evaluated using immunocytochemical analyses to detect cell surface markers. RPCs were injected through needle cannulas of different gauges (31G vs. 41G) using different storage temperatures (room temp vs. 0 C) after varying lengths of storage time (10 min vs. 2 hr). Cell viabilities were determined using a tetrazolium (MTS) assay and an automatic cell counter. All conditions were tested in three independent experiments in triplicate using cells isolated from five patients. Statistical analysis of three or more groups were performed using one-way ANOVA with post-hoc testing.
RPCs were isolated from iPSC-derived neurospheres using various dissociation conditions; the papain solution at 37 C yielded the highest cell count and viability when compared to the conditions using dispase. Data suggest that the neurospheres and isolated cells stain positively for multiple retinal cell surface markers (e.g., PAX6, MITF). There was a significant decrease in cell viability when dissociated iPSC-derived RPCs were injected in vitro using the 41G cannula compared to no cannula or to the 31G cannula. Specifically, the MTS assay showed that 79.33+/-6.52% of cells survived the 31G injection compared to 53.32+/-3.12% of cells using the 41G cannula (p<0.01). Our data also suggest that neither temperature nor transport time affect the RPC viability.
RPCs can be isolated from iPSC-derived neurospheres, and the viability of these cells is improved with a larger sized needle cannula.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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