Abstract
Purpose :
Previously we have shown that human retinal ganglion cells (hRGC) can be derived from human embryonic stem cells using organoid approach. These neurons survive and grow neurites following subretinal transplantation in mice. The translation of cell therapy relies on the opportunity to cryopreserve large numbers of cells to make them accessible for pre-clinical/clinical studies and further application. There are no gentle protocols available for banking of organoid-derived RGCs.
Methods :
Here we selected 7 commercially available DMSO-free cryopreservation agents (CPAs) [Bambanker(BB), ReproCryo, FREEZEstem, Stem-cellbanker(SC), CryoSoFree, CryoScarless, and pZerve) and tested if they can maintain the viability of human stem-cell-derived RGCs and astroglia cells after freeze-thaw. Two DMSO-free (SC and BB) and one DMSO-based [Cellbanker-1(CB)] formulations were used for further investigation. Cell viability, cell recovery, proportion of cells with neurites and neurite length after re-plating in vitro was recorded 48 hours post-thaw. Immediate transplantation of RGCs after thawing was conducted with histological assessment at 3 days for cell quantification and neurite analysis. Non-frozen hRGCs (RGC media) were used as a positive control in all experiments.
Results :
Relative to RGC media, the cell count per well was higher in DMSO-based CPA compared to DMSO-free CPA: 147% (CB), 73% (SC), and 65% (BB). Post-thaw RGC viability, however, was comparable across all three CPAs: 54% (BB), 41% (CB), 39% (SC) and 70% (RGC media). Furthermore, a comparable proportion of RGCs recovered their ability to grow neurites when cryopreserved using DMSO-free CPAs or DMSO-based CPA: 80% (SC), 66% (BB), 73% (CB) and 62% (RGC media). Neurite length at 48 hours post-thaw was comparable among all groups:161 μm (SC), 134 μm (BB), 181 μm (CB), and 146 μm (RGC media). Lastly, the xenotransplantation studies highlighted that cryopreserved RGCs survive and grow neurites: RGC media (100% engraftment rate; 64% neurite growth rate), both CB and BB (67% engraftment rate; 33% neurite growth rate), and SC (33% engraftment rate; 0% neurite growth rate).
Conclusions :
Early results signify that DMSO-based and DMSO-free CPAs could be an acceptable alternative for RGC cryopreservation as these protocols maintain high cell viability upon recovery and engrafted RGC extend neurites following transplantation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.