Improved cryopreservation of cultured RPE with addition of a silver iodide/ alginate mixture

Abstract

Purpose : Retinal cell therapeutics in early clinical trials have shown promising results. Supply chains from a cGMP cell-manufacturing site to multiple clinical transplantation centers will rely on optimal storage and transport conditions, possibly under cryopreservation. Here we investigated an optimized freezing protocol for cultured RPE.

Methods : Mesenchymal stem cells and ARPE19 cultures were grown to pre-confluence in DMEM/F12 media supplemented with 10% fetal calf serum, 1% Penicillin/ Streptomycin and FGF-2. The cells were then trypsinized from plates and frozen in said culture media with 2-3% DMSO in aliquots of 5x10⁵ cells/ 1ml at a rate of 1°C/ minute at -80°C for 24h and then in liquid nitrogen at -197°C for another 24h. Aliquots were split into cryovials without coating, with alginate coating and coated with a mixture silver iodide/alginate mixture. Subsequently, the cryovials were thawed in a water bath at 37°C and centrifuged. Their vitality was then assessed using the Live-Dead-Assay in a NucleoCounter®. All experiments were performed in triplicate or more.

Results : The viability of mesenchymal stem cells following cryopreservation was 35% (SD 9%) for uncoated, 42% (SD 18%) for alginate coated and 70% (SD 4%) for silver iodide/ alginate mixture coated cryovials. The viability of ARPE19 cells was 85% (SD 6%) for uncoated, 92% (SD 6%) for alginate coated and 95% (SD 2%) for silver iodide/ alginate mixture coated cryovials.

Conclusions : Our pilot data suggest the addition of the silver iodide/ alginate mixture to significantly improve and more accurately predict viability of frozen ARPE19 cell suspensions. Future experiments will aim at reducing the DMSO concentration and be expanded to other RPE cell sources, including pluripotent stem cells.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.