Abstract
Purpose :
Conventional tissue cryopreservation methods require the use of both cell permeating and biologically reactive cryoprotectants (e.g., DMSO) and liquid nitrogen facilities, raising numerous practical and technical concerns. To overcome these challenges in supply chain management for allogenic transplantation of corneal limbal stem cells (LSC) and iPSC derived retinal pigment epithelial (RPE) tissues, we developed and investigated the efficiency of a biocompatible cryopreservation medium, OdinSol®, which removes the need for any cell permeating cryoprotectant and enables long-term tissue cryostorage in regular -80oC freezers.
Methods :
The efficacy of OdinSol®, a unique polysaccharide cocktail that significantly reduces ice crystal size in the proximity of the cell membrane to the nano meter scale, was tested by tissue cryostorage at -80oC. In brief, each of four paired donor corneas (ages 40-65 yrs; Mid-America Transplant, St. Louis, MO) was divided into two pieces, with each piece immersed separately into a standard sterile 15ml cryovial (NalgeneTM) containing 10ml of either OdinSol® or DMEM medium containing 10% DMSO and 10% FBS, and then stored in a -80oC freezer. Human iPSC derived RPE (Phenocell, Grasse, France) monolayers were cryopreserved using the same protocol. After one to six months of storage, frozen tissues were thawed and analyzed using in vitro assays, including TUNEL or immunochemical staining, transmission electron microscopy (TEM), and ex vivo expansion of LSC outgrowth.
Results :
OdinSol® showed promising efficacy in maintaining the cell viability in both tissue types. For limbal tissues, TUNEL staining and TEM demonstrated well preserved LSC (viability > 90% after six months of storage) in the basal limbus. For RPE tissues, immunochemical staining illustrated significantly improved cell viability as well as unaltered cell function in comparison to the use of conventional cryopreservation media.
Conclusions :
OdinSol® effectively cryopreserves cell viability and function of LSCs and RPE cells inside their donor or bioartificial tissues, respectively. Its eliminating the need for cell permeating cryoprotectants, FBS, liquid nitrogen facilities is highly advantageous in addressing various technical, practical, and regulatory challenges related to biosafety and implementation of novel cell-based therapies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.