Alternative cryoprotective agent for corneal stroma-derived mesenchymal stromal cells for clinical applications

Abstract

AbstractCryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute. The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing—Dulbecco’s modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation. The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested. Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO.