Abstract
Purpose :
Human cornea stroma stem cells (CSSC)can be isolated from corneal limbal stroma for restoring collagen fibril organization and transparency of cornea. In view of this, preparation and storage of CSSC are important for cell therapy and tissue engineering applications. In this study, we compared three different cryopreservation protocols for CSSC.
Methods :
To investigate three different freezing protocols using isolated human CSSC lines from different donor tissue: protocol 1, 2% human serum+10% dimethyl sulfoxide (DMSO)+CSSC medium;protocol 2: 4% human serum+10% DMSO+ CSSC medium; protocol 3, 90% human serum+10% DMSO. The thawing protocol for all CCCS is same. Outcome measures are post-thawing cells morphology, recovery rate, cell proliferation and doubling time, genes expression analysis of CSSC markers (ABCG2, SOX2, NANOG, PAX6, SIX3) and anti-inflammatory response (TNFAIP6).
Results :
Cell lines keep the same morphology as the freshly isolated cell after frozen/thawing with all protocols tested . Cell recovery rate after frozen/thawing were higher with protocol 1 and 2 than protocol 3(p<0.05). There was no difference of doubling time in unfrozen and frozen CSSC with three protocols . ABCG2, NANOG, PAX6, SIX3 and TNFAIP6 genes expression analysis showed a similar pattern with unfrozen cells after frozen with the 3 protocols . SOX2 gene expression significantly increased after frozen with the three freezing protocols(p<0.05).
Conclusions :
All 3 protocols maintain CSSC morphology, the expression of CSSC markers and anti-inflammatory response pattern. Protocol 1 and 2 which showed a better recovery rate ,may more suitable for cryopreservation of human CSSC.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.