Endogenous viable cells in lyopreserved amnion retain differentiation potential and anti-fibrotic activity in vitro

Human amniotic membrane (AM) has intrinsic anti-inflammatory, anti-fibrotic and antimicrobial properties. Tissue preservation methods have helped to overcome the short shelf life of fresh AM allowing "on demand" use of AM grafts. Cryopreserved AM that retains all native tissue components, including viable cells, has clinical benefits in treating chronic wounds. However, cryopreservation requires ultra-low temperature storage, limiting the use of cryopreserved products. To overcome this limitation, a new lyopreservation method has been developed for ambient storage of living tissues. The goal of this study was to investigate the viability and functionality of AM cells following lyopreservation. Fresh AM and devitalized lyopreserved AM (DLAM) served as positive and negative controls, respectively. Using live/dead staining, we confirmed the presence of living cells in viable lyopreserved AM (VLAM) and showed that these cells persisted up to 21 days in culture medium. The functionality of cells in VLAM was assessed by their differentiation potential and anti-fibrotic activity in vitro. With osteogenic induction, cells in VLAM deposited calcium within the membrane, a marker of osteogenic cells, in a time-dependent manner. The migration of human lung fibrotic fibroblasts in a scratch wound assay was reduced significantly in the presence of VLAM-derived conditioned medium. Quantitative PCR analyses indicated that VLAM reduced the expression of pro-fibrotic factors such as type I collagen and increased the expression of anti-fibrotic factors such as hepatocyte growth factor and anti-fibrotic microRNA in fibrotic fibroblasts. Taken together, these results demonstrate that endogenous cells in VLAM remain viable and functional post-lyophilization. STATEMENT OF SIGNIFICANCE: This study, for the first time, provides direct evidence showing that tissue viability and functional cells can be preserved by lyophilization. Similar to fresh amniotic membrane (AM), viable lyopreserved AM (VLAM) retains viable cells for extended periods of time. More importantly, these cells are functional and maintain their osteogenic differentiation potential and anti-fibrotic activity. Our results confirmed that the novel lyophilization method preserves tissue viability.