In Vitro Characterization of Human Lyopreserved Amniotic Membrane with Living Cells

Human amniotic membrane (AM) has intrinsic anti-inflammation, anti-fibrosis, and antimicrobial properties. The cryopreserved amniotic membranes that retain all native tissue components including viable cells shows clinical benefit in treating chronic wounds. However, cryopreservation requires ultra-low temperature storage and distribution, which limits the use of cryopreserved products.

To overcome this limitation, a lyopreservation method has been developed to preserve the viable tissue for ambient storage. In this study, we confirmed the presence of living cells in the viable lyopreserved AM (VLAM). Fresh AM and devitalized lyopreserved AM (DLAM) served as positive and negative controls, respectively. Scanning electron microscopy analysis showed that the native tissue structure is preserved in VLAM. Using live/dead staining, we showed that the viable cells were present and persisted for up to 21 days in the culture medium in fresh AM and VLAM but not in DLAM. The functionality of cells in VLAM was measured by their differentiation potential and anti-fibrotic activity. With osteogenic induction, cells in VLAM, but not in DLAM, deposited calcium within the membrane in an induction-dependent and time-dependent manner.

The migration of human lung fibrotic fibroblasts in a wound-scratch assay was reduced in the presence of VLAM but not DLAM. RNA array and quantitative PCR analyses of fibrotic lung fibroblasts indicated that the presence of VLAM reduced expression of pro-fibrotic genes such as type I collagen and alpha smooth muscle actin and increased expression of anti-fibrotic growth factors and cytokine such as hepatocyte growth factor, TGF-b3, and IL-1b. Altogether, the results from this study demonstrate that endogenous cells in VLAM remained viable and functional.