In Vitro Characterization Of Human Lyopreserved Amniotic Membrane With Living Cells
Yong Mao1, Tyler Hoffman2, Amit Singal1, Sandeep Dhall2, Malathi Sathyamoorthy2, Alla Danilkovitch2, Joachim Kohn1.
1Rutgers University, Piscataway, NJ, USA, 2Osiris Therapeutics, Inc., Columbia, MD, USA.
BACKGROUND Human amniotic membrane (AM) has intrinsic anti-inflammation, anti-fibrosis and antimicrobial prosperities. The cryopreserved amniotic membrane 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. This study is to determine if the viable lyopreserved AM (VLAM) retains the viable and functional cells. METHODS Live-dead staining and imaging such as scanning electron microscopy (SEM) and confocal microscopy were used to detect the viable cells in AM and the native structures of AM. Osteogenic differentiation of VLAM was used to verify the presence of living multipotent cells in VLAM. Moreover, the anti-fibrotic activity of VLAM was studied using gene expression profiling (RNA array and quantitative PCR) and scratch wound migration assays. RESULTS SEM 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 a devitalized lyopreserved AM (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 was reduced in the presence of VLAM but not DLAM. RNA array and qPCR analyses of fibrotic lung fibroblasts indicated that the presence of VLAM reduced expression of pro-fibrotic genes such as type I collagen, alpha smooth muscle actin and increased expression of anti-fibrotic growth factors and cytokine such as hepatocyte growth factor, TGF-β3 and IL-1β. CONCLUSIONS The results from this study demonstrate that endogenous cells in VLAM remained viable and functional.
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