Structural and Functional Equivalency Between Lyopreserved and Cryopreserved Chorions with Viable Cells

Abstract

<b>Objective:</b> Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast (TR)-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of TR-free chorionic membrane (viable lyopreserved chorionic membrane [VLCM]) and compare it with the viable cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed. <b>Approach:</b> Chorion immunogenicity was tested <i>in vitro</i> in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and <i>in vivo</i> in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by LIVE/DEAD cell fluorescent staining. Inhibition of tumor necrosis factor α secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and proangiogenic properties, respectively. An <i>in vivo</i> rabbit abdominal adhesion model was used to evaluate the antifibrotic properties. <b>Results:</b> Chorionic membrane without trophoblast (CM) was shown to be nonimmunogenic. Tissue architecture, growth factors, and cell viability of fresh CM were maintained in VLCM and VCCM. <i>In vitro</i> studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Furthermore, VLCM prevents formation of postsurgical adhesions in a rabbit abdominal surgical adhesion model. <b>Innovation:</b> Characterization of structural and functional properties of VLCM is reported for the first time. <b>Conclusion:</b> Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors, and viable cells. <i>In vitro</i> and <i>in vivo</i> models demonstrate that VLCM is anti-inflammatory, proangiogenic and antifibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.