Immunogenicity of wild-type pig tissues after ice-free cryopreservation in genetically engineered recipients

DESCRIPTION provided by applicant There is an inadequate supply of donated human soft tissues for a variety of surgical applications Use of the patientandapos s own tissues is often effective but is associated with morbidity or not available due to prior use or inadequate due to pre existing disease We have been developing new cryopreservation methods that avoid ice formation which damages the extracellular tissue matrices and permit low cost storage and shipping using minus C freezers and dry ice respectively Published allogeneic large animal studies have demonstrated a significant decrease in immunoreactivity and superior outcomes compared with traditionally frozen cryopreserved control tissues Recently we discovered that the process developed for cardiovascular tissues results in a significant decrease in the innate cellular immune response of human peripheral blood mononuclear cells to porcine tissue as well as human tissue in vitro These observations suggest that porcine tissues processed by our ice free cryopreservation technology may be employed in humans This proposal evaluates a major hurdle previously encountered in vivo for porcine tissues and organs in humans and other old world primates the hyperacute immune response to the galactose a galactose antigen Gal found on porcine cells We plan performance of histological immunohistochemistry evaluation of wild type and control Gal knockout porcine tissues fresh cryopreserved by freezing and ice free cryopreserved before and after subcutaneously implantation in Gal knockout recipients Gal knockout pigs have preformed circulating antibodies to Gal just like humans so these genetically engineered pigs can be used to evaluate processing effects on Gal recognition by ice free cryopreservation without recourse to primate implant models The tissues will be explanted after and weeks and the cellular infiltrates of the implants will be characterized using routine stains Immunohistochemistry will also be performed to compare the recipientandapos s response to different processed tissue groups with emphasis on macrophage phenotype proinflammatory phenotype M versus the phenotype M associated with tissue repair and constructive remodeling If ice free cryopreservation of wild type porcine tissues results in a significant decrease in M proinflammatory macrophages and increased M repair response we will employ wild type donor pigs in future phases of product research and development If ice free cryopreservation does not impact Gal recognitions in genetically engineered pigs with anti Gal antibodies future product development will instead focus on use of Gal knockout tissue donors PUBLIC HEALTH RELEVANCE We have developed a new tissue preservation technology that minimizes the cellular immune response T cell proliferation and cytokine production to allogeneic and xenogeneic tissues Allogeneic tissues are limited by supply and are often not very effective Xenogeneic tissues from non primate species such as pigs are not limited by supply They are however limited by the hyperacute immune rejection of xenogeneic tissue galactose a galactose antigens in humans and other old world primates This proposal evaluates whether or not the recognition of this xenogeneic antigen is minimized by our ice free preservation technology The outcome of this proposal will determine whether wild type or genetically engineered pigs will provide cardiovascular dermal and orthopedic soft tissues for the estimated million plus potential clinical procedures annually in need of better products