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

 描述（由申请人提供）：用于各种外科应用的捐献人体软组织供应不足。使用患者自身的组织通常有效 但与发病率有关或由于先前使用而不可用或由于预先存在的疾病而不足。我们一直在开发新的冷冻保存方法，避免冰的形成，这会破坏细胞外组织基质，并允许分别使用零下80°C的冷冻机和干冰进行低成本的储存和运输。已发表的同种异体大动物研究表明，与传统冷冻保存的对照组织相比，免疫反应性显著降低，结局上级。最近，我们发现，心血管组织开发的过程中，在人外周血单核细胞的先天性，细胞免疫反应显着降低猪组织以及体外人体组织。这些观察结果表明，我们的无冰冷冻保存技术处理的猪组织可用于人类。该提案评估了人类和其他旧世界灵长类动物中猪组织和器官先前在体内遇到的主要障碍，即对猪细胞上发现的半乳糖-a（1，3）-半乳糖抗原（Gal）的超急性免疫应答。我们计划在皮下植入Gal敲除受体之前和之后对野生型和对照Gal敲除猪组织（新鲜、冷冻保存和无冰冷冻保存）进行组织学/免疫组织化学评价。Gal敲除猪已经形成了针对Gal的循环抗体，就像人类一样，因此这些基因工程猪可以用于通过无冰冷冻保存来评估处理对Gal识别的影响，而无需求助于灵长类动物植入模型。在2周和4周后对组织进行染色，并使用常规染色对植入物的细胞浸润进行表征。还将进行免疫组织化学，以比较受体对不同处理组织组的反应，重点是巨噬细胞表型、促炎表型（M1）与组织修复和建设性重塑相关的表型（M2）。如果野生型猪组织的无冰冷冻保存导致M1促炎性巨噬细胞显著减少和M2修复反应增加，我们将在未来的产品研发阶段使用野生型供体猪。如果无冰冷冻保存不影响具有抗Gal抗体的基因工程猪的Gal表达，则未来的产品开发将转而关注Gal敲除组织供体的使用。

 DESCRIPTION (provided by applicant): There is an inadequate supply of donated human soft tissues for a variety of surgical applications. Use of the patient'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 80°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(1,3)-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 2 and 4 weeks and the cellular infiltrates of the implants will be characterized using routine stains. Immunohistochemistry will also be performed to compare the recipient's response to different processed tissue groups with emphasis on macrophage phenotype, proinflammatory phenotype (M1) versus the phenotype (M2) associated with tissue repair and constructive remodeling. If ice-free cryopreservation of wild-type porcine tissues results in a significant decrease in M1 proinflammatory macrophages and increased M2 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.