DataSheet1_Immunosuppressed Miniswine as a Model for Testing Cell Therapy Success: Experience With Implants of Human Salivary Stem/Progenitor Cell Constructs.DOCX

An urgent need exists to develop large animal models for preclinical testing of new cell therapies designed to replace lost or damaged tissues. Patients receiving irradiation for treatment of head and neck cancers frequently develop xerostomia/dry mouth, a condition that could one day be treated by cell therapy to repopulate functional saliva-producing cells. Using immunosuppression protocols developed for patients receiving whole face transplants, we successfully used immunosuppressed miniswine as a suitable host animal to evaluate the long-term stability, biocompatibility, and fate of matrix-modified hyaluronate (HA) hydrogel/bioscaffold materials containing encapsulated salivary human stem/progenitor cells (hS/PCs). An initial biocompatibility test was conducted in parotids of untreated miniswine. Subsequent experiments using hS/PC-laden hydrogels were performed in animals, beginning an immunosuppression regimen on the day of surgery. Implant sites included the kidney capsule for viability testing and the parotid gland for biointegration time periods up to eight weeks. No transplant rejection was seen in any animal assessed by analysis of the tissues near the site of the implants. First-generation implants containing only cells in hydrogel proved difficult to handle in the surgical suite and were modified to adhere to a porcine small intestinal submucosa (SIS) membrane for improved handling and could be delivered through the da Vinci surgical system. Several different surgical techniques were assessed using the second-generation 3D-salivary tissue (3D-ST) for ease and stability both on the kidney capsule and in the capsule-less parotid gland. For the kidney, sliding the implant under the capsule membrane and quick stitching proved superior to other methods. For the parotid gland, creation of a tissue “pocket” for placement and immediate multilayer tissue closure were well tolerated with minimal tissue damage. Surgical clips were placed as fiduciary markers for tissue harvest. Some implant experiments were conducted with miniswine 90 days post-irradiation when salivation decreased significantly. Sufficient parotid tissue remained to allow implant placement, and animals tolerated immunosuppression. In all experiments, viability of implanted hS/PCs was high with clear signs of both vascular and nervous system integration in the parotid implants. We thus conclude that the immunosuppressed miniswine is a high-value emerging model for testing human implants prior to first-in-human trials.

开发用于测试旨在替代缺失或受损组织的新型细胞疗法的大型动物模型，已成为一项迫切需求。接受头颈部癌症放射治疗的患者常并发口干症（xerostomia，俗称口干），未来有望通过细胞疗法重建功能性唾液分泌细胞，以此治疗该病症。本研究采用专为全脸移植患者开发的免疫抑制方案，成功将免疫抑制小型猪作为适宜宿主动物，对负载包埋人唾液腺干/祖细胞（human salivary stem/progenitor cells，hS/PCs）的基质修饰型透明质酸（hyaluronate，HA）水凝胶/生物支架材料的长期稳定性、生物相容性及细胞与材料的体内归宿展开评估。研究首先在未处理的小型猪腮腺中开展了初步生物相容性测试。后续实验则采用负载hS/PCs的水凝胶，于手术当日启动免疫抑制方案，在受试动物体内开展。植入位点选择肾被膜以检测细胞活性，以及腮腺以测试最长达8周的生物整合效果。通过对植入位点周围组织的分析可知，所有受试动物均未出现移植排斥反应。初代植入体仅包含水凝胶包裹的细胞，在手术室中难以操作，因此对其进行改良，使其附着于猪小肠黏膜下层（porcine small intestinal submucosa，SIS）膜以提升操作性，且可通过达芬奇手术系统（da Vinci surgical system）进行递送。研究针对第二代3D唾液组织（3D-salivary tissue，简称3D-ST），评估了多种不同手术操作技术在肾被膜及无被膜腮腺中的易用性与稳定性。在肾脏部位，将植入体推至被膜下方并快速缝合的操作方法，效果优于其他方式。在腮腺部位，通过制备组织"口袋"以放置植入体并即刻进行多层组织缝合的操作，耐受性良好且组织损伤极小。术中放置手术夹作为组织取材的定位标记。部分植入实验选用放疗90天后唾液分泌量显著下降的小型猪开展，这类小型猪仍保留足够的腮腺组织以完成植入操作，且可良好耐受免疫抑制方案。所有实验中，植入的hS/PCs均保持较高活性，且腮腺植入体已出现明确的血管与神经系统整合迹象。综上，免疫抑制小型猪是一种极具应用价值的新型模型，可用于人体植入体进入首次人体临床试验前的相关测试。