Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada.
Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.
Diabetologia. 2018 Sep;61(9):2016-2029. doi: 10.1007/s00125-018-4672-5. Epub 2018 Jul 3.
AIMS/HYPOTHESIS: Islet transplantation is a treatment option that can help individuals with type 1 diabetes become insulin independent, but inefficient oxygen and nutrient delivery can hamper islet survival and engraftment due to the size of the islets and loss of the native microvasculature. We hypothesised that size-controlled pseudoislets engineered via centrifugal-forced-aggregation (CFA-PI) in a platform we previously developed would compare favourably with native islets, even after taking into account cell loss during the process.
Human islets were dissociated and reaggregated into uniform, size-controlled CFA-PI in our microwell system. Their performance was assessed in vitro and in vivo over a range of sizes, and compared with that of unmodified native islets, as well as islet cell clusters formed by a conventional spontaneous aggregation approach (in which dissociated islet cells are cultured on ultra-low-attachment plates). In vitro studies included assays for membrane integrity, apoptosis, glucose-stimulated insulin secretion assay and total DNA content. In vivo efficacy was determined by transplantation under the kidney capsule of streptozotocin-treated Rag1 mice, with non-fasting blood glucose monitoring three times per week and IPGTT at day 60 for glucose response. A recovery nephrectomy, removing the graft, was conducted to confirm efficacy after completing the IPGTT. Architecture and composition were analysed by histological assessment via insulin, glucagon, pancreatic polypeptide, somatostatin, CD31 and von Willebrand factor staining.
CFA-PI exhibit markedly increased uniformity over native islets, as well as substantially improved glucose-stimulated insulin secretion (8.8-fold to 11.1-fold, even after taking cell loss into account) and hypoxia tolerance. In vivo, CFA-PI function similarly to (and potentially better than) native islets in reversing hyperglycaemia (55.6% for CFA-PI vs 20.0% for native islets at 500 islet equivalents [IEQ], and 77.8% for CFA-PI vs 55.6% for native islets at 1000 IEQ), and significantly better than spontaneously aggregated control cells (55.6% for CFA-PI vs 0% for spontaneous aggregation at 500 IEQ, and 77.8% CFA-PI vs 33.4% for spontaneous aggregation at 1000 IEQ; p < 0.05). Glucose clearance in the CFA-PI groups was improved over that in the native islet groups (CFA-PI 18.1 mmol/l vs native islets 29.7 mmol/l at 60 min; p < 0.05) to the point where they were comparable with the non-transplanted naive normoglycaemic control mice at a low IEQ of 500 IEQ (17.2 mmol/l at 60 min).
CONCLUSIONS/INTERPRETATION: The ability to efficiently reformat dissociated islet cells into engineered pseudoislets with improved properties has high potential for both research and therapeutic applications.
目的/假设：胰岛移植是一种可以帮助 1 型糖尿病患者实现胰岛素独立性的治疗选择，但由于胰岛的大小和原代微血管的丧失，低效的氧和营养输送会阻碍胰岛的存活和植入。我们假设，通过我们之前开发的平台，通过离心强制聚集（CFA-PI）工程化的大小可控的假胰岛，即使考虑到在该过程中细胞损失，也将与天然胰岛相媲美。
将人胰岛在我们的微井系统中分离并重新聚集为均匀的、大小可控的 CFA-PI。在一系列大小范围内评估其体外和体内性能，并与未经修饰的天然胰岛以及通过常规自发聚集方法形成的胰岛细胞簇进行比较（其中分离的胰岛细胞在超低附着板上培养）。体外研究包括膜完整性、细胞凋亡、葡萄糖刺激的胰岛素分泌测定和总 DNA 含量测定。体内功效通过链脲佐菌素处理的 Rag1 小鼠肾包膜下移植来确定，每周三次监测非禁食血糖，并在第 60 天进行 IPGTT 以检测葡萄糖反应。通过恢复性肾切除术，在完成 IPGTT 后，切除移植物以确认疗效。通过胰岛素、胰高血糖素、胰多肽、生长抑素、CD31 和血管性血友病因子染色的组织学评估分析结构和组成。
CFA-PI 与天然胰岛相比，均匀性显著提高，葡萄糖刺激的胰岛素分泌也显著改善（即使考虑到细胞损失，也增加了 8.8 倍至 11.1 倍），并且对缺氧的耐受性也提高了。在体内，CFA-PI 在逆转高血糖方面的作用与天然胰岛相似（在 500IEQ 时，CFA-PI 为 55.6%，天然胰岛为 20.0%，在 1000IEQ 时，CFA-PI 为 77.8%，天然胰岛为 55.6%），并且明显优于自发聚集的对照细胞（在 500IEQ 时，CFA-PI 为 55.6%，自发聚集为 0%，在 1000IEQ 时，CFA-PI 为 77.8%，自发聚集为 33.4%；p<0.05）。CFA-PI 组的葡萄糖清除率优于天然胰岛组（CFA-PI 组在 60 分钟时为 18.1mmol/l，天然胰岛组为 29.7mmol/l；p<0.05），在低 IEQ（500IEQ）时，与未移植的正常血糖对照小鼠相当（CFA-PI 组在 60 分钟时为 17.2mmol/l）。
结论/解释：将分离的胰岛细胞高效地重新格式化为具有改善性能的工程化假胰岛的能力，具有很高的研究和治疗应用潜力。
