Del Guerra S, Bracci C, Nilsson K, Belcourt A, Kessler L, Lupi R, Marselli L, De Vos P, Marchetti P
Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, Italy.
Biotechnol Bioeng. 2001 Dec 20;75(6):741-4. doi: 10.1002/bit.10053.
Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies.
在不进行慢性免疫抑制的情况下，对胰岛进行免疫保护以实现成功的同种异体或异种移植，是一种治疗1型糖尿病的有吸引力但仍难以实现的方法。最近的研究表明，即使不存在纤维化过度生长，其他因素，主要是胰岛核心营养不足，也是腹腔内微囊化胰岛移植长期存活的主要障碍。使用分散的细胞可能有助于解决这个问题，因为可以想象细胞更容易获得营养支持。在本研究中，通过胶原酶消化和密度梯度纯化制备的纯化牛胰岛，以及通过胰蛋白酶和脱氧核糖核酸酶获得的分散牛胰岛细胞（活力>90%），被包埋在2%（w/v）海藻酸钠（常用于包封目的）或（仅用于分散胰岛细胞）大孔明胶微载体（CulthiSpher-S，常用于动物细胞生产生物制品）中。在制备不同系统后的1周内和1个月后，进行了葡萄糖刺激下的胰岛素释放研究，结果表明海藻酸钠微胶囊内的分散牛胰岛细胞无法感知葡萄糖浓度变化。相反，包埋在CulthiSpher-S微载体中的牛胰岛细胞在葡萄糖浓度升高的刺激下，仍保持增加胰岛素分泌的能力。在这种情况下，胰岛素释放量约为海藻酸钠微胶囊内完整牛胰岛释放量的60%。含胰岛细胞微载体的MTT和苏木精-伊红染色显示，在整个研究期间都存在存活且代谢活跃的细胞。这些令人鼓舞的功能数据促使我们测试微载体是否可以进行免疫隔离，以用于潜在的移植。微载体被包埋在3%的海藻酸钠中，然后覆盖一层聚-L-赖氨酸层和一层最终的外层海藻酸钠层。观察到该系统的胰岛素分泌功能得以维持，这增加了在实验性移植研究中使用含有分散胰岛细胞的微囊化CulthiSpher-S微载体的可能性。
