Department of Physiology, Christian Medical College, Vellore, India.
Department of Biochemistry, Velammal Medical College Hospital and Research Institute, Madurai, India.
Indian J Med Res. 2024 May;159(5):519-526. doi: 10.25259/ijmr_851_22.
Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium - Hank's Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.
尽管在该领域取得了进展，但用于糖尿病研究和临床胰岛移植的功能性胰岛的分离仍然是一个巨大的挑战。在这种情况下，缺乏人体/动物组织是胰岛研究的主要障碍之一，而胰岛研究具有巨大的转化潜力。本研究探讨了使用牛胰腺作为替代来源来分离胰岛并评估其用于体外研究的功能的可行性。方法：从注册屠宰场采集牛胰腺，并在冰冷的 Hank's 平衡盐溶液（HBSS）中运输到实验室。通过连续胶原酶消化，然后通过两步过滤和密度梯度分离方法进行纯化来分离胰岛。分离后，用二硫嗪染色鉴定胰岛，并在体外评估胰岛功能，以监测葡萄糖刺激的胰岛素分泌（GSIS），以响应低和高葡萄糖。还使用染色技术了解牛胰腺的细胞结构。结果：胰岛的产率为每克胰腺 157±23 个胰岛，且具有活力。冷缺血时间缩短至 60-75 分钟。胰岛在葡萄糖刺激下释放胰岛素。用高葡萄糖（28mM）刺激时观察到的胰岛素释放比用低葡萄糖（2.8mM）时更多。二硫嗪染色证实了分离后的胰岛存在，并且胰岛的大小范围从 50 到 600µm。在培养中也观察到带有腺组织的覆盖胰岛（带有胰岛组织的胰岛）。苏木精和伊红（H&E）和醛-固红显示胰岛散布在牛胰腺的腺组织中。特殊染色比常规染色更能定义胰岛。用胰岛素、胰高血糖素和生长抑素进行荧光和二氨基联苯胺（DAB）染色显示了每个胰岛中细胞的排列。β细胞主要存在于胰岛核心，α细胞散布在周围的δ细胞中。结论：本研究中描述的分离程序产生了可用于体外研究的有活力的胰岛，这些胰岛对葡萄糖挑战有不同的反应，证实了它们的活力。我们提供了一种从牛胰腺中分离功能性胰岛的简单且可重复的方法。该模型为初学者提供了胰岛实验的实践机会，并有助于重复该过程，该过程也可以推广到其他胰腺组织，并扩展糖尿病研究。
