Moogk Duane, Hanley Stephen, Ramunas John, Blaylock April, Skorepova Jana, Rosenberg Lawrence, Jervis Eric
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Biotechnol Bioeng. 2007 Aug 1;97(5):1138-47. doi: 10.1002/bit.21335.
A means of expanding islet cell mass is urgently needed to supplement the limited availability of donor islets of Langerhans for transplant. Live cell imaging of human islets in culture has the potential to identify the specific cells and processes involved in islet expansion. A novel imaging chamber was developed to facilitate long-term three-dimensional imaging of human islets during transformation. Islets have been induced to transform into duct-like epithelial cystic structures and revert back to glucose responsive endocrine cells under appropriate conditions (Jamal et al. Cell Death Differ. 2005 12:702-712). Here we aim to further our understanding by characterizing the process at a single cell level over time-essentially constructing a high resolution recorded history of each cell and its progeny during transformation and reversion. The imaging chamber enables high resolution imaging of three-dimensional islets while maintaining the structure of the islet cells and intercellular matrix components. A mathematical model was developed to validate the imaging chamber design by determining the required chamber dimensions to avoid introduction of oxygen and nutrient transport limitations. Human islets were embedded in collagen in the imaging chamber and differential interference contrast time course images were obtained at 3 min intervals. Immunofluorescent imaging confirmed that islet phenotype was maintained for at least 5 days during imaging. Analysis of the time courses confirms our ability to identify and track individual cells over time and to observe cell death and phenotype transformation in isolated human islets.
迫切需要一种扩大胰岛细胞团的方法，以补充用于移植的供体胰岛朗格汉斯细胞数量有限的问题。对培养中的人胰岛进行活细胞成像有可能识别参与胰岛扩大的特定细胞和过程。开发了一种新型成像室，以促进人胰岛在转化过程中的长期三维成像。在适当条件下，胰岛已被诱导转化为导管样上皮囊性结构，并恢复为对葡萄糖有反应的内分泌细胞（贾马尔等人，《细胞死亡与分化》，2005年，第12卷：702 - 712页）。在这里，我们旨在通过在单细胞水平上随时间表征该过程来进一步加深理解——本质上是构建每个细胞及其后代在转化和恢复过程中的高分辨率记录历史。该成像室能够对三维胰岛进行高分辨率成像，同时保持胰岛细胞和细胞间基质成分的结构。通过确定所需的室尺寸以避免引入氧气和营养物质运输限制，开发了一个数学模型来验证成像室设计。将人胰岛嵌入成像室的胶原蛋白中，并以3分钟的间隔获得微分干涉对比时间进程图像。免疫荧光成像证实，在成像过程中胰岛表型至少维持了5天。对时间进程的分析证实了我们随时间识别和追踪单个细胞以及观察分离的人胰岛中细胞死亡和表型转化的能力。
