A Novel Beta Cell Specific Microfluidic Perifusion and Imaging Device for Islet P

描述（由申请人提供）：胰岛移植是唯一一种无需外源性胰岛素即可实现血糖控制的I型糖尿病微创治疗方法。然而，胰岛移植显示出不同的成功率，主要是由于人类胰岛制剂的质量不一致。胰岛移植要成为FDA许可的生物制品，需要证明一个完善的胰岛制备过程和产品制造的一致性。联邦法规规定，每批生物制品在投入临床使用前都要进行效力测试。目前，尚无可靠的人胰岛效价检测方法。我们假设适当的胰岛效价测试必须是β细胞特异性的，同时评估与胰岛生理学相关的关键因素，包括葡萄糖刺激的线粒体电位变化、钙内流和动态胰岛素分泌。为了验证这一假设，研究人员利用微流控芯片技术开发了一种具有功能活体显微镜的创新胰岛灌注系统，可以同时测量葡萄糖诱导的线粒体电位、钙内流和动态胰岛素分泌的变化。初步结果表明，该系统能充分区分低效和高效的人胰岛制剂。本项目将重点研究以下目标：(1)通过改进芯片设计，进一步提高微流控系统的分辨率。具体而言，我们的建议侧重于：a.通过减少微流控芯片内腔室的体积来提高时间分辨率，b.通过在芯片中添加流体混合器来改善流动动力学控制并增加易用性，c.建立葡萄糖斜坡来评估胰岛素动力学，d.在一个电动平台上将多个灌注腔整合到芯片中，以增加可评估的人类胰岛的样本量，并提供更好的最终胰岛产品代表。E.开发快速胰岛素分泌测量方法。(2)在人类胰岛细胞移植的临床前裸鼠模型上验证微流控系统，并建立预测移植后胰岛移植物功能的“胰岛效能指数”。简而言之，多变量回归模型将确定哪些胰岛细胞特征与体内结果显著相关，并将这些特征用于计算指数。(3)在临床胰岛移植试验中测试微流控系统，探讨胰岛效能指数预测胰岛移植功能的有效性。该提案将测试一种创新的微流体系统，该系统提供胰腺β细胞生理学的详细分析。在未来，解决无限胰岛细胞来源和新的免疫保护策略的进一步发展可能会使胰岛移植适用于更大比例的糖尿病人群。这种微流控系统可以为当前和未来的胰岛替代疗法以及糖尿病新疗法的研究提供可靠的胰岛效价测定。

DESCRIPTION (provided by applicant): Islet transplantation is the only minimally invasive therapy for Type I diabetes that is able to achieve glycemic control without exogenous insulin. However, islet transplantation shows variable success rates, mainly due to the inconsistent quality of human islet preparations. For islet transplantation to become a FDA licensed biologic product, a well- established islet preparation process and product manufacturing consistency will need to be demonstrated. Federal regulations mandate that each biologic product lot be tested for potency before being released for clinical use. At present, there is no reliable potency test available for human pancreatic islets. We hypothesize that an appropriate islet potency test must be beta-cell specific and simultaneously assess key factors associated with islet physiology, including glucose- stimulated changes in mitochondrial potentials, calcium influx and dynamic insulin secretion. To test this hypothesis, an innovative islet perfusions system with functional, live microscopy was developed using microfluidic chip technology to enable simultaneous measurement of glucose-induced changes in mitochondrial potentials, calcium influx and dynamic insulin secretion. Preliminary results indicate that this system can adequately distinguish low potency from high potency human islet preparations. This project will focus on the following aims: (1) To further improve the resolution of the microfluidic system through modification of the chip design. Specifically, our proposal focuses on: a. improving temporal resolution by reducing the volume of the chamber within the microfluidic chip, b. improving flow dynamic control and increasing ease of use by adding a fluid mixer into the chip, c. establishing glucose ramps to evaluate insulin kinetics, d. integrating multiple perfusion chambers into the chip on a motorized platform to increase the sample size of human islets that can be evaluated and provide a better representation of the final islet product. e. developing a rapid insulin secretion measurement. (2) To validate the microfluidic system in a pre-clinical nude mouse model using human islet cell grafts and develop an "Islet Potency Index " predictive of post-transplant islet graft function. Briefly, multivariable regression modeling will determine which islet cell characteristics are significantly associated with in vivo outcome and these will be used to calculate the index. (3) To test the microfluidic system in setting of a clinical human islet transplant trial and investigate the validity of the Islet Potency Index to predict islet graft function. This proposal will test an innovative microfluidic system that provides detailed analysis of pancreatic beta-cell physiology. In the future, it is likely that further developments addressing an unlimited islet cell source and new immunoprotective strategies will make islet transplantation available to a broader proportion of the diabetic population. This microfluidic system could represent a reliable islet potency assay for current and future islet replacement therapies, as well as for the study of new diabetes therapies in general. PUBLIC HEALTH RELEVANCE: An innovative microfluidic system will be optimized and standardized for human islet potency testing. If successful, the microfluidic-based assay could be used for islet product release testing, which represents a yet unmet regulatory requirement for FDA biologic licensure of islet transplants.