Functional Genomics, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK.
Emerging Innovations Unit, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK.
SLAS Discov. 2023 Mar;28(2):3-12. doi: 10.1016/j.slasd.2023.01.002. Epub 2023 Jan 13.
Identification of novel compounds to selectively induce pancreatic beta-cell proliferation has the potential to restore functional beta-cell mass and insulin secretory demand in type 2 diabetes. The rarity of islet cell clusters (comprising of only 1% of the total pancreas mass) makes such a discovery a challenge. To address this obstacle a high throughput, 384 well, plate-based multi-parametric imaging assay was developed to capture ex vivo primary islet proliferation, allowing positive identification of compounds that can selectively enhance islet beta-cell proliferation. The use of microscopy-based, high-content imaging technology enables acquisition of additional multi-parametric information such as proliferating populations in the islet beta and non beta-cells, insulin intensity, and cell counts, improving understanding of on and off target effects in primary tissue. The protocol requires access to a high-throughput microscopy platform for automated image acquisition of treated islet cells in assay plates. High content image analysis software is required to extract multiparametric cellular features and aid identification of therapeutically relevant small molecules and perturbants. Several putative beta-cell proliferative compounds have validated in this high throughput assay format, including the pleiotropic hormone prolactin [1] and the small molecule DYRK1A inhibitor harmine [2]. It is recommended to include one, or both, as positive controls to provide a reference for image analysis, give confidence in assay performance and capture potential assay variability during experimental runs. The protocol outlined specifically focuses on the multiparametric assessment of betacell proliferation in mouse and rat ex vivo islets and provides the methodology required for the collection of high quality cellular material. The high throughput, plate based assay can additionally be adapted to evaluate and quantify other disease relevant endpoints by high content microscopy and be applied to other downstream measurements. One of the caveats of a high-throughput, 384 microplate beta-cell proliferative assay is its limitations to facilitate human beta-cell proliferation detection, especially for weak activators. Adult human beta-cell proliferation is an extremely rare biological event and assessment experimentally can be donor dependent. In addition lower human islet beta-cell subpopulations require large numbers of cells for accurate rare event measurement.
鉴定新型化合物以选择性地诱导胰岛β细胞增殖，具有恢复 2 型糖尿病患者功能性β细胞数量和胰岛素分泌需求的潜力。胰岛细胞簇（仅占胰腺总质量的 1%）的稀有性使得这一发现具有挑战性。为了解决这一障碍，开发了一种高通量、384 孔板的多参数成像测定法，以捕获离体原代胰岛增殖，从而能够鉴定出能够选择性增强胰岛β细胞增殖的化合物。基于显微镜的高通量成像技术的使用能够获取额外的多参数信息，例如胰岛β细胞和非β细胞中的增殖群体、胰岛素强度和细胞计数，从而提高对原代组织中靶内和靶外效应的理解。该方案需要访问高通量显微镜平台，以便在测定板中自动获取处理后的胰岛细胞的图像。需要使用高内涵图像分析软件来提取多参数细胞特征，并有助于鉴定有治疗意义的小分子和扰动剂。几种假定的β细胞增殖化合物已经在这种高通量测定格式中得到验证，包括多效激素催乳素[1]和 DYRK1A 小分子抑制剂 harmine[2]。建议包含一个或两个作为阳性对照，为图像分析提供参考，增强实验运行期间测定性能的可信度，并捕获潜在的测定变异性。该方案专门针对离体鼠和大鼠胰岛的β细胞增殖的多参数评估进行了概述，并提供了收集高质量细胞材料所需的方法。高通量、384 微孔板β细胞增殖测定法还可以通过高内涵显微镜来评估和量化其他与疾病相关的终点，并应用于其他下游测量。高通量、384 微孔板β细胞增殖测定法的一个限制是其难以促进人β细胞增殖的检测，尤其是对于弱激活剂。成人胰岛β细胞增殖是一种极为罕见的生物学事件，其评估在实验上可能依赖于供体。此外，较低的人胰岛β细胞亚群需要大量细胞才能进行准确的稀有事件测量。
