Pillar and perfusion well plate platforms for reproducible organoid culture from iPSC

项目概要/摘要 迫切需要改进的体外疾病建模以快速推进治疗药物的开发。 用于临床前评价或优先考虑潜在的环境毒物。最近 在体外疾病模型方面取得了重大进展， 从多能干细胞（PSC）和祖细胞（也称为，类器官）、生物打印的人体组织 具有从患者获得的细胞的构建体（也称为，3D生物打印），以及 微流体芯片（也称为，芯片上器官）。然而，这些新的创新技术仍然缺乏 足够的通量和用户友好性，能够快速识别高质量的治疗药物 候选人，特别是当疾病涉及多个器官相互作用时。解决这些 挑战，我们建议利用我们独特的"微型三维（3D）生物打印" 技术和相关的柱/灌注板平台，包括具有侧壁的384柱板 和狭缝（384PillarPlate）以及底部透明的384深孔板（384DeepWellPlate）， 静态类器官培养物以及具有侧壁和狭缝的36柱板（36PillarPlate）和具有侧壁和狭缝的36柱板（36PillarPlate）。 用于基于灌注的类器官的具有储器和微通道的灌注孔板（36PerfusionPlate） 文化我们提出的柱/灌注板平台将"3D生物打印"与"微流体样"相结合 这些特征提供了优于更传统的3D细胞培养模型的几个独特优势， 微流体模型特别地，柱/灌注板与标准384孔板兼容。 板和现有的高通量筛选（HTS）设备（例如，自动荧光 显微镜和微量滴定板读数器），这将显著降低 进入商业化的障碍。在拟议的研究中，人脑类器官（HBOs） 来源于诱导的多能干细胞（iPSC）的细胞被选择作为模型系统以开发用于治疗癌症的药物。 包括阿片类药物在内的化合物的发育神经毒性（DNT）预测评估工具 和酒精。我们的核心假设是：（i）柱/灌注板上的生物打印的HBO可以维持 通过控制和模拟体内微环境， （ii）柱/灌注板上的HBO可以模拟细胞功能， 药物和环境毒物对神经发育障碍的影响。的具体目标 拟开展的研究工作有：（1）通过微型3D技术提高类器官培养的可重复性 生物打印技术;（2）在柱板上建立高通量的原位全类器官成像， 预测性化合物筛选;（3）在柱板上建立类器官的冷冻保存。我们 设想柱/灌注板平台上的生物打印的人类类器官可以用作 用于筛选治疗药物的有前景的疾病模型，同时最大限度地减少药物中动物的使用， 发现过程。

Project Summary/Abstract There is a critical need for improved in vitro disease modeling to rapidly advance therapeutic drug candidates to preclinical evaluation or to prioritize potential environmental toxicants. Recently, there have been significant advances made in in vitro disease models, including human mini-tissues derived from pluripotent stem cells (PSCs) and progenitor cells (a.k.a., organoid), bioprinted human tissue constructs with cells obtained from patients (a.k.a., 3D bioprinting), and multi-layered cells in microfluidic chips (a.k.a., organ-on-chip). These new and innovative technologies, however, still lack enough throughput and user friendliness to enable rapid identification of high-quality therapeutic candidates, particularly when a disease involves multiple organ interactions. To address these challenges, we propose to leverage our unique “miniature three-dimensional (3D) bioprinting” technology and associated pillar/perfusion plate platforms, including a 384-pillar plate with sidewalls and slits (384PillarPlate) and a clear-bottom, 384-deep well plate (384DeepWellPlate) developed for static organoid culture as well as a 36-pillar plate with sidewalls and slits (36PillarPlate) and a 36- perfusion well plate with reservoirs and microchannels (36PerfusionPlate) for perfusion-based organoid culture. Our proposed pillar/perfusion plate platforms combining “3D bioprinting” with “microfluidic-like” features offer several distinctive advantages over more conventional 3D cell culture models and microfluidic models. In particular, the pillar/perfusion plates are compatible with standard 384-well plates and existing high-throughput screening (HTS) equipment (e.g., automated fluorescence microscopes and microtiter well plate readers) already familiar to users, which will significantly lower barriers to entry for commercialization. In the proposed research, human brain organoids (HBOs) derived from induced pluripotent stem cells (iPSCs) are selected as a model system to develop a predictive assessment tool for developmental neurotoxicity (DNT) by compounds including opioid drugs and alcohol. Our core hypotheses are: (i) bioprinted HBOs on the pillar/perfusion plates can maintain key tissue biomarkers by controlling and mimicking in vivo microenvironments and enable high- throughput, high-content cell function analysis; (ii) HBOs on the pillar/perfusion plates can model the influence of drugs and environmental toxicants to neurodevelopmental disorders. The specific aims of the proposed research are to: (1) improve reproducibility of organoid culture via miniature 3D bioprinting technology; (2) establish in situ whole organoid imaging on a pillar plate for high-throughput, predictive compound screening; (3) establish cryopreservation of organoids on the pillar plate. We envision that bioprinted human organoids on the pillar/perfusion plate platforms can be used as promising disease models for screening therapeutic drugs while minimizing the use of animals in drug discovery processes.