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

项目摘要/摘要 迫切需要改进的体外疾病模型来快速发展治疗药物 参加临床前评估或确定潜在环境毒物优先顺序的候选人。最近，那里 在体外疾病模型方面取得了重大进展，包括人类微小组织来源 从多能干细胞(PSCs)和祖细胞(也称为有机体)，生物打印的人体组织 构建了从患者获得的细胞(也称为3D生物打印)，并在 微流控芯片(也称为芯片上器官)。然而，这些新的和创新的技术仍然缺乏 足够的吞吐量和用户友好性，能够快速识别高质量的治疗 候选人，特别是当一种疾病涉及多个器官相互作用的时候。要解决这些问题 挑战，我们建议利用我们独特的“微型三维生物打印” 技术和相关的柱/灌注板平台，包括带侧壁的384柱板 和缝隙(384柱板)和一个透明底部、384深的井板(384深井板) 静态有机体培养以及带有侧壁和狭缝的36柱培养板(36柱培养板)和36- 含储罐和微通道的灌流孔板(36个灌流孔板) 文化。我们建议的柱状/灌注板平台结合了“3D生物打印”和“微流控” 与更传统的3D细胞培养模型相比，这些功能提供了几个独特的优势 微流体模型。特别是，柱子/灌注板与标准384孔兼容 平板和现有的高通量筛选(HTS)设备(例如，自动荧光 显微镜和微型滴度井板阅读器)已经为用户所熟悉，这将显著降低 商业化的准入门槛。在这项拟议的研究中，人脑有机化合物(HBOS) 从诱导多能干细胞(IPSCs)来源的细胞作为模型系统，建立了 包括阿片类药物在内的化合物的发育神经毒性(DNT)预测性评估工具 还有酒。我们的核心假设是：(I)柱/灌注板上生物打印的HBOS可以保持 通过控制和模拟体内微环境来实现关键组织生物标记物，并使高 吞吐量、高含量细胞功能分析；(Ii)柱/灌注板上的HBOS可以模拟 药物和环境毒物对神经发育障碍的影响。的具体目标 本研究的目的是：(1)利用微型3D技术提高有机物培养的重现性 生物印花技术；(2)在柱板上原位建立整体有机物成像以实现高通量， 预测化合物的筛选；(3)建立柱板上有机物的超低温保存。我们 设想在柱子/灌注板平台上生物打印的人体有机物可以用作 在筛选治疗药物的同时最大限度地减少动物使用药物的有希望的疾病模型 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.