Hatching Organoids for Continuous Tissue Production Pipelines

用于连续组织生产管道的孵化类器官

• 批准号: 10433762
• 负责人: Mark A. Skylar-Scott
• 金额: $ 31.48万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433762
• 关键词: 3-Dimensional; Address; Alginates; Autologous; Automobile Driving; Benign; Biological; Bioreactors; Blood Vessels; CD31 Antigens; CRISPR interference; Cell Culture Techniques; Cell Differentiation process; Cells; Chronic Disease; Complex; Coupling; Cues; Derivation procedure; Development; Encapsulated; Endothelial Cells; Endothelium; Engineering; Enzymes; Farm; Fibroblasts; Future; Generations; Genes; Genetic; Gold; Growth Factor; Harvest; Immune; Industrialization; Ink; Laboratories; Lyase; Medicine; Methods; Microscopy; Optics; Organ; Organ Donor; Organoids; Patients; Pharmacology; Population; Printing; Process; Production; Protocols documentation; Research; Safety; Series; Source; System; Techniques; Technology; Therapeutic; Thick; Time; Tissue Engineering; Tissues; Up-Regulation; Vision; Work; base; biofabrication; bioprinting; capsule; cell type; cohort; cost; density; directed differentiation; experimental study; flasks; hatching; in vivo; induced pluripotent stem cell; manufacturing process; morphogens; overexpression; screening; stem cell differentiation; stem cells; success; transcription factor; virtual

PROJECT SUMMARY Our evolving ability to bioprint cells to generate complex tissues and organs promises to revolutionize medicine by overcoming donor organ shortages and immune rejection. However, a major limiting factor faced by the bioprinting field is the complexity and cost in generating the billions to trillions of differentiated cells from induced pluripotent stem cells (iPSCs) to yield the necessary quantities of patient-specific cells for organ-scale bioprinting. We posit that organoids, owing to their mature cellular makeup, microarchitecture, and function, could serve as ideal building blocks for bioprinting organ-scale tissues. However, typical organoid protocols generate only 10-1,000 organoids, and their therapeutic potential is limited by batch-to-batch variability. While we have previously demonstrated that organoids can be rendered into printable and densely cellular bio-inks, organ scale bioprinting would require the synthesis of over ~1 million organoids. An optimal process for generating millions of organoids for bio-inks would A) be driven by cell-intrinsic mechanisms not requiring expensive exogenous growth and differentiation factors, B) would allow the temporal and spatial co- differentiation of stem cells to the different fates that normally cooperate in vivo resulting in organoids more likely to have the requisite functions to serve as optimal bio-inks and C) would be a continuous (i.e. batch-free) differentiation process with no down-time or batch-to-batch variability, wherein new cells are continuously added and mature organoids would be continuously extracted. To address the issue of media cost and co- differentiation, our preliminary work has yielded transcription factor overexpression for driving coordinate differentiation to divergent cell types in a growth factor-free fashion to yield mixed cell type organoids for bioprinting. To apply this process at the million-organoid scale, we propose here to develop an ‘organoid farm’, the first continuous organoid derivation process to generate millions of organoids in a continuous culture bioreactor system. Differentially fate-specific programmed iPSCs will be inserted into alginate capsules, continuously introduced into the culture, and developed to mature organoids. The input iPSCs will be programmed to spontaneously ‘hatch’ upon maturation via maturation stage-dependent expression of alginate lyase, a benign alginate-degrading enzyme, thus liberating the mature organoid in a form that is easily harvested from the ongoing culture. While the proof-of-concept experiments proposed herein utilizes mixtures of iPSCs programmed towards the endothelial and fibroblast fates that comprise the vascular tissue, this approach should be applicable to the generation of any organoid type for bioprinting virtually any tissue or organ. Further downstream applications of our organoid farm and hatching organoid techniques include automated organoid purification and pooled genetic or pharmacological screening.

项目摘要 我们不断发展的生物构图细胞产生复杂组织和器官的能力，有望彻底改变医学 通过克服供体器官短缺和免疫反应。但是，面临的主要限制因素 生物打印场是从诱导的数十亿到数万亿个分化细胞产生数十亿美元的复杂性和成本 多能干细胞（IPSC）产生了必要数量的患者特异性细胞以进行器官尺度 生物打印。我们肯定的是，由于器官成熟的细胞构，微结构和功能， 可以用作生物打印器官大小组织的理想基础。但是，典型的器官方案 仅产生10-1,000个类器官，其治疗潜力受批次之间变异性的限制。尽管 我们以前已经证明，可以将类器官渲染到可打印和垂直的细胞生物墨水中， 器官尺度的生物打印将需要综合约100万个器官。一个最佳过程 生成数百万个的生物墨水的器官将a）不需要细胞内部机制驱动 昂贵的外源生长和分化因子，b）将允许临时和空间共同 干细胞与通常在体内协调的不同命运的分化，可能会导致类器官 具有必要的功能来充当最佳的生物墨水，而c）将是连续的（即无批量） 没有停机时间或批处理变异性的分化过程，其中不断添加新单元格 和成熟的类器官将连续提取。解决媒体成本和共同的问题 差异化，我们的初步工作产生了转录因子过表达以驱动坐标 以无生长因子方式区分不同的细胞类型，以产生混合细胞类型的类器官 生物打印。为了在百万 - 核尺度上应用此过程，我们在这里建议开发一个“器官农场”， 在连续培养物中产生数百万个类器官的第一个连续类器官推导过程 生物反应器系统。差异性脂肪特异的编程IPSC将插入藻酸盐胶囊， 连续引入培养物，并发展为成熟的器官。输入IPSC将是 通过成熟阶段依赖性算法表达成熟后，编程为赞助商 - 超支“孵化” 裂解酶，一种良性算法降解酶，因此以容易收获的形式释放成熟的类器官 来自正在进行的文化。本文提出的概念验证实验利用IPSC的混合物 针对构成血管组织的内皮和成纤维细胞命运的编程，该方法应 适用于生成任何类型类型的生物涂纸几乎任何组织或器官。更远 我们的类器官农场和孵化器官技术的下游应用包括自动化器官 纯化和合并的遗传或药理筛查。