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.

项目概要 我们不断发展的生物打印细胞以生成复杂组织和器官的能力有望彻底改变医学 然而，克服供体器官短缺和免疫排斥是其面临的一个主要限制因素。 生物打印领域的关键在于通过诱导产生数十亿至数万亿分化细胞的复杂性和成本 多能干细胞（iPSC）产生器官规模所需数量的患者特异性细胞 我们认为类器官由于其成熟的细胞构成、微结构和功能， 可以作为生物打印器官规模组织的理想构建模块然而，典型的类器官方案。 仅产生 10-1,000 个类器官，并且其治疗潜力受到批次间差异的限制。 我们之前已经证明类器官可以转化为可打印的密集细胞生物墨水， 器官规模的生物打印需要合成超过 100 万个类器官。 产生数百万个用于生物墨水的类器官将 A）由细胞内在机制驱动，不需要 昂贵的外源生长和分化因子，B）将允许时间和空间共同 干细胞分化为通常在体内协同作用的不同命运，更有可能产生类器官 具有作为最佳生物墨水所需的功能，并且 C) 将是连续的（即无批次） 分化过程没有停机时间或批次之间的差异，但新细胞会不断添加 并不断提取成熟的类器官，以解决媒体成本和合作问题。 分化，我们的初步工作已经产生了用于驱动协调的转录因子过表达 以无生长因子的方式分化为不同的细胞类型，产生混合细胞类型的类器官 为了在百万个类器官规模上应用这一过程，我们在此建议开发一个“类器官农场”， 第一个连续类器官衍生过程，可在连续培养中产生数百万个类器官 差异命运特异性编程的 iPSC 将被插入藻酸盐胶囊中， 不断引入培养物中，并发育成成熟的类器官。 通过海藻酸盐的成熟阶段依赖性表达，编程为在成熟时自发“孵化” 裂解酶，一种良性藻酸盐降解酶，从而以易于收获的形式释放成熟的类器官 虽然本文提出的概念验证实验利用了 iPSC 的混合物。 针对构成血管组织的内皮细胞和成纤维细胞的命运进行编程，这种方法应该 适用于生成任何类器官类型，用于几乎任何组织或器官的生物打印。 我们的类器官农场和孵化类器官技术的下游应用包括自动化类器官 纯化和汇总遗传或药理学筛选。