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: 10667497
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667497
• 关键词: 3-Dimensional; Address; Alginates; Autologous; Automobile Driving; Benign; Biological; Bioreactors; Blood Vessels; CD31 Antigens; CRISPR interference; Cell Culture Techniques; Cell Differentiation Induction; Cell Differentiation process; Cells; Chronic Disease; Complex; Coupling; Cues; Derivation procedure; Development; Encapsulated; Endothelial Cells; Endothelium; Engineering; Enzymes; Farm; Fibroblasts; Future; Generations; Genes; Genetic; Growth Factor; Harvest; Immune; Industrialization; Laboratories; Lyase; Medicine; Methods; Microscopy; Optics; Organ; Organ Donor; Organoids; Patients; Pluripotent Stem Cells; Population; Printing; Process; Production; Protocols documentation; Research; Safety; Series; Source; System; Techniques; Technology; Therapeutic; Thick; Time; Tissue Engineering; Tissues; Up-Regulation; Vascularization; Vision; Work; base; biofabrication; bioink; bioprinting; capsule; cell type; cohort; cost; density; directed differentiation; experimental study; flasks; hatching; in vivo; induced pluripotent stem cell; large scale production; manufacturing process; morphogens; overexpression; pharmacologic; screening; self assembly; 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.

项目总结

项目成果

Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

• DOI: 10.1002/adhm.202201138
• 发表时间: 2022-12
• 期刊: ADVANCED HEALTHCARE MATERIALS
• 影响因子: 10
• 作者: Ho, Debbie L. L.;Lee, Stacey;Du, Jianyi;Weiss, Jonathan D. D.;Tam, Tony;Sinha, Soham;Klinger, Danielle;Devine, Sean;Hamfeldt, Art;Leng, Hope T. T.;Herrmann, Jessica E. E.;He, Mengdi;Fradkin, Lee G. G.;Tan, Tze Kai;Standish, David;Tomasello, Peter;Traul, Donald;Dianat, Noushin;Ladi, Rukmini;Vicard, Quentin;Katikireddy, Kishore;Skylar-Scott, Mark A. A.
• 通讯作者: Skylar-Scott, Mark A. A.