Trillion cell culture to fuel organ biofabrication

万亿细胞培养为器官生物制造提供燃料

• 批准号: 10473259
• 负责人: Mark A. Skylar-Scott
• 金额: $ 141.66万
• 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: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10473259
• 关键词: 3-Dimensional; Affect; Autologous; Bioreactors; Blood Vessels; Cardiac; Cardiac Myocytes; Cell Culture Techniques; Cell Differentiation process; Cell Maintenance; Cells; Cre lox recombination system; Custom; Development; EFRAC; Electric Stimulation; Endothelial Cells; Engineering; Fibroblasts; Future; Goals; Growth Factor; Growth Factor Receptors; Human; Human Engineering; Laboratories; Maintenance; Mechanical Stimulation; Methods; Modeling; Myocardial; Organ; Organ Transplantation; Organ failure; Organoids; Patients; Perfusion; Production; Protocols documentation; Research; Research Personnel; Suspension Culture; Therapeutic; Thick; Vascularization; Vision; biofabrication; bioink; bioprinting; body system; cell type; cost; induced pluripotent stem cell; novel; stem cell growth; stem cells; synthetic biology; transcription factor

PROJECT SUMMARY The convergence of human induced pluripotent stem cells (hiPSCs), organoids, synthetic biology and 3D bioprinting promises a future of patient-specific lab-grown organs for patients suffering from organ failure. However, to realize this organ engineering vision, biofabrication researchers sorely need thousand-liter-scale cultures of hiPSCs to generate enough material to begin high-throughput experimentation. Solving the myriad challenges in organ construction, vascularization, maintenance, maturation, and characterization will require decades of painstaking research. Yet, deriving patient-specific cells at this scale remains two orders of magnitude too expensive for academic laboratories due, in large part, to the expensive growth factors required for hiPSC maintenance and differentiation. Furthermore, existing protocols to generate organoids from stem cells are cumbersome, slow, and inefficient, limiting the number of organoids that can be derived for 3D bioprinting applications. In these proposed studies, we detail novel methods to dramatically reduce the cost of stem cell maintenance and increase the scale of organoid production. To reduce the costs of large-scale hiPSC growth by two orders of magnitude, we propose to engineer growth factor-free hiPSCs by programming them to express constitutively-active growth factor receptors which can be excised prior to differentiation. To enhance the scale and throughput in generating multicellular cardiac organoids, we propose engineering hiPSCs to undergo simultaneous multicellular differentiation without requiring growth factors. To achieve this, we propose a novel stochastic Cre-lox recombination system to upregulate one-of-three transcription factors, EOMES, Nkx3.1, or ETV2, to generate tri-cellular synthetic cardiac organoids containing cardiomyocytes, fibroblasts, and endothelial cells, respectively. By culturing millions of these synthetic cardiac organoids in suspension culture, we will derive therapeutically-relevant quantities of densely cellular myocardial bioink for 3D bioprinting. We will next use synthetic cardiac organoid bioink to derive a human-scale, thick-walled, and vascularized ventricle model. These bioprinted ventricles will be housed in a custom perfusion bioreactor for studying how mechanical and electrical stimulation can maintain vascular perfusion, enhance cardiomyocyte maturation and alignment, and affect organ- scale contractility and ejection fraction. The highly scalable stem cell and organoid culture methods presented here are applicable across many organ systems, and could revolutionize the scale and pace of organ biofabrication research.

项目摘要 人类诱导的多能干细胞（HIPSC），器官，合成生物学和3D的收敛性 生物打印有望为患有器官衰竭的患者的患者特异性实验室器官的未来。 但是，为了实现这种器官工程愿景，生物制造研究人员非常需要千分表 HIPSC的培养物生成足够的材料以开始高通量实验。解决无数 器官构建，血管化，维护，成熟和表征的挑战将需要 数十年来艰苦的研究。然而，以此规模得出患者特异性细胞仍然是两个阶 对于学术实验室而言，由于所需的昂贵增长因素，学术实验室太昂贵了 用于HIPSC维护和差异化。此外，现有的方案从干细胞中生成类器官 繁琐，缓慢且效率低下，限制了可用于3D生物打印的类器官的数量 申请。在这些提出的研究中，我们详细介绍了大幅降低干细胞成本的新方法 维护和增加器官生产的规模。降低大规模HIPSC增长的成本 通过两个数量级，我们建议通过编程来设计无增长因子的HIPSC来表达 组成型活性生长因子受体，可以在分化之前切除。提高规模 和生成多细胞心脏器官的吞吐量，我们建议工程hipsc进行 同时进行多细胞分化，而无需生长因子。为了实现这一目标，我们提出了一本小说 随机CRE-Lox重组系统，以上调三个转录因子，EOMES，NKX3.1或 ETV2，生成含有心肌细胞，成纤维细胞和内皮的三个细胞合成心脏器官 细胞分别。通过在悬浮培养中培养数百万这些合成心脏器官，我们将得出 与治疗相关的密集量，可用于3D生物打印。我们接下来使用 合成心脏器官生物互联，以推导人尺度，厚壁和血管化心室模型。这些 生物打印的心室将安装在定制灌注生物反应器中，用于研究机械和电气 刺激可以维持血管灌注，增强心肌细胞的成熟和比对，并影响器官 比例收缩性和射血分数。提出的高度可扩展的干细胞和类器官培养方法 这里适用于许多器官系统，可以改变器官的规模和步伐 生物制造研究。