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 人类诱导多能干细胞（hiPSC），类器官，合成生物学和3D的融合 生物打印为患有器官衰竭的患者提供了患者特异性实验室生长器官的未来。 然而，为了实现这一器官工程愿景，生物制品研究人员迫切需要千升规模的 培养hiPSC以产生足够的材料来开始高通量实验。解决无数 器官构建、血管化、维持、成熟和表征的挑战将需要 几十年的艰苦研究。然而，在这种规模下获得患者特异性细胞仍然需要两个数量级的时间。 对于学术实验室来说，这在很大程度上是由于所需的昂贵的生长因子， 用于hiPSC维持和分化。此外，从干细胞产生类器官的现有方案 繁琐，缓慢和低效，限制了可用于3D生物打印的类器官的数量 应用.在这些拟议的研究中，我们详细介绍了新的方法，以显着降低干细胞的成本， 维持和增加类器官生产的规模。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表达无生长因子的hiPSC， 组成型活性生长因子受体，其可以在分化前切除。为了提高规模 和生产能力，我们提出工程化hiPSCs， 同时进行多细胞分化而不需要生长因子。为了实现这一点，我们提出了一个新的 随机Cre-lox重组系统上调三种转录因子之一，EOMES，Nkx3.1，或 ETV 2，以产生含有心肌细胞、成纤维细胞和内皮细胞的三细胞合成心脏类器官 单元格。通过在悬浮培养中培养数百万个这种合成的心脏类器官，我们将获得 用于3D生物打印的治疗相关量的致密细胞心肌生物墨水。我们下次将使用 合成的心脏类器官生物墨水，以获得人类规模的厚壁和血管化的心室模型。这些 生物打印的心室将被安置在一个定制的灌注生物反应器中，用于研究机械和电气 刺激可以维持血管灌注，增强心肌细胞成熟和排列，并影响器官- 鳞片收缩性和射血分数。高度可扩展的干细胞和类器官培养方法 这些都适用于许多器官系统，并可能彻底改变器官移植的规模和速度。 生物织物研究