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.

项目概要 人类诱导多能干细胞 (hiPSC)、类器官、合成生物学和 3D 的融合 生物打印有望为患有器官衰竭的患者提供针对患者的实验室培养器官的未来。 然而，为了实现这一器官工程愿景，生物制造研究人员迫切需要千升规模的 hiPSC 培养物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、维护、成熟和表征方面的挑战将需要 几十年的潜心研究。然而，以这种规模获得患者特异性细胞仍然需要两个数量级 对于学术实验室来说过于昂贵，这在很大程度上是由于所需的昂贵的生长因子 用于 hiPSC 的维护和分化。此外，现有的从干细胞生成类器官的方案 繁琐、缓慢且低效，限制了可用于 3D 生物打印的类器官的数量 应用程序。在这些拟议的研究中，我们详细介绍了大幅降低干细胞成本的新方法 维持并增加类器官生产规模。降低大规模 hiPSC 生长的成本 通过两个数量级，我们建议通过对无生长因子的 hiPSC 进行编程来表达它们 组成型活性生长因子受体，可以在分化前切除。为提升规模 和生成多细胞心脏类器官的吞吐量，我们建议对 hiPSC 进行工程设计以进行 无需生长因子即可同时进行多细胞分化。为了实现这一目标，我们提出了一部小说 随机 Cre-lox 重组系统上调三个转录因子 EOMES、Nkx3.1 之一或 ETV2，生成含有心肌细胞、成纤维细胞和内皮细胞的三细胞合成心脏类器官 细胞，分别。通过在悬浮培养物中培养数百万个此类合成心脏类器官，我们将得出 用于 3D 生物打印的治疗相关量的致密细胞心肌生物墨水。我们接下来会用到 合成心脏类器官生物墨水，以获得人体规模、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定制的灌注生物反应器中，用于研究机械和电气如何 刺激可以维持血管灌注，增强心肌细胞的成熟和排列，并影响器官 鳞片收缩力和射血分数。提出了高度可扩展的干细胞和类器官培养方法 这些适用于许多器官系统，并且可以彻底改变器官的规模和速度 生物制造研究。