Trillion cell culture to fuel organ biofabrication

项目总结 人类诱导多能干细胞、有机体、合成生物学和3D的融合 生物打印为器官衰竭患者承诺了特定于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1000升规模的 培养HiPSCs以产生足够的材料来开始高通量实验。解决无数的问题 器官构建、血管化、维持、成熟和表征方面的挑战将需要 几十年的艰苦研究。然而，在这种规模下提取患者特有的细胞仍然是两个数量级的 对于学术实验室来说，数量级太昂贵了，这在很大程度上是因为所需的昂贵的增长因素 用于HiPSC的维持和分化。此外，现有的从干细胞产生有机类化合物的方案 繁琐、缓慢和低效，限制了可用于3D生物打印的有机类化合物的数量 申请。在这些拟议的研究中，我们详细介绍了显著降低干细胞成本的新方法。 保持和提高有机化合物生产规模。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不含生长因子的HiPSCs，使其表达 具有结构性活性的生长因子受体，可在分化前切除。提升规模 在产生多细胞心脏器官类化合物方面，我们建议工程hPSCs进行 同时多细胞分化，不需要生长因子。为了实现这一点，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中的一个，Eome，Nkx3.1，或 ETV2，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器官 细胞。通过在悬浮培养中培养数百万个这样的合成心脏器官，我们将衍生出 用于3D生物打印的致密细胞心肌生物墨水的治疗相关数量。我们接下来将使用 合成心脏有机体生物墨水，以获得人体大小、厚壁和血管化的脑室模型。这些 生物打印的脑室将被安置在定制的灌流生物反应器中，用于研究机械和电气 刺激可以维持血管灌流，促进心肌细胞成熟和排列，并影响器官- 鳞片收缩能力和射血分数。提供了高度可伸缩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的规模和速度 生物制造研究。

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.