Hatching Organoids for Continuous Tissue Production Pipelines

项目摘要 我们不断发展的生物打印细胞以生成复杂组织和器官的能力有望彻底改变医学 克服捐赠器官短缺和免疫排斥。然而，一个主要的限制因素所面临的 生物打印领域的最大挑战是从诱导的细胞中产生数十亿至数万亿分化细胞的复杂性和成本。 多能干细胞（iPSC），以产生用于器官规模移植的必要数量的患者特异性细胞。 生物打印我们认为，类器官，由于其成熟的细胞组成，微结构和功能， 可以作为生物打印器官规模组织的理想构建块。然而，典型的类器官方案 仅产生10- 1，000个类器官，并且它们的治疗潜力受到批次间差异的限制。而 我们先前已经证明类器官可以被制成可印刷的和密集的细胞生物墨水， 器官规模的生物打印将需要合成超过100万个类器官。一种最佳工艺， 产生数百万个用于生物墨水的类器官将A）由细胞内在机制驱动， 昂贵的外源性生长和分化因子，B）将允许时间和空间的共同作用， 干细胞分化为不同的命运，这些命运通常在体内合作，更可能导致类器官 具有用作最佳生物油墨的必要功能，并且C）将是连续的（即无批次的） 没有停机时间或批次间变异性的分化过程，其中连续添加新细胞 并且成熟的类器官将被连续提取。为了解决媒体成本和合作问题， 分化，我们的初步工作已经产生了转录因子过表达的驱动坐标 以无生长因子的方式分化为不同的细胞类型，以产生混合细胞类型的类器官， 生物打印为了将这一过程应用于百万个类器官规模，我们在这里建议开发一个“类器官农场”， 第一个连续的类器官衍生过程，在连续培养中产生数百万个类器官 生物反应器系统不同命运特异性编程的iPSC将被插入藻酸盐胶囊中， 连续引入培养物中，并发育成成熟的类器官。输入的iPSC将是 通过藻酸盐的成熟阶段依赖性表达， 裂解酶，一种良性的藻酸盐降解酶，从而以容易收获的形式释放成熟的类器官 从正在进行的文化。虽然本文提出的概念验证实验利用iPSC的混合物， 对于包括血管组织在内的内皮细胞和成纤维细胞的命运，这种方法应该 可应用于产生用于生物打印几乎任何组织或器官的任何类器官类型。进一步 我们的类器官农场和孵化类器官技术的下游应用包括自动化类器官 纯化和合并的遗传或药理学筛选。

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.