Hatching Organoids for Continuous Tissue Production Pipelines

项目总结 我们生物打印细胞以产生复杂组织和器官的进化能力有望给医学带来革命性的变化 通过克服供体器官短缺和免疫排斥。然而，美国政府面临的一个主要限制因素是 生物打印领域是从诱导的细胞中产生数十亿到数万亿分化细胞的复杂性和成本 多能干细胞(IPSCs)产生器官规模所需数量的患者特异性细胞 生物打印。我们假设有机化合物，由于它们成熟的细胞组成，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，典型的有机体方案 仅产生10-1,000个有机化合物，其治疗潜力受到批次之间变异性的限制。而当 我们之前已经证明，有机化合物可以制成可打印的密集细胞生物墨水， 器官规模的生物打印需要合成100多万种有机化合物。一种最佳的生产工艺 产生数百万种用于生物墨水的有机物将A)由细胞内在机制驱动，而不需要 昂贵的外源生长和分化因素，B)将允许时间和空间协同 干细胞分化为体内正常合作的不同命运，更有可能产生有机物 具有用作最佳生物油墨的必要功能，C)将是连续的(即无批次) 没有停机时间或批次之间变化的差异化过程，其中不断添加新的单元 而成熟的有机化合物将被连续提取。为了解决媒体成本问题并共同- 分化，我们的初步工作已经产生了驱动坐标的转录因子的过表达 以无生长因子的方式分化为不同类型的细胞，以产生混合细胞型有机体 生物打印。为了在百万级有机化合物规模上应用这一过程，我们建议在这里开发一个“有机化合物农场”， 第一个在连续培养中产生数百万个有机化合物的连续衍生过程 生物反应器系统。不同命运特异性编程的IPSCs将被插入到藻酸盐胶囊中， 不断地引入培养物，并发展成成熟的有机化合物。输入的IPSC将是 通过成熟阶段依赖的海藻酸盐表达，编程在成熟时自发‘孵化’ 裂解酶，一种良性的藻酸盐降解酶，从而以一种容易获得的形式释放成熟的类有机物 从持续的文化中解脱出来。虽然这里提出的概念验证实验使用了IPSC的混合物 对构成血管组织的内皮细胞和成纤维细胞命运进行编程，这种方法应该 适用于生物打印几乎任何组织或器官的任何有机类型的生成。进一步 我们的有机农场和孵化有机技术的下游应用包括自动有机 纯化和联合遗传或药理学筛选。

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.