High-Throughput Culture Method of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Cells.
High-Throughput Culture Method of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Cells.
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DOI：
10.1089/ten.tec.2021.0174
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发表时间：
2021-12-01
期刊：
影响因子：
--
通讯作者：
Ott, Harald C
中科院分区：
文献类型：
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作者：
Becerra, David;Wu, Tong;Ott, Harald C
肺再生依赖于肺祖细胞的可用性。需要大量的自我更新的、患者特异性的诱导多能干细胞衍生的肺泡上皮细胞（iPSC-AEC）来充分地再细胞化整个器官构建体。产生功能性iPSC-AEC的现有方法对于大规模细胞生产是不可行的。我们提出了一种新的方案来生产iPSC-AEC，这是可扩展的整个器官再生。用具有荧光报告基因的遗传修饰的iPSC进行iPSC的分化，所述iPSC在模拟肺发育的逐步方案中经历分化。将细胞纯化、分选并包埋在液体基质胶前体中以形成粘附液滴或形成载有细胞的基质胶球状体，随后将其转移到具有作为漂浮液滴的培养基的转瓶中。培养后，分离iPSC-AEC的单层球体以形成单细胞悬浮液。将来自两种培养条件的相等数量的iPSC-AEC接种到脱细胞肺支架中。在漂浮液滴中培养的IPSC-AEC比在粘附液滴中培养的IPSC-AEC增殖显著更快，细胞总数和Ki 67表达显著更高。在两种培养条件下均观察到远端肺标志物的等效表达。从两个培养组再细胞化的肺具有相似的组织学外观。采用浮动液滴法更换培养基所需时间显著缩短，且更具成本效益。与先前的培养方法相比，浮动液滴培养方法表现出增强的增殖能力、稳定的远端肺上皮表型和减少的资源。在这项研究中，我们提供了一种用于全肺构建体再生的iPSC-AEC生产的方法。我们描述了一种新的培养方法，用于诱导多能干细胞衍生的肺泡上皮细胞（AEC）扩增，与先前描述的方法相比，具有增强的增殖能力和减少的资源需求。该方法可扩展用于人类全肺再生生物工程，或者可以自动化用于商业细胞生产。这种培养方法可能对I型AEC与II型AEC的区分有影响。
Lung regeneration is dependent on the availability of progenitor lung cells. Large numbers of self-renewing, patient-specific induced pluripotent stem cell-derived alveolar epithelial cells (iPSC-AECs) are needed to adequately recellularize whole-organ constructs. Prior methods to generate functional iPSC-AECs are not feasible for large-scale cell production. We present a novel protocol to produce iPSC-AECs, which is scalable for whole-organ regeneration. Differentiation of iPSCs was performed with genetically modified iPSCs with fluorescent reporters, which underwent differentiation in a stepwise protocol mimicking lung development. Cells were purified, sorted, and embedded in a liquid Matrigel precursor either to form adherent droplets or to form cell-laden Matrigel spheroids, which were subsequently transferred to spinner flasks with media as floating droplets. After culture, monolayer spheres of iPSC-AECs were isolated to form single cell suspensions. Equal numbers of iPSC-AECs from the two culture conditions were seeded into decellularized lung scaffolds. IPSC-AECs cultured in floating droplets were significantly more proliferative than those in adherent droplets, with significantly higher total cell counts and Ki67 expression. Equivalent expression of the distal lung markers was observed for both culture conditions. Lungs recellularized from both culture groups had similar histological appearance. Media changes took significantly less time with the floating droplet method and was more cost effective. The floating droplet culture method demonstrated enhanced proliferative capacity, stable distal lung epithelial phenotype, and reduced resources compared with prior culture methods. In this study, we provide a means for iPSC-AEC production for regeneration of whole-lung constructs. Impact statement We describe a novel culture method for induced pluripotent stem cell-derived alveolar epithelial cell (AEC) expansion with enhanced proliferative capacity and reduced resource requirements compared with previously described methods. This method is scalable for human whole-lung regeneration bioengineering or could be automated for commercial cell production. This culture method may have implications for the differentiation of type I AECs from type II AECs.