Institute of Active Polymers and Berlin-Brandenburg Centre for Regenerative Therapies, Helmholtz-Zentrum Hereon, Teltow, Germany.
Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany.
Clin Hemorheol Microcirc. 2021;79(1):217-230. doi: 10.3233/CH-219111.
Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70% confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90% confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.
人诱导多能干细胞（hiPSCs）是一种很有前途的细胞来源，可以生成患者特异性的肺类器官，因为它们具有优越的分化潜力。然而，目前的 3D 细胞培养方法繁琐且耗时，成功率低，批次间差异大。在这里，我们通过系统地调整初始细胞汇合度和细胞分布的均一性来探索肺芽类器官的建立。比较了单细胞和细胞团接种的效率。我们没有采用传统的 3D 培养方法，而是建立了 2.5D 类器官培养方法，通过显微镜直接监测内部结构。结果发现，诱导前的细胞汇合度和分布是两个关键参数，它们强烈影响 hiPSC 的分化轨迹。在单细胞接种组中，当 hiPSC 以 70%的汇合度均匀分布（SC_70%_hom）或在细胞团接种组中以 90%的汇合度不均匀分布（CL_90%_het）时，可观察到具有 NKX2.1 阳性表达的肺芽类器官，早在诱导后 9 天即可观察到。这些结果表明，在以单细胞接种 hiPSCs 时，成功形成肺芽类器官需要适度的细胞汇合度和均匀分布，而当以细胞团接种 hiPSCs 时，高汇合度将是促进肺类器官形成的一个显著因素。具有明确培养条件的 2.5D 类器官可能成为一种简单、高效和有价值的工具，有助于药物筛选、疾病建模和个性化医疗。
