Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15–18%, without any detrimental effect on cell viability. Despite utilizing 10–50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC₅₀ values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 μM and 25.4 ± 8.3 μM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

中文翻译：

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通过微阵列 3D 生物打印在柱板平台上可重复生成人类肝脏类器官 (HLO)

人类肝脏类器官（HLO）在重现体内肝脏组织的结构和功能方面具有巨大的潜力。然而，在 6/24 孔板中形成基质胶圆顶的 HLO 传统培养方法存在技术局限性，例如在临床和药理学实验室环境中用于预测评估化合物的基于类器官的测定中成本高且通量低。为了解决这些问题，我们开发了一种独特的微阵列 3D 生物打印方案，在具有侧壁和狭缝的柱板上仿生水凝胶中的祖细胞，再加上透明底部的 384 深孔板，用于 HLO 的放大生产。微阵列 3D 生物打印是一种基于液滴的打印技术，用于在柱板上生成大量小类器官，用于预测肝毒性测定。将由人 iPSC 分化而来的前肠细胞与基质胶混合，然后快速均匀地打印在柱板上，导致变异系数 (CV) 值在 15-18% 范围内，且对细胞活力没有任何不利影响。尽管与 6/24 孔板中基质胶圆顶中的对应细胞培养物体积相比，使用的细胞培养体积小 10-50 倍，但在柱板上分化的 HLO 表现出相似的形态和卓越的功能，这可能是由于营养物和氧气在规模小。第 25 天的 HLO 在支柱板上的活力、白蛋白分泌、CYP3A4 活性和药物毒性测试方面表现强劲且功能齐全，所有这些都具有较低的 CV 值。根据三项独立的原位评估试验，计算出索拉非尼和他莫昔芬的IC ₅₀值分别为 6.2 ± 1.6 μM 和 25.4 ± 8.3 μM。因此，我们独特的 3D 生物打印和柱板上的微型类器官培养可用于放大、可重复生成 HLO，只需最少的手动干预即可对化合物肝毒性进行高通量评估。