Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

从肝脏分离出的原代肝细胞与体内非常相似天然肝肝细胞，但它们在2D培养中具有寿命有限的缺点。尽管可以使用夹层培养和具有间充质干细胞（MSC）作为有吸引力的辅助细胞来源来延长寿命的3D类器官，但它不能完全复制体内结构。此外，由于低氧应激，长期3D培养会导致细胞死亡。因此，为了克服2D和3D类器官的缺点，我们尝试使用将藻酸盐水凝胶与原代肝细胞和MSC结合使用的3D打印技术。分离的肝细胞的活力超过90％，并且细胞存活7天，而MSC的3D肝结构没有形态变化。与2D系统相比，在3D肝脏结构中，功能性肝基因和蛋白质的表达水平长达7天。这些结果表明，MSC分泌的3D生物打印技术和旁分泌分子均支持肝细胞的长期培养而无形态变化。因此，该技术在形成多细胞聚集体的同时允许细胞的广泛扩增，可以应用于药物筛选，并且可以是开发人造肝脏的有效方法。