He Yuting, Liu Qin, Zhou Yanyan, Bao Ji
Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, NHC, West China Hospital, Sichuan University, No. 37, Guoxue Alley, Wuhou District, Chengdu, 610041, Sichuan Province, China.
Department of Pathology, West China Second Hospital, Sichuan University, Chengdu, 610041, Sichuan Province, China.
Tissue Eng Regen Med. 2025 Jul 14. doi: 10.1007/s13770-025-00736-7.
Porcine primary hepatocytes are vital for liver therapy due to their procurement ease and robust functions. However, they rapidly dedifferentiate in vitro, challenging large-scale maintenance. This study aims to enhance the long-term survival and self-renewal of primary porcine hepatocytes by generating spheroids using a rocker system and optimizing conditions with HUVECs and Roof plate-specific spondin 1 (RSPO1).
Primary hepatocytes were co-cultured with HUVECs in a rocker system using serum-free medium to form spheroids, mimicking their native microenvironment. RSPO1 was added to the media to promote hepatocyte signaling and proliferation. Then pheroids were generated with HUVECs overexpressing RSPO1 (R-HUVECs). The effects of these conditions on the viability, hepatic function, and proliferation of hepatocytes were evaluated.
The 3D environment and RSPO1 synergistically enhanced hepatocyte proliferation and maintained essential liver functions long-term. Co-culture with HUVECs and R-HUVECs promoted spheroid formation, with spheroids surviving and functioning for 28 days.
Large-scale cultured hepatocyte + R-HUVEC spheroids address in vitro challenges of scale, yield, and functional sustainability, promising advances in liver therapeutics and drug development.
猪原代肝细胞因其易于获取和强大的功能，对肝脏治疗至关重要。然而，它们在体外会迅速去分化，这对大规模培养构成挑战。本研究旨在通过使用摇床系统生成球体，并与脐静脉内皮细胞（HUVECs）和顶板特异性促分泌素1（RSPO1）优化培养条件，来提高猪原代肝细胞的长期存活率和自我更新能力。
原代肝细胞与HUVECs在摇床系统中使用无血清培养基共培养以形成球体，模拟其天然微环境。将RSPO1添加到培养基中以促进肝细胞信号传导和增殖。然后用过量表达RSPO1的HUVECs（R-HUVECs）生成球体。评估这些条件对肝细胞活力、肝功能和增殖的影响。
三维环境和RSPO1协同增强了肝细胞增殖，并长期维持基本肝功能。与HUVECs和R-HUVECs共培养促进了球体形成，球体存活并发挥功能达28天。
大规模培养的肝细胞+R-HUVEC球体解决了体外培养在规模、产量和功能可持续性方面的挑战，有望推动肝脏治疗和药物开发的进展。
