Enhancing Long-Term Survival and Self-Renewal of Primary Hepatocytes via Rapid Spheroid Formation Using Rocker System through Co-Culturing with HUVEC Over-Expressing RSPO1.

BACKGROUND

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).

METHODS

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.

RESULTS

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.

CONCLUSION

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.