Enhancing Long-Term Survival and Self-Renewal of Primary Hepatocytes via Rapid Spheroid Formation Using Rocker System...

Area of Science:

• Hepatology
• Cell Biology
• Biotechnology

Background:

• Porcine primary hepatocytes are crucial for liver therapy due to accessibility and function.
• In vitro dedifferentiation limits large-scale maintenance of primary hepatocytes.
• This study addresses the challenge of maintaining hepatocyte viability and self-renewal in culture.

Purpose of the Study:

• To enhance long-term survival and self-renewal of primary porcine hepatocytes.
• To develop scalable hepatocyte culture methods for therapeutic applications.
• To optimize spheroid culture conditions using HUVECs and RSPO1.

Main Methods:

• Primary porcine hepatocytes were co-cultured with HUVECs in a rocker system to form spheroids.
• Serum-free media and Roof plate-specific spondin 1 (RSPO1) were used to optimize conditions.
• Hepatocytes were also cultured with HUVECs overexpressing RSPO1 (R-HUVECs) to assess effects on viability, function, and proliferation.

Main Results:

• The 3D spheroid environment combined with RSPO1 synergistically boosted hepatocyte proliferation and sustained liver functions.
• Co-culture with HUVECs and R-HUVECs facilitated spheroid formation.
• Spheroids demonstrated survival and functionality for up to 28 days.

Conclusions:

• Large-scale cultured hepatocyte + R-HUVEC spheroids overcome in vitro limitations in scale, yield, and functional sustainability.
• This approach offers promising advancements for liver therapeutics and drug development.
• Optimized spheroid culture enhances the potential of primary hepatocytes in regenerative medicine.