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Background: <p>Porcine primary hepatocytes are vital for liver therapy due to their procurement ease and robust functions. However, they rapidly dedifferentiate <i>in vitro</i>, 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).</p> Methods: <p>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.</p> Results: <p>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&#xa0;days.</p> Conclusion: <p>Large-scale cultured hepatocyte + R-HUVEC spheroids address <i>in vitro</i> challenges of scale, yield, and functional sustainability, promising advances in liver therapeutics and drug development.</p>

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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

  • 期刊论文

  • 发表日期：

    2025年7月14日

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  • Yuting He,
  • Qin Liu,
  • Yanyan Zhou,
  • Ji Bao

摘要

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.