Paracrine Factor Local Gradient‐Generating System for Engineering Perfusable Vascularized Hepatocyte Tissues with Perfusion‐Induced Proliferation

Donor organ shortages drive an urgent need for engineered hepatocyte tissues; however, functional vascular integration remains a major bottleneck in liver tissue engineering. Current vascularization strategies struggle to achieve perfusable microvessels that penetrate hepatocyte tissue. Furthermore, recapitulating hepatic regeneration in vitro remains challenging. This study presents a paracrine factor local gradient (PFLG)-generating system that constructs vascularized, perfusable hepatocyte tissues and recapitulates the perfusion-mediated proliferative activity of primary hepatocytes. The PFLG-generating platform integrates fibroblast-loaded cryogels with a microfluidic device to direct angiogenesis prior to hepatocyte seeding, thereby enabling microvessels to penetrate 3D hepatocyte tissue. Within the vascularized constructs, microvessels directly penetrated the hepatocyte parenchyma, recapitulating the intimate hepatocyte-microvessel contacts observed in vivo. These constructs enhanced hepatocyte polarity and promoted functional bile canaliculi formation. Importantly, perfusion culture induced robust hepatocyte proliferative activity, as evidenced by a significant increase in Ki67-positive hepatocytes, including mitotic cells, without loss of cellular polarity. By contrast, this proliferative response was minimal under static conditions. Time-lapse imaging and functional assays confirmed perfusion through penetrating microvessels. These findings demonstrate that perfusion-mediated cues are essential for inducing hepatocyte cell-cycle re-entry while maintaining functional polarity. This modular and programmable culture platform lays the foundation for scaling-up toward transplantable liver tissues.