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

Donor organ shortages drive the urgent need for engineered hepatocyte tissues, yet functional vascular integration remains a major bottleneck in liver tissue engineering. Current vascularization strategies struggle to achieve perfusable microvessels that penetrate hepatocyte tissue. Furthermore, in vitro recapitulation of hepatic regeneration remains a major challenge. This study presents a paracrine factor local gradient (PFLG)-generating system that constructs vascularized, perfusable hepatocyte tissues and recapitulates the perfusion-mediated proliferative capacity 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 three-dimensional hepatocyte tissue. Within the vascularized constructs, microvessels directly penetrated the hepatocyte parenchyma, recapitulating intimate hepatocyte-microvessel contact in vivo. These constructs enhanced hepatocyte polarity and functional bile canaliculi. Importantly, under perfusion culture, robust hepatocyte proliferation was induced, with Ki67-positive hepatocytes increasing significantly, including mitotic cells while preserving 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 proliferation while maintaining functional polarity. This modular and programmable culture platform lays the foundation for scale-up toward transplantable liver tissues.