Microneedle array facilitates hepatic sinusoid construction in a large-scale liver-acinus-chip microsystem

The liver is essential for metabolism of blood glucose and drugs. To study liver structure–function at the microscale, researchers distinguish hepatic lobule and hepatic acinus. The hepatic acinus, centered on the portal area and defined by both structural and functional properties, features a dual blood supply via the portal vein (PV) and hepatic artery (HA) with outflow to the central vein (CV), generating oxygen and nutrient gradients that drive zonated differences in metabolism, gene expression, and sinusoid morphology. Conventional animal models and 2D/3D cultures have limitations due to interspecies differences and inability to reproduce in vivo-like 3D microenvironments and perfusable vasculature. Organ-on-a-chip platforms better mimic physiological microenvironments, yet liver chips still face challenges in constructing peripheral vascular systems and especially hepatic sinusoids, and in reproducing the tri-vascular HA–PV–CV microcirculation and resulting gradients. The authors aim to construct perfusable hepatic sinusoids within a large-scale liver-acinus-chip that incorporates a dual blood supply, evaluate the impact on flows, cell viability, tissue microstructure, and hepatocyte metabolism, and preliminarily examine effects of oxygen and glucose gradients and drug testing applicability.

Early liver chips achieved only 2D dynamic endothelial cell culture with media flowing over cell layers. Subsequent approaches fabricated sinusoid-like scaffolds using photolithography, laser cavitation, dielectrophoresis, and magnetic induction, but were limited by coarse resolution (>100 µm) or inability to make complex 3D structures. Self-assembly of endotheliocytes via growth-factor gradients or flow produced more physiological sinusoids but was slow, had low probability of forming perfusable channels, and suffered from unordered growth and poor reproducibility. Many liver chips neglected the tri-vascular structure and dynamic flow initially; later single-flow pathway systems supplied nutrients and removed waste but could not reproduce physiological oxygen/nutrient gradients or scale to larger tissues. Dual-vascular designs (artery–tissue–vein) formed microvascular networks and some gradients but did not recreate the liver’s double blood supply and physiologically similar gradients. Previous work by the authors implemented dual blood supply tri-vascular designs for single and multiple hepatic lobules; although sinusoid-like structures and some perfusable sinusoids were achieved, device complexity was high and perfusable sinusoid formation was unsatisfactory for large-scale culture.

Design and device: A microneedle-assisted hepatic acinus chip (mHAC) was designed inspired by hepatic acinus geometry. Each culture unit is a triangular prism representing 1/6 of a hepatic lobule (1/2 acinus). PV pathways occupy two edges, HA between them, and CV along the lower edge. Hepatic sinusoids are oriented perpendicular to the top surface across the culture area. The chip comprises three PMMA layers: Layer 1 for CV, Layer 2 for the culture area (isosceles trapezoid) with a porous membrane, and Layer 3 for PV and HA, with silicone membranes separating/sandwiching layers. The assembled chip connects via PTFE/silicone tubing to an external culture system and can operate stably for more than 14 days. Microneedle array and sinusoid formation: 3D-printed microneedle arrays (needle radii 100, 150, 200 µm; micropost volume fraction approximating sinusoid porosity) were fabricated. A photocurable extracellular matrix (GelMA) either cell-free or cell-laden was cast in Layer 2 with the microneedle array and auxiliary mold inserted. After 30 s UV photocuring, the microneedle array was demolded to create primary sinusoid pathways; the auxiliary mold was then removed. HHSEC and HepaRG cells were loaded within the GelMA matrix per schematic, and the device was assembled and cultured at 37°C, 5% CO2 with dual perfusion through PV and HA and outflow to CV. Characterization and flow studies: Confocal laser scanning microscopy (CLSM) visualized and measured the sinusoid pathways in cell-free and cell-laden GelMA following demolding. Computational simulations evaluated interstitial flow directions and the flow rate across the culture area as a function of sinusoid radius. Dye perfusion experiments verified flow directions and assessed mixing among PV, HA, and CV pathways. Functional assays: Hepatocyte metabolic function was assessed by measuring secreted albumin (ALB), blood urea nitrogen (BUN), and total bile acids (TBA) over 14 days in microneedle-assisted groups (r = 100, 150, 200 µm) versus microneedle-unused control (r = 0 µm). Activities of cytochrome P450 enzymes CYP3A4 and CYP1A2 were assayed at Day 7. Cell viability and microstructure formation were evaluated by live/dead staining and imaging, and evidence of secondary sinusoid formation (flow-driven angiogenesis) was documented.

- Microneedle demolding created primary sinusoid pathways throughout the GelMA ECM; most pathways were intact in both cell-free and cell-laden matrices. Pathway radii were slightly larger than microneedle radii, attributed to GelMA dehydration during CLSM. - Simulations showed fluid flow predominantly from PV and HA toward CV via the formed sinusoids, with increased interstitial flow across the culture area as sinusoid radius increased. For 150 µm sinusoids, simulated flow matched physiologically relevant interstitial flow values reported in literature. - Dye perfusion confirmed directional flow from PV/HA to CV with negligible mixing of HA and PV outside the culture area, indicating maintained separation suitable for establishing differing oxygen/nutrient conditions. - Hepatocyte metabolic outputs (ALB, BUN, TBA) were significantly higher in microneedle-assisted groups than in the no-microneedle control across early (Days 1–4), mid (Days 5–9), and late (Days 10–14) culture periods. The 150 µm microneedle group yielded the highest levels, consistent with more physiological interstitial flow. - CYP3A4 and CYP1A2 activities at Day 7 were significantly higher in microneedle-assisted groups than in controls. - Cell viability was higher in induced (microneedle) groups, with the 150 µm group highest. Primary sinusoids were clearly visible throughout the tissue, and secondary sinusoids formed via flow-driven angiogenesis, enhancing nutrient delivery to regions distant from primary channels.

The study addresses the challenge of constructing perfusable hepatic sinusoids in liver chips by using a demolded microneedle array within a hepatic acinus-inspired, dual blood supply microfluidic architecture. The formed primary sinusoids, along with spontaneously developed secondary sinusoids, substantially enhance interstitial flow from PV and HA to CV, mirroring physiological microcirculation and enabling maintenance of distinct supply streams. These fluidic improvements correlate with increased cell viability, formation of liver-like microstructures, and enhanced hepatocyte metabolic function (ALB, BUN, TBA) and drug-metabolizing enzyme activities (CYP3A4, CYP1A2). The 150 µm sinusoid size provided the most physiologically relevant flows and best functional outcomes among tested sizes. The ability to maintain separate PV and HA inputs supports the establishment of oxygen and glucose gradients, which the study preliminarily demonstrates can modulate hepatocyte function, underpinning the device’s relevance for modeling acinar zonation and for drug testing.

A microneedle-assisted strategy effectively constructs perfusable hepatic sinusoids within a large-scale liver-acinus-chip featuring a dual blood supply. The approach yields primary and flow-induced secondary sinusoid networks that enhance interstitial transport, boost hepatocyte viability, support microstructure development, and elevate metabolic and enzymatic functions, with a 150 µm sinusoid radius performing optimally. The system maintains separate PV and HA streams, enabling physiologically relevant oxygen and nutrient gradients and demonstrating preliminary applicability in drug testing. This work advances biofabrication of more biomimetic, fully functionalized large-scale liver bioreactors and provides a platform for studying acinar zonation and hepatotoxicity. Future research can optimize sinusoid architecture, refine gradient control, extend culture longevity, and expand multi-cellular co-culture complexity.

- Occasional small breakages in sinusoid pathways occurred due to microneedle demolding, though most pathways remained intact. - The demonstration of oxygen and glucose gradient effects on hepatocyte functions was preliminary. - Detailed affiliations for some collaborators and certain long-term performance metrics beyond 14 days are not provided in the presented text excerpt.