Geltrex‐Enhanced Two‐Dimensional Culture as a Viable Alternative to Primary Rat Hepatocyte Sandwich Models

Primary hepatocytes rapidly lose viability and function in conventional two‐dimensional (2D) cultures due to the absence of a physiologically relevant extracellular matrix (ECM). The collagen sandwich method improves polarization and function but creates a diffusion barrier that limits nutrient and signal exchange. This study investigates whether daily supplementation of a diluted, non‐gelling Geltrex layer can sustain hepatocyte function and viability in 2D culture, offering a practical alternative to the sandwich method. Primary rat hepatocytes were cultured for 15 days under four conditions: monolayer (ML), monolayer with Geltrex (ML + GT), sandwich (SW), and sandwich with Geltrex (SW + GT). Cell morphology, confluency, viability (CCK‐8, live/dead staining), and functionality (urea synthesis, albumin production, CYP3A4 activity) were assessed. The ML group showed significant declines in confluency, viability, and functional markers over time. Geltrex supplementation preserved confluency (~97% at day 15), improved viability, and maintained higher albumin production and CYP3A4 activity compared to ML. Functional outputs in ML + GT were comparable to SW and SW + GT groups, without the diffusion limitations of the sandwich top gel. Daily supplementation with low‐dose Geltrex creates a biochemically enriched, diffusion‐permissive microenvironment that supports long‐term viability and function of primary rat hepatocytes in 2D culture. This method represents a simple and effective alternative to traditional sandwich cultures for liver cell studies and drug testing applications.