A Simple Liquid Column System for Adjustable Hydrostatic Pressure and Depth-Dependent Hypoxia: Impact on NIH/3T3 Fibroblast Behavior In Vitro

This study aimed to develop an accessible, adjustable cell culture model utilizing varying liquid column heights to evaluate the combined microenvironmental impacts of hydrostatic pressure (HP), nutrient availability, and depth-dependent oxygen gradients on NIH/3T3 fibroblasts. The liquid column medium (LCM) system was constructed by integrating customized glass pipes with standard 48-well cell culture plates, achieving a maximum column height of 23.3 cm (equivalent to 18 mmHg of base HP). Twenty-four hours post-seeding, fibroblasts were exposed to four distinct LCM heights: 0.3 cm (control), 7.7, 15.5, and 23.3 cm, and analyzed after 1 and 3 days of exposure. First-order mathematical modeling of oxygen diffusion predicted that increasing medium depth concurrently restricts pericellular oxygen availability over extended culture durations. Exposure to LCM heights induced marked cell spreading, elevated cell density along the well margins, and significantly reduced cell proliferation in a height-dependent manner. By day 3, the highest cell count was observed in the control group (16 × 10³ ± 850 cells/well), whereas total cell counts decreased progressively under increasing LCM heights (13 × 10³ ± 720, 10 × 10³ ± 610, and 6.5 × 10³ ± 430 cells/well at 7.7, 15.5, and 23.3 cm, respectively). Furthermore, LCM exposure expanded mean nuclear area and increased the proportion of binucleated cells. Overall, these findings demonstrate that manipulating medium column height provides a versatile, low-cost in vitro model for exploring cell behaviors under coupled mechanical pressure and fluid depth conditions.