P10 - Optimization of Porcine-Derived Hydrogel Formulation for Mechanical Stability and Biocompatibility for 3D Endothelial Culture

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

LP

Lucy(Seohyeon) Park

Undergraduate Student
Bucknell University
Lewisburg, Pennsylvania, United States

Co-Author(s)

Andie McCullough

Undergraduate Researcher
Bucknell University
Ebensburg, Pennsylvania, United States

Primary Investigator(s)

Olivia Boerman

Assistant Professor of Biomedical Engineering
Bucknell University
Lewisburg, Pennsylvania, United States

Introduction: : This study aimed to develop a porcine-derived hydrogel with a target stiffness of approximately 100 kPa to serve as a physiologically relevant substrate for evaluating endothelial response to mechanical stimulus. Hydrogels are used for mimicking physiologically relevant tissue properties in vitro, particularly when assessing mechanobiological cellular responses to stimuli such as ultrasound. Key formulation variables in this study included varying gelatin concentration, crosslinker exposure, and preparation conditions (sterile vs. non-sterile). Hydrogels were cast at 4 mm thickness to reduce substrate effects during mechanical characterization. While sterile gels were designed for future biocompatibility assays, only non-sterile gels were used for stiffness measurements to avoid contamination and preserve sterile conditions for subsequent cell seeding.

Materials and

Methods: : Porcine gelatin powder was mixed with distilled water to produce hydrogels at 20% w/w. The mixture was stirred intermittently with a sterile spatula during a 20 minute incubation in a 70°C water bath until fully dissolved. Hydrogels were cast into well plates and cooled at 4°C for 2–3 hours. Crosslinking was performed by incubating hydrogels overnight in 0.2% glutaraldehyde, followed by sequential PBS washes (after 30 minutes, 1 hour, 2 hours, and 17 hours) to remove residual non-crosslinked glutaraldehyde. Samples were incubated at 37°C and stored in PBS for 1 and 4 days.
Mechanical characterization was conducted using the Activelife Biodent microindenter with a 1.56 mm cylindrical tip. Each of the three independently fabricated hydrogel replicates underwent five indents, each consisting of five cycles of sinusoidal displacement (300 µm amplitude, 1 Hz). Force–displacement data were analyzed with a custom Python script to calculate loading and unloading reduced moduli. Stiffness was derived from the slope of force–displacement curves in the elastic region and normalized by probe tip diameter. Script outputs were validated using known stiffness standards and comparison with prior instrument software values.
Human umbilical vein endothelial cells (HUVECs) were seeded at 30,000 cells/cm² and incubated at 37°C, 5% CO₂, and 95% humidity. A brightfield microscope was used to image cells seeded on the hydrogels after 96 hours of incubation. Three biological replicates were prepared and imaged in multiple regions to assess attachment and viability. A two-way t-test was used for statistical analysis, and significance was determined by p < 0.05.

Results, Conclusions, and Discussions:: After 1 day of incubation, replicate 1 approached the target modulus, with an average loading modulus (LM) of 99.91 kPa and average unloading modulus (UM) of 95.67 kPa. In contrast, replicate 2 (LM: 68.66 kPa; UM: 67.38 kPa) and 3 (LM: 71.79 kPa; UM: 72.94 kPa) exhibited lower initial stiffness, potentially due to uneven mixing, incomplete early crosslinking, or temperature fluctuations during fabrication. After 4 days of incubation, all three replicates exceeded or neared the 100 kPa threshold with LM of 108.22 kPa (A), 102.05 kPa (B), and 105.20 kPa (C), and corresponding UM of 102.94 kPa (A), 98.34 kPa (B), and 102.98 kPa (C), suggesting a gradual completion of glutaraldehyde-mediated crosslinking and stabilization of the hydrogel network structure in physiological conditions. These findings, summarized in Figure 1, support the material’s long-term mechanical stability and tunability toward the target modulus. Cell imaging of sterile gels revealed regions of both sparse and dense cell attachment (Figure 2). This heterogeneity may result from a concave gel surface, caused by surface tension between the gel and well walls, potentially affecting local cell seeding density and focus. The presence of adherent, viable cells across multiple regions of the hydrogel confirms that the crosslinked scaffold is cytocompatible after incubation and washing; the hydrogel surface supported cell attachment and survival despite possible curvature-induced variations in seeding density. This indicates that the glutaraldehyde-crosslinked scaffold is not acutely cytotoxic and remains biofunctional after 96 hours of incubation, supporting its suitability for further testing in endothelial cell-based applications for physiologically relevant experiments.

Acknowledgements and/or References (Optional): :