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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session D

Poster H14 - Optimization of HUVEC Network Angiogenesis in 3D Collagen Matrices

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The goal of this work was to develop a 3D endothelial culture on a collagen scaffold to conduct physiologically relevant in vitro studies. 3D experiments are critical for mimicking physiologic conditions, especially in considering mechanobiological effects of substrates. Endothelial cells line blood vessel walls and regulate internal fluid flow as well as exchange of substances across the vessel membrane. These cells play a crucial role in wound healing via angiogenesis, the creation of new vasculature (Félétou 2011). A 3D in vitro model containing endothelial cells in a tissue-like membrane is critical for evaluating endothelial cell response to low-frequency (20 kHz), low-intensity (100 mW/cm^2) ultrasound (LFLI US). This research varied cell seeding techniques, resuspension concentrations, and incubation periods to develop a robust 3D model for endothelial neovasculature growth. This model seeded human umbilical vein endothelial cells (HUVECs) within a collagen scaffold. A collagen scaffold was selected due to its mechanical and chemical resemblance to the extracellular matrix (Fan et. al 2023). Confocal microscopy was utilized to document neovasculature throughout the scaffolds under each set of culture conditions to determine an optimal protocol.

Materials and

Methods:  Human umbilical vein endothelial cells (HUVECs) between passages 4 and 9 were cultured in vascular media until 70-80% confluency. HUVECs were then resuspended in vascular media in a ratio of 600,000 cells per 100 uL. Collagen-based Spongostan MS0005 gelatin scaffolds (1.0 cm x 1.0 cm x 1.0 cm) were cut into four slices, and placed into individual wells in a 12 well plate. Each scaffold slice was saturated with 100 uL of vascular media for 15 minutes to initiate fluid absorption and then removed. 50 uL of resuspended cell solution were then added to the superior side of each scaffold. After 15 minutes, the scaffolds were flipped and 50 uL of cell solution were added to the other side. Scaffolds incubated at 37°C for 2 hours to permit HUVEC adherence before 2 mL of vascular media were added to each well. Scaffolds incubated for 96, 120, 144, or 168 hours with vascular media changes every 36 hours, and were soaked in 4% paraformaldehyde at the end of their incubation period. Fluorescent staining utilizing BSA, DAPI, and FITC was performed for isolation of cellular structures in confocal microscopy. A Leica TCS SP5 confocal microscope was used with the LAS AF software to acquire images using a 40x objective. Images were analyzed using the ImageJ Angiogenesis Analysis toolkit software to observe angiogenic results based upon average quantity of vascular junctions, vascular length, and master segment length for each incubation period. ANOVA testing was utilized to determine statistical significance.

Results, Conclusions, and Discussions: These data suggest that the most optimal seeding protocol for neovascularization observation using confocal microscopy was seeding 600,000 cells per scaffold, seeding from both sides of the scaffold, and incubating for 120 hours after seeding. A cell density of less than 600,000 cells per 100 uLs yielded a cell density too sparse for networking visualization. Conversely, concentrations exceeding 800,000 cells per 100 uL resulted in oversaturation of the scaffolds with cell nuclei which obscured vascularization. Furthermore, when the full 100 uL of resuspended HUVECs were added to one side of the scaffold, the cells did not fully migrate through the scaffold and yielded a visibly more dense network at higher z-positions and more sparse networking at lower z-positions within the matrix. This resulted in the protocol adjustment to include double-sided seeding with half of the solution (50 uL) on each side for more even network distribution. In assessment of incubation period effects on angiogenesis, increasing vascular junction quantities were demonstrated as incubation time increased (Figures 1a-1e). However, analysis of total vascular and master segment length indicated an increase between 96 and 120 hour incubation periods, plateauing by the end of the 144 hour incubation period (Figures 1f, 1g). The simultaneous increase in quantity of junctions and plateau of master segment length development suggested a decline in interconnected vascularization in favor of disconnected branching, as the master segment length quantity is indicative of the amount of branches that connect multiple junctions (Savkovic et al. 2021). The 120 hour incubation period yielded the greatest ratio of developed master segment length relative to the quantity of junctions, with an average of 1331.6 µm of vascular segmentation connecting 15 junctions. These data trends suggested that under the aforementioned 3D HUVEC culture conditions, the optimal incubation period for maximized angiogenic potential is between 96 and 144 hours. Each set of the above observations was conducted across three experimental trials containing a minimum of three samples per testing group. These results demonstrate a robust seeding protocol for culturing HUVECs in a 3D scaffold to conduct more physiologically relevant experiments.

Acknowledgements (Optional):