E9-a - Optimization of Cell Seeding in a Cylindrical Vascular Microfluidic Device

Saturday, October 11, 2025

10:00 AM - 12:00 PM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

JZ

James Zhang

High School Student Researcher
Bucknell University, Pennsylvania, United States

Co-Author(s)

Jack Glennon

Undergraduate Researcher
Bucknell University
Erie, Pennsylvania, United States

Primary Investigator(s)

Olivia Boerman

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

Introduction: : Chronic wounds affect over 6.5 million people, incurring costs exceeding $25 billion a year in the US. Low-intensity, low-frequency ultrasound (LILF US) has been shown to advance chronic wound healing (Samuels et al., 2013). While current treatments primarily focus on alleviating symptoms, LILF US promotes healing; however, its biological mechanisms are not well understood. Chronic wounds are associated with reduced blood flow, consequently lowering shear stress which negatively impacts angiogenesis and impedes wound healing. While studies conducted in 3D scaffolds can provide insight on how ultrasound modulates cell behavior, it is more physiologically accurate to conduct in vitro experiments in the presence of shear stress or fluid flow. To model how cells would act in a simulated wound environment, a microfluidic device that is capable of enacting shear stress on cells through fluid flow is required.

A reliable protocol for seeding cells in cylindrical flow chambers must be developed. Determining the most optimal concentrations of cell suspensions as well as seeding techniques are necessary for viable experiments. Moreover, due to the cylindrical curvature of the substrate, more advanced imaging techniques will be required. With successful seeding and imaging techniques, it would be possible to move forward in using a microfluidic device for in vitro experiments to elucidate the effects of LILF US on key biologics and ultimately enhance ultrasound technology for advanced healing. This study aims to develop a robust cell seeding protocol for a cylindrical microfluidic device capable of fluorescent imaging.

Materials and

Methods: : Pasteur pipettes, chosen as an initial proof-of-concept model for their thin walls, were modified by removing their thicker ends (Fig. 1A). 600 μL of fibronectin was pipetted into the sterile pasteur pipettes using a custom pipette system. The pipettes were then capped with silicon caps to ensure a watertight seal and rotated at 10 RPM for 45 minutes at 37° C. After draining the fibronectin, 250,000 HUVECs/mL human umbilical vein endothelial cells (HUVECs) were seeded into the fibronectin coated pipette. After the cells were rotated at 10 RPM for 4 hours in the incubator, adhered cells were stained with DAPI to stain the nuclei of the cells. After staining, z-stack fluorescent images were captured to visualize the cells seeded on the curved surface.

Next, a cytotoxicity assay following ISO 10993 was performed on the resin (AnyCubic Clear) that was to be used in the microfluidic device, to confirm its biocompatibility. 250,000 and 350,000 HUVECs/mL were seeded in a volume of 200 uL in the resin and gelatin fabricated microfluidic device to determine the most optimal cell seeding density.

After covering and incubating the seeded HUVECs for 4 hours at 37° C, they were stained with DAPI and FITC (to label cell nuclei and cytoskeleton respectively) and imaged using the Echo Revolve Fluorescent microscope.

Results, Conclusions, and Discussions:: HUVECs seeded on a curved surface of a pasteur pipette were successfully stained and imaged using a fluorescent microscope (Fig. 1B).The cytotoxicity assay showed no statistically significant difference between resin and negative control (Fig. 1C), confirming biocompatibility of the resin to be used for microfluidic fabrication. The resin was used to successfully fabricate a microfluidic device with a curved gelatin bed for cells (Fig. 1D). The 350,000 HUVECs/mL group cells’ adherence to the substrate and stained nuclei indicated their viability (Fig. 1E and F) whereas minimal to no cells were detected in the 250,000 HUVECs/mL group. Notably, the surface of the substrate itself showed background noise, indicating that the gelatin may be autofluorescent. Overall, these results demonstrated that these seeding, incubation, and imaging protocols were successful for a cylindrical surface.

This study establishes a reliable cell seeding and imaging protocol for cylindrical microfluidic devices. The demonstrated feasibility of endothelial cell culture and imaging on curved substrates supports future use of such devices as physiologically relevant models for studying LILF US effects on chronic wound healing under shear stress conditions.

Acknowledgements and/or References (Optional): : Samuels JA, Weingarten MS, Margolis DJ, Zubkov L, Sunny Y, Bawiec CR, Conover D, Lewin PA. Low-frequency ( < 100 kHz), low-intensity ( < 100 mW/cm(2)) ultrasound to treat venous ulcers: a human study and in vitro experiments. J Acoust Soc Am. 2013 Aug;134(2):1541-7. doi: 10.1121/1.4812875. PMID: 23927194; PMCID: PMC3745491.