Poster C6 - Characterization of Extracellular Matrix Obtained from Cell-Based Hollow Fiber Membranes

Thursday, October 24, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Alexandra Trzpis (she/her/hers)

Biosystems & Biosignals Engineering Undergraduate Student
University of Rochester
Pittstown, New Jersey, United States

Co-Author(s)

CS

Chris Slavin

Graduate Student
University of Arkansas, United States
AC

Anelisse Claros

Graduate Student
University of Arkansas
Fayetteville, Arkansas, United States

Primary Investigator(s)

Jeffrey Wolchok, PhD

Primary Investigator
University of Arkansas, Arkansas, United States

Introduction: Volumetric muscle loss (VML) is a significant loss of skeletal muscle tissue that leads to functional and regenerative impairment of that muscle area. This acute, catastrophic condition can stem from combat-related injuries such as bullet wounds or extensive surgical procedures that remove large amounts of muscle such as radical tumor resections. Layering cell-derived extracellular matrix (ECM) fibers and autogenic minced muscle paste into the anterior tibialis injury sites of mouse models improved wound healing to an extent comparable with the muscle mass restoration results of tissue-sourced ECM scaffolds. Given that cell-derived ECM would reduce reliance on tissue donors and alleviate supply limitations, this study aims to investigate what aspects of the cell culture process maximize ECM production to optimize repair in VML injuries.

Materials and

Methods: A polysulfone dope solution and an N-Methylpyrrolidone (NMP) bore solution in deionized water were extruded through a spinneret using pressurized nitrogen into an air gap before precipitating in a water bath via nonsolvent-induced phase separation. The collected rope of polymer scaffold was cut into 4 cm segments, sterilized in 70% ethanol, and incubated in bovine plasma fibronectin before being seeded with foreskin and bladder cells individually. Some of the foreskin cells received TGF-beta in their media, and others did not. These were cultured at 1, 3, and 6-week intervals. After these time points, the polymer scaffold was dissolved with 3 NMP washes over 3 days to isolate the ECM fiber, which was then weighed to determine the ECM yield. Furthermore, we determined the hollow fiber diameters using a scanning electron microscope and intend to measure the pore diameters via evapoporometry.

Results, Conclusions, and Discussions: To determine the point at which ECM production plateaus, we will create a time-dependent yield curve of ECM weights per 4 cm of fiber at 1, 3, and 6-week intervals. We will also determine if there is a statistically significant difference in ECM yield between 3-week foreskin cell fibers cultured with and without the protein TGF-Beta based on their measured weights. To determine the impact of cell type on ECM production, we weighed the ECM yield of 3-week fibers cultured with bladder cells versus foreskin cells, but we have yet to confirm these results with proteomics. Currently, we know the mass of foreskin fibroblasts at 3 weeks of culturing is 2.74 mg with a 0.09786 yield per cm and 0.0326 yield per cm per week. We also know the mass of bladder fibroblasts at 3 weeks of culturing is 3.44 mg with a 0.1433 yield per cm and 0.04778 yield per cm per week. Finally, we will determine the fiber diameter via scanning electron microscopy and measure the fiber pore diameter via evapoporometry to better understand the physical properties of the fibers that may influence ECM yields. Currently, our evapoporometry results show that a majority of the pore sizes lie between 32.19 and 33.19 nm. Thus, this study may reveal that culture time, cell type, and TGF-beta inclusion impact the growth rate of ECM, but we have not collected all of our results yet. Ideally, this data could be used to expedite the development of cell-based hollow fiber membranes in future experiments involving volumetric muscle loss injuries.

Acknowledgements (Optional): National Science Foundation Grant: EWFD 2243953