4 - Engineering an In Vitro System to Study the Effects of Mechanical Stretch on Human MyometrialTissue Contractility

Saturday, October 26, 2024

1:00 PM – 1:15 PM EST

Location: Room 341

Presenting Author(s)

Aava Abedinpour (she/her/hers)

Undergraduate Researcher
University of Southern California
Scottsdale, Arizona, United States

Co-Author(s)

Alisa Peshina (she/her/hers)

Biomedical Engineering PhD Student
University of Southern California
Culver City, California, United States
Antonina Maxey

Doctoral Candidate
University of Southern California
Los Angeles, California, United States

Primary Investigator(s)

Megan McCain

Associate Professor & Chonette Early Career Chair
Alfred E. Mann Department of Biomedical Engineering, United States

Introduction: The myometrium, the middle layer of the uterine wall, mainly consists of uterine smooth muscle cells. During menstruation and pregnancy, the myometrium is responsible for contractions. Overdistension of the uterus is one of the causes of preterm labor, which results in preterm birth, the leading cause of death in children under 5. Preterm labor occurs in 10% of all pregnancies. However, there are no treatments that can delay contractions for longer than 48 hours (Barnett et al., 2023). The lack of effective model systems is a barrier to developing safe and sustainable treatments for uterine disorders. Therefore, the effects of mechanical stretch on the uterus must be further investigated. Previous studies have used rat and human tissue strips as uterine model systems to study the effects of stretch on the myometrium. However, these models did not study mechanoadaptation and are limited by low reproducibility, which makes it challenging to conduct systematic studies (Maxey, 2021). The goal of our study is to engineer an in vitro system that is compatible with obtaining functional outputs through traction force microscopy and can be used to evaluate the effects of stretch on human uterine smooth muscle cell (HUtSMC) contractility.

Materials and

Methods: Polyacrylamide (PA) gels can be fabricated at different elastic moduli by changing the relative concentrations of acrylamide and bis-acrylamide. PA gels with a stiffness of 1 kPa, a physiologically relevant elastic modulus, were fabricated on transparent silicone membranes that were chemically activated with a 10% benzophenone solution in acetone. Benzophenone generates free radicals upon UV exposure, ensuring gel adherence to the membrane. Before UV treatment, plasma-treated 12 mm glass coverslips were used to flatten the PA gel solution on the center of the benzophenone-treated membranes. A UV intensity of 57.11 mW/cm2 was applied to the samples for 170 seconds, and the 1 kPa gels fully crosslinked 30 minutes post-UV treatment (Fig 1A). Fibronectin island micropatterns were transferred to the gel surface by microcontact printing using a stamp-off approach. Isotropic polydimethylsiloxane (PDMS) stamps were coated with biotinylated fibronectin, and stamps with an array of 200 μm x 100 μm island patterns were UVO treated. Upon stamp-to-stamp contact, the micropattern transfers to the isotropic stamp, which is immediately used to stamp the 1 kPa PA gels. Fibronectin was stained with a 1° anti-fibronectin antibody followed by a 2° anti-rabbit antibody and
imaged with a Nikon Eclipse TS100 inverted microscope (Fig 1B). Uncured and spin-coated PDMS was used to attach Curibio’s cytostretcher chambers (12 mm x 12 mm) to the membranes. To ensure cell adherence to fibronectin, HUtSMCs were seeded onto engineered stretch chambers and fixed 48 hours later, before immunostaining with Phalloidin, DAPI, and anti-fibronectin (Fig 1C).

Results, Conclusions, and Discussions: We successfully fabricated 1 kPa PA gels on silicone membranes. However, the UV-mediated benzophenone treatment required further troubleshooting to successfully graft the gels onto the membranes. Due to their softness, 1 kPa gels that were not thoroughly crosslinked were susceptible to the formation of cracks around the perimeter of the gel after coverslip removal. Waiting 30 minutes after UV treatment allowed the gels to fully crosslink. We also discovered that plasma-treating coverslips prior to their use helped to avoid the formation of cracks in the
cross-linked gels during coverslip removal. After solidifying the gel fabrication process, we successfully micropatterned fibronectin islands by using light pressure when applying a PDMS stamp to the gel. To further validate membrane-gel strain transfer, island aspect ratios will be measured at 0%, 10%, and 15% strains using 3D printed frames (Fig 1D), which will allow the islands to be stretched longitudinally (Fig 1B) at the specified strains. ImageJ software will be used to measure width-to-length ratios of individual islands at the three different strains. We expect the results to be consistent with the linear segment of the stress-strain curve, suggesting that 100% of the strain is transferred from the silicone membrane to the gel. After immunostaining, we also validated that HUtSMCs successfully adhered to the fibronectin islands in the engineered stretch chambers (Fig 1D). For future directions, traction force microscopy will be implemented to obtain functional data on the forces generated by cells upon exposure to stretch. Using our engineered system, we will be able to examine the way stretching forces experienced during pregnancy affect uterine smooth muscle cell contractility to find treatments for preterm labor.

Acknowledgements (Optional):