(I-354) Engineering a Micropatterned Stretch Chamber to Evaluate the Effects of Mechanical Strain on Uterine Smooth Muscle Tissue Contractility

Introduction:: The myometrium, which is the middle of the three uterine tissue layers, is primarily composed of uterine smooth muscle cells. The fundamental role of the myometrium is to contract in response to hormonal stimulation. In its healthy state, the myometrium can adapt to relatively diverse mechanical environments; however, stretching forces (e.g., pregnancy) are hypothesized to have various effects on uterine smooth muscle cell contractility. Additionally, prolonged exposure to mechanical strain can inflict changes on the cellular microenvironment and may contribute to the pathology of uterine disorders, such as preterm labor and pre-eclampsia. These and many other women’s health conditions are vastly unexplored and lack effective model systems. Thus, our goal is to develop a functional and physiologically relevant in vitro cell culture system that can be used to evaluate the effects of stretch on uterine smooth muscle cell contractility. To achieve this, we are engineering a micropatterned stretch chamber that enables tuning of substrate stiffness and cell alignment to mimic different features of native uterine smooth muscle tissue. These chambers can be seeded with uterine smooth muscle cells and then subjected to static mechanical strain at defined rates using a cytostretcher. We also designed our system to be compatible with functional assays, such as calcium imaging and traction force microscopy (TFM), for obtaining physiological measurements of uterine smooth muscle tissue in response to strain or other stimuli.




Materials and Methods:: (1) Polyacrylamide (PA) gel attachment to polydimethylsiloxane (PDMS) membranes: PDMS silicone strips were treated with benzophenone, a UV-reactive photoinitiator that promotes strong gel adhesion. PA gels of 13 kPa doped with fluorescent beads were fabricated on the surface of PDMS strips and the samples were exposed to UV light. (2) Microcontact printing (stamp-off): PDMS stamps with an array of rectangular “island” patterns (200 µm x 100 µm) were used to imprint fibronectin islands on the surface of PA gels (Fig. 1A). To validate patterning, fibronectin was stained with a primary anti-fibronectin antibody (F3638, Sigma) followed by a secondary anti-rabbit antibody (A-11035, ThermoFisher). Images of individual fibronectin islands were captured at 20X on a Nikon Eclipse TS100 inverted microscope (Fig. 1C). (3) Stretch chamber platform assembly: The underside silicone layers of Curibio’s cytostretcher chambers (12 mm x 12 mm) were removed and discarded. Uncured PDMS was spin-coated and used as an adhesive that, once cured, formed a bond between the base of the cytostretcher chamber and the PDMS–PA gel sample. To enable visualization on a microscope stage while inducing mechanical strains of 10% and 15%, stretch-inducing frames were designed in 3-D CAD software and 3-D printed (Fig. 1B).


Results, Conclusions, and Discussions:: We fabricated and bonded PA gels of physiologically relevant elastic moduli (1 kPa to 13 kPa) to stretchable PDMS membranes. PA gels can be adapted to the appropriate elastic modulus by adjusting the relative concentrations of acrylamide (monomer) and bis-acrylamide (cross-linking agent). We also micropatterned arrays of fibronectin rectangles onto the PA gel surface to confine cell adhesion to the fibronectin-positive areas on the gel such that we can use these substrates to engineer and stretch aligned uterine micro-tissues in future studies. The percent strain transfer from the PDMS membrane to the gel surface was estimated by measuring length:width aspect ratios of individual islands at 0, 10, and 15% strains once mounted on the 3-D printed stretch-inducing frames. We imaged a single island when exposed to 0% and 15% strains, respectively, and found the length:width ratio to decrease from 1.97 to 1.45 (R² = 0.98), which is consistent with the linear portion of the stress-strain curve. (Fig. 1C). Together, microcontact printing onto PA gels fabricated on stretchable PDMS membranes enable independent control over tissue architecture, matrix stiffness, and strain, respectively, which allows us to systematically investigate uterine smooth muscle cell mechanobiology.

Our stretch chamber platform is also compatible with functional assays, such as calcium imaging and TFM. In future studies, we will use these approaches to identify molecular mechanosensors, such as stretch-activated calcium ion channels, to identify new therapeutic targets for conditions such as preterm labor.


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