Quantifying Hormone-Mediated Traction Forces Generated by Engineered Uterine Smooth Muscle Microtissues

Introduction:: The myometrium is the middle layer of the uterus and consists of smooth muscle tissue that is responsible for uterine contractility. Myometrial smooth muscle cells contract in response to oxytocin, a natural hormone that is released by the pituitary gland during labor. When the contractile forces generated by the myometrium are delayed or excessively weak, a synthetic version of oxytocin is often administered to induce contractions. Otherwise, the patient may require surgical intervention (C-section) to prevent harm to the neonate or post-partum hemorrhage. By contrast, early onset of labor can be mitigated via tocolytics, such as progesterone and magnesium sulfate (MgSO₄), that relax the muscle. However, tocolytics have low efficacy and many off-target effects. Together, these examples highlight the need for more strategies to modulate the contractility of the myometrium, which requires methods for robustly quantifying the amount of force generated by human myometrium in response to perturbations at medium-throughput. However, current models are limited to animal models, which lack human relevance, or tissue strips isolated from patients, which are difficult to access and maintain long-term and are limited by patient variability. Thus, our objective is to establish and validate an in vitro assay for quantifying contractile forces generated by engineered human myometrial tissue. To achieve this, we used microcontact printing to engineer aligned human myometrial microtissues on compliant polyacrylamide hydrogels doped with fluorescent beads to facilitate traction force microscopy.

Materials and Methods:: We fabricated 1 kPa polyacrylamide gels doped with high density fluorescent microparticles and streptavidin-acrylamide. To engineer aligned microtissues, we micropatterned arrays of 200 x 100 µm biotinylated fibronectin islands onto the gels. Gels were seeded with primary human uterine smooth muscle cells, incubated for one day, serum starved for 12-24 hours, then transferred to a temperature controlled inverted fluorescence microscope. Fluorescent bead displacements were then measured in response to a bolus of oxytocin (0.2-2 mM) at t=0 for 12 minutes (induced force), then trypsin after t=12 for 6 minutes (basal force), with one image captured per minute. Tissues exposed to tocolytics were treated with magnesium sulfate (3.5 mM) for 2 hours or progesterone (0.016 µM) for 12 hours during the serum starvation period. Given the isotropic and elastic nature of the gels, the displacements were converted to traction forces using a previously established Matlab script¹.

Results, Conclusions, and Discussions:: Results and Discussion: While microcontact printing fibronectin onto polyacrylamide gels with elastic modulus less than 5 kPa has previously been difficult to achieve², we successfully printed islands with high fidelity and attained robust myometrial cell adhesion and alignment (Figs. 1A,B). Although this rigidity is softer than typical uterine tissue, it enables deformation by myometrial smooth muscle cells. An image of the fluorescent beads prior to the addition of oxytocin shows displacement of beads, indicating basal forces applied to the substrate (Figs. 1C,D). Induced forces increased with oxytocin concentrations (Fig E), as expected. Pre-incubation with magnesium sulfate or progesterone, reduced the induced forces generated after oxytocin addition (Fig. 1E). Pre-treatment with magnesium sulfate or progesterone also slightly reduced basal force (Fig. 1F).

Conclusion: We engineered human uterine micro-tissues on a compliant matrix and found that micro-tissues have a graded contractile response to oxytocin. Micro-tissues also exhibit reduced contractile forces in response to tocolytics, as expected. We will leverage this assay to evaluate contraction in response to other compounds, including caffeine, a compound that is believed to cause uterine relaxation. This assay will also be used to evaluate potential mechanobiological pathways that can be targeted for new drugs to alter contractile activity and can be integrated with patient-derived cells to evaluate patient-specific responses to drugs.


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References (Optional): : 1. McCain ML, Lee H, Aratyn-Schaus Y, Kléber AG, Parker KK. Cooperative coupling of cell-matrix and cell-cell adhesions in cardiac muscle. Proc Natl Acad Sci U S A 2012;109:9881-6
2. Pasqualini FS, Agarwal A, O'Connor BB, Liu Q, Sheehy SP, Parker KK. Traction force microscopy of engineered cardiac tissues. PLoS One 2018;13:e0194706