Identifying Mechano-Modulators of Myometrial Contractility in a Scalable Hydrogel Culture Platform.

Abstract

Maternal and fetal health during pregnancy is closely tied to contractile modulation of the uterine myometrium, as dysfunctional contractions underlie pregnancy complications such as preterm labor and postpartum hemorrhaging. Structural abnormalities increase the risk for pregnancy complications, yet the complex interplay between biochemical and biomechanical cues in the myometrial microenvironment is not well defined. Significant gaps in our understanding of myometrial physiology and the lack of physiologically relevant research tools to enable systematic investigation have resulted in a largely ineffective and nonspecific therapeutic landscape for myometrial contractile modulation during pregnancy. We address this unmet need by developing a widely accessible approach for in vitro hydrogel platform fabrication to advance the study of agonist-mediated calcium responses in myometrial cells. We directly fabricated polyacrylamide hydrogels in polypropylene multiwell plates and Petri dishes and validated their mechanical properties and use as a mechanically tunable cell culture substrate. Using "physiological" matrix conditions during pregnancy, a fluorescent calcium mobilization assay was implemented in a plate-reader-based workflow to determine the dose sensitivity of myometrial cells to the endogenous agonist oxytocin. Using hydrogels with elastic moduli spanning physiological, pathological, and supraphysiological conditions in the myometrium, we assessed stiffness-mediated effects in myometrial cell behavior, including agonist-mediated calcium responses. We observed the mechanosensitive modulation of calcium response curve amplitude in oxytocin-stimulated myometrial cells. Myometrial cell behavior on mechanically tunable substrates was compared against widely used tissue culture plastic, and the observed changes to myometrial cell morphology and calcium responses highlight the significant influence of supraphysiological substrates. To explore complex pathological microenvironments, myometrial cells were exposed to the proinflammatory trigger lipopolysaccharide (LPS) before agonist stimulation. In our studies, LPS exposure altered calcium responses during oxytocin stimulation in cells cultured on supraphysiological substrates. Altogether, we present an easily adoptable in vitro hydrogel platform with scalable fabrication and versatile application use, including new directions in myometrial mechanobiology.Impact StatementWe investigated mechanosensitive modulation of agonist-mediated calcium responses in myometrial cells during pregnancy using a high-throughput in vitro polyacrylamide hydrogel platform. Polypropylene labware enabled scalable fabrication of an in vitro hydrogel platform without being costly or labor-intensive. Our studies found that oxytocin-stimulated calcium responses in myometrial cells are sensitive to nonphysiological matrix conditions. This work highlights the importance of using physiologically relevant engineered microenvironments and represents new directions for understanding myometrial mechanobiology. Our platform can help accelerate the identification of agonists and antagonists to counteract dysfunctional myometrial contractions and can be used more broadly in other tissues that exhibit mechanosensitive pathologies.