Biomimetic Micropatterned Adhesive Surfaces To Mechanobiologically Regulate Placental Trophoblast Fusion

Abstract

The placental syncytiotrophoblast is a giant multinucleated cell that forms a tree-like structure and regulates transport between mother and baby during development. It is maintained throughout pregnancy by continuous fusion of trophoblast cells, and disruptions in fusion are associated with considerable adverse health effects including diseases such as preeclampsia. Developing predictive control over cell fusion in culture models is hence of critical importance in placental drug discovery and transport studies, but this can currently be only partially achieved with biochemical factors. Here, we investigate whether biophysical signals associated with budding morphogenesis during development of the placental villous tree can synergistically direct and enhance trophoblast fusion. We use micropatterning techniques to manipulate physical stresses in engineered microtissues and demonstrate that biomimetic geometries simulating budding robustly enhance fusion and alter spatial patterns of synthesis of pregnancy-related hormones. These findings indicate that biophysical signals play a previously unrecognized and significant role in regulating placental fusion and function, in synergy with established soluble signals. More broadly, our studies demonstrate that biomimetic strategies focusing on tissue mechanics can be important approaches to design, build, and test placental tissue cultures for future studies of pregnancy-related drug safety, efficacy, and discovery.

Mechanobiological regulation of placental trophoblast fusion and function through extracellular matrix rigidity

“…Following our previous protocols, cells were fixed in 4% (w/v) paraformaldehyde in PBS for 15 minutes at room temperature; and embedded in a thin layer of porous polyacrylamide hydrogel (100 Pa stiffness) to prevent detachment of the weakly-adhered syncytial patches after fusion. This process was previously confirmed to have no effect on the immunostaining results 67 . Substrates were washed twice with PBS, permeabilized in 0.1% (v/v) triton X-100 in PBS for 15 minutes, and washed twice with PBS again.…”

“…Human primary vCTBs, were obtained from term placentas from vaginal delivery of uncomplicated pregnancies, and were isolated using previously used methods 67 . Briefly, placental tissue was consecutively digested in trypsin and DNase; after which each of the supernatants were collected, pooled, and separated via a Percoll gradient.…”

“…All vCTB preparations were at least 95% pure after cell sorting 68,69 . vCTBs were mixed in a 1:10 solution of DMSO in FBS cryoprotectant, frozen overnight at −80 °C, and stored in liquid nitrogen 67 . Primary vCTBs were plated at 1.6 × 10 6 cells/mL, allowed to attach for 4 hours, and gently rinsed with warm media to wash away mononuclear syncytial fragments 69,70 .…”

“…immunostaining. Staining techniques for fused BeWo cells on patterned and hydrogel substrates were previously established in our lab 67 . Following our previous protocols, cells were fixed in 4% (w/v) paraformaldehyde in PBS for 15 minutes at room temperature; and embedded in a thin layer of porous polyacrylamide hydrogel (100 Pa stiffness) to prevent detachment of the weakly-adhered syncytial patches after fusion.…”

“…Samples were blocked in 2.5% (v/v) goat serum in PBS for 2 hours at room temperature to prevent non-specific binding. As per previous protocols for indirect staining 67 , cells were incubated with anti-E-cadherin antibody (1:200, Abcam, ab1416; overnight, 4 °C) or anti-β-hCG antibody (1:500, Thermo Fisher, #14-6508-82; overnight, 4 °C) in goat serum. For secondary staining, cells were washed twice with PBS and incubated with goat anti-mouse IgG H&L (Alexa Fluor.…”