A straightforward cell culture insert model to incorporate biochemical and biophysical stromal properties into transplacental transport studies.

Introduction: The placental extracellular matrix (ECM) dynamically remodels over pregnancy and in disease. How these changes impact placental barrier function is poorly understood as there are limited Methods: Uniform polyacrylamide (PAA) gels were polymerized within cell culture inserts by (re)using the insert packaging to create a closed, controllable environmental chamber. PAA pre-polymer solution was added dropwise via a syringe to the cell culture insert and the atmosphere was purged with an inert gas. Transport and cell culture studies were conducted to validate the model. Results: We successfully incorporated and ECM functionalized uniform PAA gels to cell culture inserts enable cell adhesion and monolayer formation. Imaging and analyte transport studies validated gel formation and expected mass transport results and successful cell studies confirmed cell viability, monolayer formation, and that the model could be used transplacental transport studies. Detailed methods and validation protocols are included. Discussion: It is well appreciated that ECM biophysical and biochemical properties impact cell phenotype and cell signaling in many tissues including the placenta. The incorporation of a PAA gel within a cell culture insert enables independent study of placental ECM biophysical and biochemical properties in the context of transplacental transport. These straightforward and low-cost methods to build three dimensional cellular models are readily adoptable by the wider scientific community.