Assistant Professor Arizona State University, United States
Introduction:: Despite being a key organ during pregnancy, the human placenta is poorly understood. Ethical concerns of early human pregnancy research and poor homology of animal models constrain the study of the human placenta. Further, simple 2D in vitro models and many placenta-on-a-chip designs poorly recapitulate the native placenta microenvironment and early placental development (Turco and Moffett 2019). To close this critical gap, we have designed a microfluidic platform to replicate the placenta at early gestation stages. We use stereolithography -3D printing for rapid fabrication of complex geometric chip design (Hart et al. 2020) modeling fetal and maternal vasculature structures and use 3D hydrogel culture of placental cells to mimic early placental development. Further, we evaluate the biocompatibility of commercially available resins with a trophoblast cell line, evaluate hydrogel systems to generate placental organoid-like structures within the placenta-on-a-chip, and characterize the transport kinetics of molecules between the fetal and maternal compartments.
Materials and Methods:: Microfluidic chips were designed in Solidworks, and 3D printed with a Form 3 printer. Resin compatibility: Clear, Biomed Clear, and Surgical Guide resin (Formlabs, Sommerville, USA) were evaluated for biocompatibility with the JAR cell lines by treating well plate surfaces. Select resins were additionally treated over 24 hr. with a saline soak. Cells were cultured 72 hr. in direct contact with resin or within a 3D hydrogel (poly (ethylene glycol) (PEG) -maleimide + RGD +, a degradable VPM crosslinker) and evaluated for metabolic activity (Alamar Blue) and live/dead confocal imaging. Hydrogel selection: JAR cells were cultured in PEG-maleimide (VPM crosslinker) (5% w/v), PEG-DBCO (PEG-Azide crosslinker) (10% w/v), and alginate (Calcium crosslinked) (1.5% w/v) hydrogels for 7 days and assessed as described above. Molecular transport on chip: Fluorescent molecules of varied molecular weights (10-150 kDa) were infused on the fetal vascular side and transport across hydrogel to maternal compartment measured with fluorescent imaging and image analysis.
Results, Conclusions, and Discussions:: Microfluidic chips to model the placenta were designed and 3D printed, and placenta structures generated using cell-laden hydrogels within the wells (Figure 1) to generate a barrier between fetal and maternal vascular structures. To ensure optimal cell viability using 3D printed resins, we tested multiple commercially available resins with cells in 2D culture (direct contact with resin) and within degradable PEG hydrogels (3D). We found all resins except for the Biomedical resin significantly reduced the viability (Figure 2C) and metabolic activity of JAR cells in both 2D, and 3D culture at 24 hr. (Figure 2A, B). Soaking resins in saline for 24 hr. (1-day tx group) did not reduce resin toxicity for all groups besides the biomed resin used in the 2D JAR incubation. Further, we evaluated JAR organoid development in alginate, PEG-maleimide (PEG-MAL), and PEG-DBCO hydrogels to identify an optimal matrix for placenta- on-a-chip formation and found alginate and PEG-maleimide to be suitable matrices to support organoid growth and viability. Finally, we have preliminarily evaluated the transport kinetics of varied molecules (10k-150kDa MW) across our placenta compartment within the chip to characterize and quantify the molecular weight cutoff of hydrogel-only and cell-laden hydrogel placenta mimics.
We have designed, and 3D printed a microfluidic chip to model transport across the human placenta and placental development in early gestation. We identified a trophoblast-compatible 3D printing resin and identified hydrogel matrices suitable to generate placenta-like structures on our chip to separate maternal and fetal vascular compartments. Finally, we have begun to use the chip to characterize molecular transport kinetics, and future studies will evaluate multicellular interactions at the fetal-maternal interface, with a focus on immune cell-trophoblast interactions.
