A Three-Dimensional Trophoblast Invasion Microfluidic Platform for Toxicological Screening

Abstract

To improve our understanding of human placental function and placental cell responses to pregnancy stressors, the development of in vitro models that better recapitulate the in vivo placental microenvironment is needed. Here, we describe a three-dimensional (3D) silicone polymer polydimethylsiloxane (PDMS) microfluidic platform for modeling human trophoblast invasion recreating a placental heterocellular microenvironment. This platform allows the formation of a cellular barrier establishing a chemical gradient and real-time evaluation of trophoblast cell invasion and heterocellular cell-to-cell interactions.

Keywords: Invasion; Microfluidics; Three-dimensional; Trophoblast.

Figure 1.. Basic instruments.

A. Silicone polymer polydimethylsiloxane (PDMS) 3D microfluidic chip. B. 3D microfluidic chip scheme depicting the following components: 1) a central compartment (red) with a central feeder line supplied by two inlet ports (i1, i2) connected to two outlet ports (o1, o2) and 2) two outer channels (blue) with two outer feeder lines supplied by two inlet ports (i3, i4) connected to two outlet ports (o3, o4). The barrier between the central compartment and the outer channels is filled with pillars (pillar spacing (PS):3 μm; total width: 50 μm). C. Utensils needed: Scissors, forceps, 1ml syringe, blunted needle hub, clamp (blue), tubing cuttings, and microfluidic chip. D. Syringe pump.

Figure 2.. Microfluidic chip priming.

A. Microfluidic chip onto Petri dish before priming. B. Desiccator containing microfluidic chip bathed in DPBS in a Petri dish. C. Desiccator connected to vacuum line. D. Microfluidic chip onto Petri dish ready to be coated with fibronectin.

Fig 3.. Checking cell seeding integrity under the microscope.

A. Microfluidic chip after cell seeding. Note that only the central compartment and the bottom outer channel have been seeded with cells. B. Microfluidic chip after cell attachment. The central compartment and both outer channels have been seeded with cells. C-D. Microfluidic chip with various air bubbles (white arrows).

Fig 4.. Fibronectin coating (part 1).

A. Microfluidic chip with a DPBS drop over i1 port for fibronectin coating. B-C. Connecting of the fibronectin solution tubing with i1 port. D. Cut the tubing connected to i1 port. E. Connecting of the fibronectin solution tubing with i4 port. F. Connecting of the fibronectin solution tubing with i3 port. Asterisk denotes drop at the end of tubing to be connected.

Fig 5.. Fibronectin coating (part 2).

A. Fibronectin solution from i2 port comes out from i1 port. B. i1 port is then clamped and fibronectin solution comes out through o1 and o2 ports. C. o1, o2 and i2 ports are then clamped and fibronectin solution from i4 port comes out o4 port. D. After clamping o4 and i4 port, the fibronectin solution from i3 port comes out of port o3. E-F Outlook of the chip after clamping of the remainder of the ports (follow text for guidance).

Figure 6.. Assembled microfluidic platform.

A. Pump loaded with three cell media syringes connected to the chip (1 central compartment and 2 outer channels) and outflow of media connected to collecting 0.75 ml tubes. B. Close up detail of the chip and collecting tubes. C. Microfluidic chip under fluorescence microscope.