Modeling the Endothelial Glycocalyx Post-Pneumonectomy in a 3D Fluidic Chip - An Approach to Fabricating a Vascular-based Organ-on-Chip System

The Mensah Lab is focused on vascular research and is developing new techniques for researching the endothelial glycocalyx and its role in vascular disease. Recent developments involve creating more advanced in vitro models for glycocalyx research and researching therapeutic strategies to target endothelial glycocalyx health and repair in diseases. Experimental challenges include incomplete in vitro models, which fail to accurately replicate healthy glycocalyx, as well as difficult quantification and measurement techniques.

Our current contribution to the field have expanded the current understanding of the endothelial glycocalyx, particularly in its role in cancer metastasis, regeneration of damaged glycocalyx, and interactions with vascular surface receptors. Our method presents an inexpensive and effective method for creating organ-on-a-chip vascular systems that express more well-developed and biomimetic endothelial glycocalyx compared to conventional techniques. To begin, weigh 10 parts silicone elastomer base in a 50-milliliter centrifuge tube.

Pour one part curing agent into the tube containing the silicone elastomer base to a final volume of 30 milliliters. Mix thoroughly to obtain a uniform solution. Now pour the well-mixed solution into the molds, then place the molds into a vacuum chamber to degas the PDMS for 30 to 60 minutes or until no bubbles remain.

Remove the degassed PDMS from the vacuum chamber and allow it to cure at room temperature for 48 hours. Extract the cured PDMS from the PLA mold. Then place two pieces of double-sided tape in each cell culture plate.

Next position each PDMS vasculature half on top of the tape in its own cell culture dish. Place several 30-microliter drops of fibronectin along the PDMS vasculature using a micropipette. Tilt the PDMS as needed, and use a pipette tip to evenly coat the surface.

After a 30-minute incubation, aspirate the fibronectin solution from the PDMS. Then let the pieces air-dry inside the cabinet. Place 3D-printed plugs at each end of the PDMS vasculature halves to prevent cell suspension leakage, using two plugs per piece.

With a micropipette, seed 300 microliters of HLMVEC cell suspension into each PDMS piece and incubate. Then return the halves to the cabinet and remove the four plugs. Add HLMVEC culture medium to submerge the PDMS vasculature halves, and incubate again for six to 10 hours.

Now aspirate the HLMVEC media from the Petri dish containing the vasculature halves. Place each PDMS half into the polycarbonate blocks, ensuring proper alignment. Apply silicone sealant to the ends of the PDMS vasculature.

Insert inch-long tubing into both ends of the bottom PDMS piece. Then flip the top acrylic block containing the top PDMS half onto the bottom acrylic block. Bolt the acrylic blocks together, ensuring the threaded block is on the bottom.

Plug the free ends of the tubing using 3D-printed plugs. Remove one plug, and gently inject 500 microliters of HLMVEC culture medium into the vasculature using a micropipette. Tilt to remove air bubbles, and then replace the plug.

Bring the assembled chip and sterile components into the biosafety cabinet. Connect all tubing and assemble the flow system, ensuring the inlet tubing connects to the bottom of the syringe reservoir to facilitate gravity flow. Now gently prime the flow system with HLMVEC medium.

Use a 50-milliliter serological pipette. Inject 30 milliliters of medium into the top of the syringe reservoir, and operate the peristaltic pump at low speed to fill tubing. Wash each PDMS vasculature half in PBS for five minutes, adding enough solution to fully submerge the vascular section.

The fluid simulation showed significantly elevated shear stress at vessel bifurcations in the pneumonectomy model compared to the control model. Brightfield microscopy confirmed uniform HLMVEC cell attachment in both static and flow conditions at 93.1 milliliters per minute after 24 hours. Fluorescence imaging of the inlet bifurcation showed intact and well distributed cell layers in both control and pneumonectomy models.

Imaging immediately after the second bifurcation revealed substantial cell loss in both the control and pneumonectomy models. Cells remained adherent and showed consistent staining in the region downstream of the second bifurcation in both models. Uniform DAPI-stained nuclei in the vascular models indicated that confluence was maintained despite flow exposure.

An early control model trial resulted in uneven cell distribution due to air bubbles introduced during system priming.