Name: Modeling the Endothelial Glycocalyx Post-Pneumonectomy in a 3D Fluidic Chip - An Approach to Fabricating a Vascular-based Organ-on-Chip System (Video) | JoVE Journal
Uploaded: 2025-09-16T13:20:00.000+00:00
Duration: 6 min 12 s
Description: Learn to construct an organ-on-chip system modeling pneumonectomy for vascular research using silicone elastomer and HLMVEC cells. This method enables investigation of endothelial glycocalyx impairment and surgical complications.

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5 minutes

The authors would like to express our deepest gratitude to the Worcester Polytechnic Institute Biomedical Engineering Department for their support and guidance. We would like to provide a special thanks to Megan Ouellette, John Martel, and Sudish Vengat for their collaborative efforts and innovative ideas that greatly enhanced the quality and viability of this research. In addition, we would like to thank Samantha Cocchiaro, and Udaya Rattan for their in-lab contributions and their essential support and assistance.

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<li>Nian, K., Harding, I. C., Herman, I. M., Ebong, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood-brain+barrier+damage+in+ischemic+stroke+and+its+regulation+by+endothelial+mechanotransduction%5BTitle%5D)+AND+%22Front.+Physiol%22%5BJournal%5D)">Blood-brain barrier damage in ischemic stroke and its regulation by endothelial mechanotransduction.</a> Front. Physiol. 11, 605398(2020).</li>
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<li>Kataoka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+imaging+of+endothelial+glycocalyx+layer+with+wheat+germ+agglutinin+using+intravital+microscopy%5BTitle%5D)+AND+%22Microsc.+Res.+Tech%22%5BJournal%5D)">Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.</a> Microsc. Res. Tech. 79, 31-37 (2016).</li>
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<li>Emde, B., Heinen, A., Gödecke, A., Bottermann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wheat+germ+agglutinin+staining+as+a+suitable+method+for+detection+and+quantification+of+fibrosis+in+cardiac+tissue+after+myocardial+infarction%5BTitle%5D)+AND+%22Eur.+J.+Histochem%22%5BJournal%5D)">Wheat germ agglutinin staining as a suitable method for detection and quantification of fibrosis in cardiac tissue after myocardial infarction.</a> Eur. J. Histochem. 58 (4), 2448(2014).</li>
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The authors declare no conflicts of interest.

Vascular modeling is an important new field for exploration. The successful creation of an accurate vascular model holds great promise for advancing the field of tissue engineering and drug development. This article outlines a novel method for generating an OOC vascular system that can be utilized to emulate a pneumonectomy or any other vascular system or geometry to study a potential link between GCX impairment and post-pneumonectomy complications. On a broader scale, this methodology can also be applicable to emulate o...

A pneumonectomy is an invasive and high-risk surgical intervention performed to extract the right or left lung1. This procedure is typically utilized as a treatment option for various severe ailments such as lung cancer, pulmonary tuberculosis, and traumatic lung injuries2. While the procedure holds promise, it poses significant risks, highlighted by increased rates of postoperative complications ranging from arrhythmias to pulmonary edemas1.
Pneumonectomies have a profound impact on cardiovascular dynamics by directing the heart's entire cardiac output into the remaining lung3. This redirection of blood flow is known to increase pressure and shear stresses within the remaining pulmonary vasculature4. With these vast alterations in blood flow patterns, the endothelial glycocalyx (GCX) plays a pivotal role in sensing these changes in the blood flow patterns5.
The GCX is a gel-like layer that regulates biochemical activities in response to changes in its environment6,7. Its expression has been shown to increase with prolonged exposure to uniform flow and decrease with exposure to disturbed flow compared to static culture8,9. The GCX exhibits a variable thickness ranging from 0.1-1 µm across the entire vasculature and functions as an essential mechanotransduction layer coating the endothelial cells of blood vessels10. The network is composed of a dense layer of sugars, lipids, and proteins, as shown in Figure 1. These sugars bind to form glycans, which can exist either freely in the bloodstream or attached to proteins or lipids, resulting in the formation of proteoglycans, glycoproteins, or glycolipids7,11. Proteoglycans are composed of glycosaminoglycan (GAG) chains, which bind to the cell membrane through plasma proteins or receptors. These GAGs include heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate, and keratan sulfate12. Heparan sulfate is the predominant GAG, making up over 50% of the total GAGs found in the GCX. These side chains can attach to embedded core proteins, which include glypicans (anchored by glycosylphosphatidylinositol) and syndecans (with a membrane-spanning domain), along with CD44 (a surface adhesion receptor). The complexes described above play a crucial role in regulating growth factors, anti-inflammatory reactions, facilitating vessel dilation, and controlling vascular permeability13.
<img alt="Endothelial cell diagram; phospholipid bilayer, integrin receptors, glycoprotein interaction." class="xfigimg" src="/files/ftp_upload/67246/67246fig1v2.jpg" data-altupdatedat="1762425657000" data-alt="Figure 1"> 
Figure 1: GCX component diagram. Schematic representing the components of the endothelial glycocalyx. This schematic displays the apical glycocalyx, which is found on the lumen of the vessel, showing its membrane-bound proteoglycans and glycoproteins. Created with BioRender.com <a href="https://www.jove.com/files/ftp_upload/67246/67246fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the precise connection between pneumonectomies and GCX damage is not well studied, there exists a plausible correlation between the altered blood flow patterns induced by pneumonectomies and the integrity of the GCX. Disturbed blood flow in the vasculature is known to inflict damage on the GCX6. We hypothesized that increased disturbed flow and changes in shear stress in the remaining vasculature following a pneumonectomy may inflict damage to the GCX over time. This accelerated deterioration may catalyze postoperative complications and morbidities, thereby increasing the inherent risks to the patient's overall health13.
This protocol aimed to model the pulmonary vasculature following pneumonectomy, following the general procedure outlined in Figure 2. Fluid simulations were conducted on a computer-aided design model replicating human pulmonary vasculature with and without pneumonectomy. The pneumonectomy models were created by designing 3D pulmonary vasculature in 3D modeling software, based on dimensions derived from CT scans and literature data, then further simplifying them for flow simulations in computational fluid dynamics (CFD) software. The simulations identified specific areas of interest, chosen by areas where shear stress was increased in the pneumonectomy models when compared to the control. These areas of interest were chosen to replicate for building an in vitro model. The in vitro model utilized a fluidic chamber design to simulate multiple sections of vasculature with normal geometry and pneumonectomy geometry to simulate the difference in flow conditions.
Human lung microvascular endothelial cells (HLMVECs) were cultured on fibronectin-coated polydimethylsiloxane (PDMS) substrates, shaped to mimic a selected bifurcation region. Microvascular endothelial cell culture media was perfused through the system using a peristaltic pump. Finally, the cells and GCX were fixed, immunostained with a GCX-staining antibody, and visualized using a fluorescent microscope to evaluate their condition after exposure to fluid flow. A general timeline of the protocol can be found in Table 1.
<img alt="CT scan to COMSOL flow simulation to Organ-on-Chip setup; PDMS molds in fluid dynamics experiment." class="xfigimg" src="/files/ftp_upload/67246/67246fig2.jpg" data-altupdatedat="1762425657000" data-alt="Figure 2"> 
Figure 2: OOC development sequence. Sequence of developing a novel OOC system to study the effects of lung pneumonectomy (1) CT scan generated of human pulmonary vasculature. (2) CT scan geometry used to identify regions of interest for 3D CAD model. (3) Fluid flow simulations to determine geometries of interest for in vitro model. (4) PDMS mold modeled containing areas of interest from simulation results (5) Mold inserted into acrylic case to fix mold for perfusion (6) Completed flow chamber setup (7) Complete flow chamber setup transferred to incubator (8) Brightfield and fluorescent images taken after exposure to shear stress. Created with BioRender.com <a href="https://www.jove.com/files/ftp_upload/67246/67246fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: General protocol timeline. General Sequence of performing steps within the protocol with average duration (h) of each step. <a href="https://www.jove.com/files/ftp_upload/67246/Table%201.xlsx" target="_blank">Please click here to download this Table.</a>

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modeling the endothelial glycocalyx post-pneumonectomy in a 3d fluidic chip - an approach to fabricating a vascular-based organ-on-chip system

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

Endothelial glycocalyx (GCX), a carbohydrate-rich layer coating the luminal surface of endothelial cells, plays a pivotal role in regulating cellular responses to stimuli. It is comprised of transmembrane proteins serving as mechanotransducers for cellular responses. While it naturally maintains its structure and stability under homeostatic conditions, exposure to high-shear stress can induce damage with numerous consequences. General shear stress effects on endothelial cells have been explored, but the extent and impact of shear stress on vascular systems, specifically post-pneumonectomy, have not been well studied. To investigate this, a comprehensive approach was undertaken, involving the creation of a CAD model of pulmonary vasculature pre- and post-pneumonectomy. Utilizing computational fluid simulation, key regions of elevated shear stress and pressure were identified and replicated in an organ-on-chip (OOC) system. Human lung microvascular endothelial cells (HLMVECs) were seeded onto a mold in the shape of the selected sections to characterize the effects of elevated shear stress in vitro. Following experimentation, HLMVECs were immunostained to qualitatively evaluate GCX health under normal and increased stresses induced by pneumonectomy. The integration of computational modeling and experimental analysis enhances our understanding of how changes in shear stress affect GCX, clarifying their effects on vascular function and post-surgical complications.

The results from the fluid simulation laminar flow steady state study showed an increase in shear stress in certain bifurcations in the pneumonectomy models when compared to the control model, shown in Figure 11. Visualized in step 2.8 of the protocol, the red areas denote areas of high shear stress, while the blue areas show areas of low shear stress.
<img alt="3D fluid dynamics simulation of aorta, pressure distribution...

Here we present a protocol for constructing an organ-on-chip (OOC) system designed to mimic pneumonectomy. This system investigates the potential correlation between glycocalyx impairment and complications following pneumonectomy.

Endothelial glycocalyx (GCX), a carbohydrate-rich layer coating the luminal surface of endothelial cells, plays a pivotal role in regulating cellular ...

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.

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Research

JoVE Journal

Bioengineering

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Learn to construct an organ-on-chip system modeling pneumonectomy for vascular research using silicone elastomer and HLMVEC cells. This method enables investigation of endothelial glycocalyx impairment and surgical complications.

Modeling the Endothelial Glycocalyx Post-Pneumonectomy in a 3D Fluidic Chip - An Approach to Fabricating a Vascular-based Organ-on-Chip System (Video) | JoVE Journal

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

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