This work was supported by the National Institutes of Health, Grant Number: GM114359 and GM134701 (M.F.K. and L.E.K.), 1F31AI164870-01 (J.C.L.), and Defense Threat Reduction Agency, Grant Number: HDTRA11910012 (M.F.K. and L.E.K.).

Heparinized human blood is obtained for neutrophil isolation from healthy adult donors (males and females, aged between 21 and 60 years old), following informed consent as approved by the Institutional Review Board of Temple University (Philadelphia, PA, USA).
1. Priming and coating the device with human fibronectin
NOTE: The bMFA has two inlet ports and two outlet ports connected to the vascular compartment. It also has one port connected to the tiss...

<ol>
	<li>Kolaczkowska, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+and+function+in+health+and+inflammation.">Neutrophil recruitment and function in health and inflammation.</a> Nature Reviews Immunology. 13 (3), 159-175 (2013).</li><li>Yang, Q., Wijerathne, H., Langston, J. C., Kiani, M. F., Kilpatrick, L. 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F., Chavakis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+interactions+in+inflammation.">Leukocyte-endothelial interactions in inflammation.</a> Journal of Cellular and Molecular Medicine. 13 (7), 1211-1220 (2009).</li><li>Sadik, C. D., Luster, A. 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R., Pardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+leukocyte-tissue+interactions.">Pathophysiology of leukocyte-tissue interactions.</a> Current Opinion in Cell Biology. 18, 491-498 (2006).</li><li>Chistiakov, D. A., Orekhov, A. N., Bobryshev, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shear+stress+on+endothelial+cells:+go+with+the+flow.">Effects of shear stress on endothelial cells: go with the flow.</a> Acta Physiologica. 219 (2), 382-408 (2017).</li><li>Prabhakarpandian, B., Shen, M. -. C., Pant, K., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvascular Research. 82, 210-220 (2011).</li><li>Zou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSGL-1+derived+from+human+neutrophils+is+a+high-efficiency+ligand+for+endothelium-expressed+E-selectin+under+flow.">PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow.</a> American Journal of Physiology-Cell Physiology. 289, 415-424 (2005).</li><li>Lamberti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+microfluidic+assay+for+in+vitro+modeling+of+leukocyte-endothelium+interactions.">Bioinspired microfluidic assay for in vitro modeling of leukocyte-endothelium interactions.</a> Analytical Chemistry (Journal). 86, 8344-8351 (2014).</li><li>Prabhakarpandian, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+microvascular+networks+for+quantitative+analysis+of+particle+adhesion.">Synthetic microvascular networks for quantitative analysis of particle adhesion.</a> Biomedical Microdevices. 10 (4), 585-595 (2008).</li><li>Soroush, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+microfluidic+assay+reveals+a+key+role+for+protein+kinase+C+delta+in+regulating+human+neutrophil-endothelium+interaction.">A novel microfluidic assay reveals a key role for protein kinase C delta in regulating human neutrophil-endothelium interaction.</a> Journal of Leukocyte Biology. 100, 1027-1035 (2016).</li><li>Roth, N. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+dynamic+neonatal+blood-brain+barrier+on+a+chip.">A novel dynamic neonatal blood-brain barrier on a chip.</a> PloS one. 10, 142725 (2015).</li><li>Soroush, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-endothelial+interactions+of+murine+cells+is+not+a+good+predictor+of+their+interactions+in+human+cells.">Neutrophil-endothelial interactions of murine cells is not a good predictor of their interactions in human cells.</a> The FASEB Journal. 34, 2691-2702 (2020).</li><li>Soroush, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Kinase+C-Delta+(PKC&#948;)+tyrosine+phosphorylation+is+a+critical+regulator+of+neutrophil-endothelial+cell+interaction+in+inflammation.">Protein Kinase C-Delta (PKC&#948;) tyrosine phosphorylation is a critical regulator of neutrophil-endothelial cell interaction in inflammation.</a> Shock. 51 (5), 538-547 (2019).</li><li>Soroush, F., Tang, Y., Zaidi, H. M., Sheffield, J. B., Kilpatrick, L. E., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKC&#948;+inhibition+as+a+novel+medical+countermeasure+for+radiation-induced+vascular+damage.">PKC&#948; inhibition as a novel medical countermeasure for radiation-induced vascular damage.</a> The FASEB Journal. 32, 6436-6444 (2018).</li><li>Pradhan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microvascularized+tumor-mimetic+platform+for+assessing+anti-cancer+drug+efficacy.">A microvascularized tumor-mimetic platform for assessing anti-cancer drug efficacy.</a> Scientific Reports. 8 (1), 3171 (2018).</li><li>Tang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomimetic+microfluidic+tumor+microenvironment+platform+mimicking+the+EPR+effect+for+rapid+screening+of+drug+delivery+systems.">A biomimetic microfluidic tumor microenvironment platform mimicking the EPR effect for rapid screening of drug delivery systems.</a> Scientific Reports. 7 (1), 1-14 (2017).</li><li>Tang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-delta+inhibition+protects+blood-brain+barrier+from+sepsis-induced+vascular+damage.">Protein kinase C-delta inhibition protects blood-brain barrier from sepsis-induced vascular damage.</a> Journal of Neuroinflammation. 15, 309 (2018).</li><li>Mondrinos, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+endothelial+protein+kinase+C-delta+(PKCd)+regulates+neutrophil+migration+in+acute+lung+inflammation.">Pulmonary endothelial protein kinase C-delta (PKCd) regulates neutrophil migration in acute lung inflammation.</a> The American Journal of Pathology. 184, 200-213 (2014).</li><li>Kilpatrick, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+sepsis-induced+lung+injury+by+selective+inhibition+of+protein+kinase+C-d+(d-PKC).">Protection against sepsis-induced lung injury by selective inhibition of protein kinase C-d (d-PKC).</a> Journal of Leukocyte Biology. 89, 3-10 (2011).</li><li>Mondrinos, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+and+efficacy+of+targeted+pulmonary+delivery+of+a+protein+kinase+C-d+inhibitory+peptide:+Impact+on+Indirect+lung+Injury.">Biodistribution and efficacy of targeted pulmonary delivery of a protein kinase C-d inhibitory peptide: Impact on Indirect lung Injury.</a> Journal of Pharmacology and Experimental Therapeutics. 355, 86-98 (2015).</li><li>Liverani, E., Mondrinos, M. J., Sun, S., Kunapuli, S. P., Kilpatrick, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+protein+kinase+C-delta+in+regulating+platelet+activation+and+platelet-leukocyte+interaction+during+sepsis.">Role of protein kinase C-delta in regulating platelet activation and platelet-leukocyte interaction during sepsis.</a> PloS one. 13, 0195379 (2018).</li><li>Smith, A. M., Prabhakarpandian, B., Pant, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+shear+adhesion+map+using+SynVivo+synthetic+microvascular+networks.">Generation of shear adhesion map using SynVivo synthetic microvascular networks.</a> Journal of Visualized Experiments: JoVE. (87), e51025 (2014).</li></ol>

The bMFA reproduces the topography and flow conditions of the in vivo microvascular networks and can be used to study leukocyte-endothelial cell interaction and endothelial function in vitro under physiologically realistic conditions. In the microvasculature of either mouse or human, the geometry of the microvascular networks are self-similar and fractal, and the Reynolds number &#60;&#60; 1, indicating that vascular geometry does not significantly impact flow patterns. Therefore, the bFMA can be used t...

Inflammation is the host response to infection and injury, and the endothelium plays an important role in the inflammatory response1,2,3. Inflammatory dysregulation is the underlying cause of a number of disease pathologies such as sepsis, cardiovascular diseases, asthma, inflammatory bowel disease, cancer&#160;and COVID-19. Leukocyte-endothelial cell interactions play a central role in these inflammatory diseases. During inflammation, the release of PAMPS (pathogen-associated molecular patterns) from pathogens or DAMPS (damage-associated molecular patterns) from injured tissues activate immune cells to release cytokines/chemokines and other proinflammatory mediators that lead to the activation of endothelium, resulting in alterations in vascular endothelium barrier function and increased permeability3,4. Increased activation of endothelial cells during inflammation results in enhanced leukocyte-endothelial cell interaction leading to excessive migration of activated leukocytes across the vascular endothelium into key organs1,5,6,7.
The recruitment of leukocytes is initiated by chemically diverse chemoattractants composed of bioactive lipids, cytokines, chemokines&#160;and complement components8,9. Leukocyte recruitment is a multi-step process that includes five discrete steps: 1) leukocyte margination and capture/attachment, 2) rolling, 3) firm arrest, 4) spreading and crawling and 5) extravasation/migration (Figure 1). Each step of this process requiring crosstalk between leukocytes and endothelial cells to orchestrate this dynamic phenomenon1,9. Ultimately, arrested leukocytes extravasate to inflamed tissues across endothelium via a multi-step process controlled by concurrent chemoattractant-dependent signals, adhesive events and hemodynamic shear forces1,9,10,11,12.
Given the central role of shear stress in regulating endothelial cell function and the significance of the leukocyte-endothelium cell interactions13, several in vitro models have been developed during the last few decades to study various aspects of the leukocyte migration cascade in a more controlled environment14. Traditional fluidic devices to study leukocyte-endothelial cell interactions can be classified into two broad categories14: a) devices for studying leukocyte rolling, adhesion&#160;and adhesion molecule expression such as parallel plate flow chambers and b) devices for studying leukocyte migration under static conditions such as transwell chambers. Systems like parallel plate flow chambers have been used to study the roles of adhesion molecules and their ligands in the adhesion cascade under shear forces15. However, a significant drawback is that these simplistic, idealized devices (e.g., straight channel) are not able to reproduce the scale and geometry of the in vivo microvasculature (e.g., successive vascular bifurcations, vascular morphology)&#160;and the resulting flow conditions (e.g., converging or diverging flows at bifurcations). As a result, these devices&#160;can only model adhesion but not transmigration. Transwell chambers can only study transmigration under&#160;static conditions without considering the in vivo geometrical features and flow conditions. Thus, these traditional models do not mimic the microenvironment of living tissues or resolve adhesion and migration cascade in a single assay6.
To address this limitation, we have developed and extensively validated a novel 3D biomimetic microfluidic assay (bMFA) (Figure 2), which realistically reproduces in vivo microvascular networks on a chip16,17,18. The protocol for microfabrication of this device has been published previously17&#160;and is only briefly described here. The microvasculature of mouse cremaster muscle was digitized using a modified Geographic Information System (GIS) approach19. Then, the synthetic microvascular network was generated on polydimethylsiloxane (PDMS) using soft-lithography processes based on the digitized microvascular network14,17,20,21,22. Briefly, the digitized network images were printed on Mylar film, which was then used as a mask to pattern a SU-8 positive photoresist on top of a silicon wafer to create the masters for fabrication. Microfabricated pillars (10 &#181;m diameter, 3 &#181;m tall) were used to create the 3 &#181;m high and 100 &#181;m wide pores, an optimum size for leukocyte migration23,24,25, connecting the vascular channels and tissue compartments. PDMS was prepared according to the manufacturer&#39;s instructions and poured over the developed masters. Further, the PDMS was degassed and allowed to cure overnight in an oven (65 &#176;C) to create complementary microchannels in PDMS. Subsequently, the cured PDMS was peeled from the SU-8 master, followed by punching ports for inlets/outlets. Then, the PMDS was plasma bonded to a glass slide. The surface of the microfluidic device comprises native glass and PDMS. In order to promote cell attachment, spreading&#160;and proliferation, extracelluar matrix (ECM) coating is required. The bMFA includes a microvascular network and a tissue compartment connected via 3 &#181;m high and 100 &#181;m wide pores (Figure 2). This microfluidic system reproduces the entire leukocyte adhesion/migration cascade in a physiologically relevant 3D environment of a complete microvascular network with interconnecting vessels and bifurcations, including circulation, margination, rolling, adhesion&#160;and migration of leukocytes into the extra-vascular tissue compartment in a single system14,16,17,21,26.
It should be noted that even when the flow rate at the inlet of bMFA is fixed, the flow conditions in the network vary at different locations and cannot be calculated by a simple mathematical formula. A computational fluid dynamics (CFD)-based model was developed to calculate different flow parameters (e.g., shear stress, shear rate, velocity) at different locations in the network. This CFD model was used to simulate the dye perfusion patterns and flow parameters in the bMFA. Cross-validation with experimental results suggested that the flow resistances across the network are well predicted by the computational model (Figure 3)17. This CFD model was then used to estimate velocity and shear rate profile in every vessel of bMFA (Figure 4), allowing analysis of the effects of shear flow and geometry on leukocyte rolling, adhesion and migration16. Leukocytes preferentially adhere near bifurcations and in low shear regions in vivo, and these spatial patterns of leukocyte adhesion were successfully demonstrated in bMFA using neutrophils (Figure 5)16. This paper describes the protocol for preparing bMFA to study leukocyte-endothelial cell interaction under inflammatory conditions using human lung microvascular endothelial cells (HLMVEC) and human neutrophils. Microphysiological systems, such as the bMFA, can be used to study endothelial cell interactions with different types of cells such as neutrophils, monocytes, lymphocytes&#160;and tumor cells18,27,28,29,30. The bMFA can be seeded with primary endothelial cells from different organs (e.g., lung vs. brain) and different species (e.g., human vs. murine endothelial cells), as well as endothelial cell lines21,27,31,32. The bMFA can be used to study multiple cellular responses, cell-cell interactions, barrier function, drug delivery&#160;and drug toxicity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomimetic microfluidic assay (bMFA)</td>
			<td>SynVivo</td>
			<td>SMN1-C001</td>
			<td>Exclusive at SynVivo</td>
		</tr>
		<tr>
			<td>Blunt needle</td>
			<td>Jensen Global</td>
			<td>JG24-0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C70-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CFDA, SE</td>
			<td>ThermoFisher</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, 250,000, Powder</td>
			<td>Spectrum Chemical Mfg. Corp</td>
			<td>DE-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Premium</td>
			<td>GE Health Care</td>
			<td>17-5442-02</td>
			<td>Leukocyte isolation media</td>
		</tr>
		<tr>
			<td>fMLP</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Fisher Scientific</td>
			<td>AAJ1692630</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Fisher Scientific</td>
			<td>33-016-015</td>
			<td>use vendor recommended ECM for different cell lines</td>
		</tr>
		<tr>
			<td>Microvascular Endothelial Cell Growth Medium-2 BulletKit</td>
			<td>Lonza</td>
			<td>cc-3202</td>
			<td>Human lung microvascular endothelial cell culture medium (HLMVEC).</td>
		</tr>
		<tr>
			<td>Human lung microvascular endothelial cells</td>
			<td>Lonza</td>
			<td>cc-2527</td>
			<td>use vedor remommended trypsin-EDTA and TNS</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti2</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>NIS-elements, 5.20.01</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PhD Ultra Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>70-3007</td>
			<td>Syringe Pump</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>02-003-763</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide clamp</td>
			<td>SynVivo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S640-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Synvivo Pneumatic Primer</td>
			<td>SynVivo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, Trypsin Neutralization Solution(TNS)</td>
			<td>Lonza</td>
			<td>cc-5034</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher Scientific</td>
			<td>50-206-8921</td>
			<td>Tubing</td>
		</tr>
	</tbody>
</table>

a microphysiological system to study leukocyte-endothelial cell interaction during inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

After 48 h of culture under shear flow in bMFA, endothelial cells covered the surface of the vascular channels in bMFA and aligned in the direction of flow (Figure&#160;6). Confocal microscopy indicated that all surfaces of the vascular channels were covered by endothelial cells, forming a complete 3D lumen in bMFA18.
Using this protocol, a neutrophil adhesion map can be acquired, showing that there is significant adhesion of neutrophils to...

Watch this Scientific Journal Video about A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation at JoVE.com

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. Inflammation dysregulation often causes altered vascular endothelium barrier function and excessive leukocyte trafficking, which can result in organ damage. The biomimetic microfluidic assay, what we call bMFA, reproduces topography and flow conditions of in-vivo microvascular networks and allows for realtime assessment of rolling, firm adhesion, spreading, and migration of neutrophils into the tissue compartment.A strength of the biomimetic microfluidic assay is the ability to use primary human cells and clinically relevant patient samples to increase clinical translation and rapidly screen potential therapeutics. Demonstrating the procedures will be Mr.Qingliang Yang, a graduate research assistant from my laboratory. Start inserting the tube of around one inch in length within the ports using fine forceps, except for one inlet port.Using jaw clamps, clamp the two outlet ports and the tissue compartment together. Dilute the fibronectin stock solution to 100 micrograms per milliliter with PBS. Load a one-milliliter syringe connected to a 24-gauge blunt needle with the diluted fibronectin solution and connect the syringe to a four-inches-long tubing.Insert the tubing into the open inlet port, then push the plunger until human fibronectin is released from another inlet port and clamp it. Repeat the process for the remaining ports until all the channels, tissue compartment, and tubing are filled with the human fibronectin solution. Remove the needle but keep the four-inch-long tubing inserted and unclamped.To perform degassing, connect the unclamped tubing to the pneumatic primer connected to a compressed nitrogen tank with a pressure of five pounds per square inch for 15 minutes. Under the microscope, check that there are no air bubbles trapped within the channel or tissue compartment and reconnect the device if the air bubbles are present. Remove the device from the pneumatic primer and incubate it at 37 degrees Celsius for one hour.Using pre-warmed human lung microvascular endothelial cell culture media, flush all the channels and tissue compartment. After harvesting the endothelial cells as described in the text manuscript, pellet the cells by centrifugation for five minutes at 150 times G.In the programmable syringe pump, mount a one-milliliter syringe and attach the tubing to the blunt needle. Draw approximately 20 microliters of the cell suspension into the tubing without letting it into the syringe barrel.Remove the clamp from the outlet port of the device. Connect the tubing to the inlet port without introducing any air bubble into the channel. Stop the pump with a flow rate of four to eight microliters per minute and observe under the microscope.Stop the pump when the channels are filled with cells. Clamp the outlet and cut the inlet tubing. Place the device in the incubator for four hours at 5%carbon dioxide and 37 degrees Celsius.After four hours of incubation, prepare another syringe and fill it with fresh cell culture media. Mount the syringe in a syringe pump and connect it to the inlet port. Remove the clamp from the outlet port.Pass the fresh media through the device for about five minutes at four to eight microliters per minute to remove the floating or unattached cells. Prepare a syringe by filling it with fresh cell culture media. Mount the syringe in a syringe pump and connect it to one inlet port while keeping the outlet port open.Place the device in an incubator at 37 degrees Celsius and 5%carbon dioxide. Program the syringe pump for culturing endothelial cells under flow as described in the text manuscript. Using a microscope, check bMFA after 48 hours of culture under flow.Confocal microscopy indicated that all surfaces of the vascular channels were covered by endothelial cells, forming a complete 3D lumen in bMFA. After preparing three different bMFA devices, load three one-milliliter syringes with cell culture media or TNF-alpha or TNF-alpha combined with PKC delta inhibitor respectively, where TNF-alpha and inflammatory cytokine is used to stimulate endothelial cells and neutrophils. PKC delta inhibitor is a novel anti-inflammatory inhibitor.Connect the three loaded syringes to three bMFA devices. Using buffer TNF-alpha or TNF-alpha with added inhibitor, treat human lung microvascular endothelial cells for four hours at 0.1 microliters per minute. After isolating the human neutrophils as described in the text manuscript, resuspend the neutrophils in 999 microliters of HEPES buffer and add one microliter of 10-millimolar CFDA SE dye stock solution to the suspension, resulting in a 10-micromolar working solution of CFDA SE and incubate it for 10 minutes at room temperature.Wash the cells twice with HEPES buffer by centrifuging the solution for five minutes at 315 times G.After counting the cells, resuspend at 2 million neutrophils per milliliter in cell culture media or TNF-alpha or TNF-alpha added with PKC delta inhibitor and incubate at room temperature for 15 minutes. Fill the syringe with one-micromolar of a chemo-attractant, fMLP, prepared in cell culture media. Open one inlet and one outlet port of the bMFA.Remove the port tubing from the tissue compartment and insert the fMLP tubing. Inject approximately 20 microliters of fMLP into the tissue compartment for all bMFA, leaving the one treated with cell culture media. Cut the tubing and clamp it.Replete the syringe with approximately 200 microliters of neutrophil suspension and mount the syringe on the syringe pump. After placing the device on an inverted microscope stage, set the flow rate at one microliter per minute and start the pump. Wait until a small drop of the neutrophil suspension comes out of the tubing and insert the tubing into the inlet port.Fluorescently-labeled neutrophils flow into the vascular channels and interact with endothelial cells and the physiologically-relevant flow conditions. After 10 minutes from starting the experiment, open the image analysis software, switch the objective lens to 10x, and use the stage joystick to center the device under the microscope. To obtain the adhesion map, go to Acquire, click Scan Large Image.A new window pops up. Choose 10x Objective lens and set the field option, for example, 5 by 3. Click Scan, click File, and save the adhesion map.To obtain the migration map for migration analysis, center the device under the microscope using the stage joystick. Click View, Acquisition Controls, ND Acquisition. A new window pops up.Set the Path to save the file and type the file name. Check Large Image function, set Scan Area, for example, 5 by 3. Check Time function, set the Interval as five minutes, and Duration as 60 minutes.Take time lapse images of the tissue compartment, with one image taken every five minutes during the next hour. CFD simulation shows the laminar flow pattern in vascular channels, except for bifurcation areas where the flow pattern is disturbed. After performing the biomimetic microfluid assay, phase contrast images revealed that the surfaces of the vascular channels were covered with endothelial cells, and aligned in the direction of shear flow after 48 hours of culture.Fluorescence imaging was carried out using confocal microscopy, indicating that the endothelial cells form a complete three-dimensional lumen in bMFA A neutrophil adhesion map was obtained, which revealed that there is significant adhesion of neutrophils to endothelial cells in bMFA. Neutrophil migration map in bMFA revealed that upon TNF-alpha activation, considerable migration of neutrophils occurs inside the tissue compartment, while no such migration was observed without TNF-alpha activation. An adhesion map correlating the spatial distribution of neutrophils with the shear rate demonstrated that neutrophil adhesion occurred preferentially in vessels with low shear rate and near bifurcation regions.Further, TNF-alpha treatment significantly increases adhesion, which was inhibited with the PKC delta inhibitor. Analyzing time lapse images indicated that TNF activation of endothelial cells increases neutrophil migration in response to fMLP, whereas treatment with the PKC delta inhibitor reduced migration compared to TNF-alpha treated cells. Thus, bMFA can be used to test the efficacy of a novel therapeutic for treating inflammatory disease.The bMFA can also be used to study the endothelium integrity by measuring variables such as permeability and trans-endothelial electrical resistance, what's called TEER, and adhesion molecule expression during inflammation. The biomimetic microfluidic assay can mimic the microenvironment of different organs and is not limited to single cell types or species, and can also model cell/cell communication critical to organ function and modeling different diseases.

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3.0K vistas.  Temple University. Las interacciones entre leucocitos y células endoteliales juegan un papel importante en enfermedades inflamatorias como la sepsis. La desregulación de la inflamación a menudo causa alteración de la función de barrera del endotelio vascular y tráfico excesivo de leucocitos, lo que puede provocar daños en los órganos. El ensayo microfluídico biomimético, lo que llamamos bMFA, reproduce la topografía y las condiciones de flujo de las redes microvasculares in vivo y permite la evaluaci...