This study was supported by grants from the Swedish Research Council (90293501, A0290401, A0290402), the European Community&#39;s Seventh Framework Programme under grant agreement n&#176;602699 (DIREKT), the NovoNordisk Foundation, the Gurli and Edward Brunnberg Foundation, Stem Therapy, Vleugel Foundation and &#197;ke Wiberg Foundation.

NOTE: Blood was drawn from healthy individuals by open system venipuncture in approval with the Ethical Review Board in Uppsala (Permit No. 2008/264).
1. Seeding Cells
<ol>
	<li>Culture primary human umbilical vein endothelial cells (HUVEC) at 37 &#176;C in 5% CO2 in T75 flasks coated with 1% gelatin in PBS pH 7.4.</li>
	<li>Remove culture medium from a T75 flask containing HUVEC (or other type of primary human endothelial cells) and wash the cells with 2.1 mM EDTA in PBS pH 7.4. Detach cells by adding 1 ml 0.25% trypsin-EDTA and incubating for 2 - 3 min in 37 &#176;C.</li>
	<li>Collect cells by rinsing the flask with 9 ml 10% FBS in PBS pH 7.4 and transfer the cell suspension to a 15 ml tube. Spin down the cells at 735 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet carefully in endothelial cell growth medium microvascular (EC GMMV) and count the cells in a B&#252;rker chamber. Seed 21,000 cells/cm2 in 1-well cell culture slides and incubate at 37 &#176;C. The HUVEC will be confluent after 1 - 2 days of culture. Monitor the cell morphology and confluency daily under a light microscope.</li>
</ol>
2. Preparation of Whole Blood
<ol>
	<li>Prepare all artificial surfaces that will be in contact with blood with a double layer of immobilized heparin (CHS) according to the manufacturer&#8217;s instructions. CHS coated materials protected from dust can be stored at room temperature up to 6 months without loss of function.</li>
	<li>Use open system venipuncture to draw blood from a healthy donor shortly (&#60;30 min) before starting the blood endothelial chamber experiment. Couple a hypodermic needle (18 G x 50 mm) to CHS coated silicon tubing (inner diameter: 2 mm) before carefully collecting blood in a CHS coated 50 ml tube. Supplement blood with unfractionated heparin to reach a final concentration of 0.75 IU/ml. Mix the blood gently by inverting the tube 2 - 3 times.</li>
</ol>
3. Blood Endothelial Chamber Model
<ol>
	<li>Fabricate blood chambers (Figure 1A; top view) in acrylic polymethyl methacrylate (PMMA) consisting of two cylinders (height 8 mm and radius 9 mm) that are glued to a PMMA slide (25 x 75 mm). Fit the edges of the cylinders facing upwards with rubber O-rings to seal the blood chamber against the glass slides with cultured endothelial cells (Figure 1B). Rotate the blood chambers connected to the culture slide with endothelial cells thereafter in a vertical position (Figure 1C).</li>
	<li>Use a CHS coated pipette tip to transfer 1.5 ml blood into each CHS coated chamber taking care not to activate the blood.</li>
	<li>Transfer 1 ml blood into microcentrifuge tubes containing 30 &#956;l 0.34 M K3EDTA to reach a final concentration of 10 mM K3EDTA for determination of initial plasma protein concentrations using an enzyme linked immunosorbent assay (ELISA). Carefully mix the blood and keep the tubes on ice.</li>
	<li>Remove the plastic walls of the cell culture glass slides according to manufacturer&#8217;s instructions and carefully wash the cells in PBS pH 7.4. Make sure to subsequently remove as much liquid as possible from the slides. Do not let the cells dry out.</li>
	<li>Place the slide on top of the blood chambers, with cells facing the blood. Secure the slide by placing a clamp around the blood endothelial cell chamber. Use a plastic slide for additional support to avoid breakage of the cell culture glass slide when placing the clamp.</li>
	<li>Fix the blood endothelial cell chamber onto a rotating wheel (40 cm in diameter) place in a 37 &#176;C water bath. Rotate each chamber on the wheel at 0.5 x g for 30 min.</li>
	<li>After incubation, dismantle the blood endothelial cell chamber. Wash the cell culture slides in PBS pH 7.4 and fix the cells in ice cold 1% paraformaldehyde (PFA) for 10 min. Transfer 1 ml blood from each chamber to microcentrifuge tubes containing 30 &#956;l 0.34 M K3EDTA to reach a final concentration of 10 mM K3EDTA and mix the tubes gently. Keep the tubes on ice.</li>
	<li>Centrifuge the tubes at 4,560 x g for 10 min in 4 &#176;C. Transfer the blood plasma to new microcentrifuge tubes and store in -70 &#176;C until further analysis.</li>
</ol>
4. Analysis of Blood Endothelial Interactions
<ol>
	<li>Perform immunofluorescent staining with mouse anti-human CD16 followed by secondary goat anti-mouse Alexa 488 and phalloidin-TexasRed. Perform each staining step for 1 hr at room temperature and wash the slides in PBS pH 7.4 between every step. Counterstain the nuclei with DAPI for 10 min at room temperature. Analyze blood components bound to the endothelial cells by fluorescence microscopy in combination with image analysis using suitable image analysis software such as CellProfiler.</li>
	<li>Quantify the reactions in the blood compartment with thrombin-antithrombin (TAT) ELISA as per manufacturer&#8217;s instructions of the plasma samples.</li>
	<li>Use appropriate statistical tools, e.g., an unpaired t test, to analyze the data.</li>
</ol>

<ol>
	<li>Mestas, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+Mice+and+Not+Men:+Differences+between+Mouse+and+Human+Immunology.">Of Mice and Not Men: Differences between Mouse and Human Immunology.</a> J Immunol. 172 (2), 2731-2738 (2004).</li><li>Seok, 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=Genomic+responses+in+mouse+models+poorly+mimic+human+inflammatory+diseases.">Genomic responses in mouse models poorly mimic human inflammatory diseases.</a> Proc Natl Acad Sci U S A. 110 (9), 3507-3512 (2013).</li><li>Hong, J., Ekdahl, K. N., Reynolds, H., Larsson, R., Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+to+study+interaction+between+whole+blood+and+biomaterials.+Studies+of+platelet+and+coagulation+activation+acid+the+effect+of+aspirin.">A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation acid the effect of aspirin.</a> Biomaterials. 20 (7), 603-611 (1999).</li><li>Doctor, A., Spinella, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Processing+and+Storage+on+Red+Blood+Cell+Function+In.">Effect of Processing and Storage on Red Blood Cell Function In.</a> Vivo. Semin Perinatol. 36 (4), 248-259 (2012).</li><li>Andersson, 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=Optimal+heparin+surface+concentration+and+antithrombin+binding+capacity+as+evaluated+with+human+non-anticoagulated+blood+in+vitro.">Optimal heparin surface concentration and antithrombin binding capacity as evaluated with human non-anticoagulated blood in vitro.</a> J Biomed Mater Res A. 67 (2), 458-466 (2003).</li><li>Ekdahl, K. N., Hong, J., Hamad, O. A., Larsson, R., Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+blood+compatibility+of+materials,+cells,+and+tissues:+basic+concepts,+test+models,+and+practical+guidelines.">Evaluation of the blood compatibility of materials, cells, and tissues: basic concepts, test models, and practical guidelines.</a> Adv Exp Med Biol. 735, 257-270 (2013).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+dynamics+of+the+endothelium.">Spatial and temporal dynamics of the endothelium.</a> J Thromb Haemost. 3 (7), 1392-1406 (2005).</li><li>Cines, D. 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=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91 (10), 3527-3561 (1998).</li><li>Kirchhofer, D., Tschopp, T. B., Hadvary, P., Baumgartner, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+stimulated+with+tumor+necrosis+factor-alpha+express+varying+amounts+of+tissue+factor+resulting+in+inhomogenous+fibrin+deposition+in+a+native+blood+flow+system.+Effects+of+thrombin+inhibitors.">Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.</a> J Clin Invest. (5), 2073-2083 (1994).</li><li>Esmon, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+circuits+in+thrombosis+and+inflammation.">Molecular circuits in thrombosis and inflammation.</a> Thromb Haemost. 109 (3), 416-420 (2013).</li><li>Rosenberg, R. D., Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular-Bed&#8211;Specific+Hemostasis+and+Hypercoagulable+States.">Vascular-Bed&#8211;Specific Hemostasis and Hypercoagulable States.</a> N Engl J Med. 340 (20), 1555-1564 (1999).</li></ol>

The authors have nothing to disclose.

Multiple interactions between the blood and vessel wall are normally maintained in a quiescent state due to non-adhesive and anti-thrombotic nature of the endothelium.7 During pathological conditions involving inflammation, the endothelium is activated with a resulting increase in adhesion receptor expression8 and a reduced ability to inhibit hemostasis.9 Activation of the cascade systems in the blood in turn amplifies the thrombogenicity of the endothelium causing further thrombosis and leukocyte recruitment.10 To gain a better understanding of this delicate interactions between endothelial cells and whole blood, we have developed a novel in vitro method where cultured endothelial cells are placed in contact with whole blood. To our knowledge, our setup is the first to show endothelial cells incubated with whole human blood with no or very little anticoagulant for longer time periods.
The sensitivity of this system is enabled by open system venipuncture in combination with a protective layer of immobilized heparin on all surfaces placed in contact with blood. The use of an open system during blood procurement reduces the activation of the cascade systems during venipuncture whilst permitting the use of a self-determined amount of anticoagulant. Commercially available evacuated blood tubes may, however, be used depending on the intended end points of the study. The final concentration of anticoagulant added to most commercially available closed system tubes may inhibit sensitive, and otherwise hard to study, interactions in the setup.6 A protective heparin surface further eliminates activation of blood through surface contact by any surfaces other than the endothelial cells.5 Indeed, incubation of whole blood supplemented with a small amount of unfractionated heparin in chamber without the protection of CHS resulted in clot formation.
The air bubble inside the chamber enables mixing of the blood during incubation with the endothelial cells. In order to assess the effect of the air bubble size, we used two different blood volumes. In this model, we measured similar TAT levels regardless of air bubble size in chambers incubated with HUVEC. TAT was, however, increased with a smaller air bubble size in the fully CHS treated chambers. This, in accordance with previously described endothelial cell properties11, indicates a regulatory effect upon the increased activation of coagulation provided by the endothelial cells that is lacking in the inert CHS chamber. The air bubble in the system creates a flow upon rotation of the chamber. The size of the bubble will affect the forces within this rotating system. This is shown by our results in Figure 2D were a smaller bubble creates higher TAT values representing a decreased force upon the blood i.e., the blood movement within the chamber is lower and thereby activation of the cascade system occurs to a higher extent. It should be mentioned that in this system we are creating a rotating flow that will be different in speed across the chamber with the highest speed along the walls and the lowest in the center. This is obviously not an optimal environment with regard to flow and shear stress for the endothelial cells, but still we have a system producing stable and repeatable results. More optimal conditions would include circulating laminar flow in the absence of turbulence. This is, however, not possible in the model represented here and to our knowledge such a system is not available to date. Although, there are several microfluidic systems available commercially that are not possible to combine with whole blood with no or low levels of anticoagulant added to the system thereby fail to enable proper sensitive evaluation of the interactions between all components present in blood and endothelial cells.
Furthermore, there was a 2-fold increase in recruitment of blood cells towards TNF&#945; activated HUVEC in combination with a doubled formation of TAT independently of the blood volume. This verifies stability of the model system and also shows the possibility to use an activated endothelium in combination with whole blood. Future on, the blood endothelial cell chamber can be used to investigate blood cell recruitment towards activated endothelial cells by a variety of markers expressed on cells under various conditions and stages of activation. Furthermore, the model may be used in combination with pharmacological agents in order to evaluate effects on inflammatory conditions.
In summary, we show the great benefit of combining a chamber model with human blood and vascular cells to create a complete human in vitro system to perform relevant investigations of vascular disease.

Methods for analyzing blood-vascular interactions in cardiovascular disease generally involve animal research experiments. Results created in experimental research animal models may, however, have little or no implication for human disease.1,2 As such, there is a need for good and reliable in vitro models to investigate cellular interactions between the blood compartment and vascular endothelial cells in a human-like system. We have therefore established a blood endothelial cell chamber model. This is based on a previously described model used to investigate interactions between biomaterials and human whole blood.3 Contrary to other in vitro setups where usually limited number of purified components, i.e., thrombocytes, leukocytes or endothelial cells are available, the present model incorporates all the components present in a blood vessel.
The setup of the blood endothelial cell chamber model is designed to enable use of freshly drawn human blood with little or no added anticoagulant. Blood stored during longer time periods acquire so called storage lesions where breakdown of erythrocytes may interfere with delicate interactions between blood and endothelial cells.4
To avoid clot formation during handling of whole blood ex vivo requires either high doses of anticoagulant or that all material in contact with blood have to be non-activating. As non-activating surfaces are rare in the laboratory environment, materials may alternatively be equipped with a protective layer of immobilized heparin. A protective layer of immobilized heparin (hereafter referred to as Corline heparin surface (CHS)) that may be applied to most materials creating a surface where blood contact can occur without causing an activation of the coagulation cascade.5 Thus, by furnishing the chambers with CHS, the blood endothelial cell chamber model enables use of very low concentrations of anticoagulant in the blood. Avoiding the addition of high concentrations of anticoagulant in the blood endothelial chamber model makes it possible to study sensitive interactions within a blood vessel that might otherwise be masked.6
The blood endothelial cell chamber model consists of two chambers formed by attaching plastic cylinders (height: 8 mm; radius: 9 mm) to a plastic microscope slide. The edges facing upward on the cylinders are equipped with grooves that are fitted with rubber O-rings used for sealing the chambers against the cell culture slide. The chambers are only partially filled with blood as the air left in the chamber keeps blood in movement when the chambers are subsequently rotated in a vertical position (Figure 1).
In order to assess the functionality of the model, we performed experiments with either a completely CHS coated chamber or Human Umbilical Vein Endothelial Cells (HUVEC). Two separate blood volumes were assayed and the formation of thrombin antithrombin (TAT) complexes (an indirect marker of coagulation) was measured with enzyme linked immunosorbent assay (ELISA). We then assessed the effect of blood volumes and tumor necrosis factor (TNF&#945;) stimulated endothelial cells on leukocyte recruitment by image analysis.
<img alt="Figure 1" src="/files/ftp_upload/52112/52112fig1highres.jpg" /> 
Figure 1. Setup of the blood endothelial cell chamber model. (A) Top view schematic drawing of the blood chamber made in PMMA. (B) Primary human endothelial cells are cultured on 1-well chamber slides. Fresh human whole blood is added to two chambers on a microscope slide. The chambers and all material used for handling the blood are treated with a protective HS coating. After removing the walls of the cell culture slide, the cells are placed facing the blood and the system is clamped shut. (C) The blood endothelial cell chambers are then incubated under rotation in 37 &#176;C. Afterwards, the reactions in the blood compartment may be analyzed by ELISA and the endothelial cells may be analyzed by microscopy.

<table><tbody><tr><td>Human Umbilican Vein Endothelial Cells (HUVEC)</td><td>PromoCell</td><td>C-12200</td><td>Any other type of primary human endothelial cell may be used.</td></tr><tr><td>Endothelial Cell Growth Medium MV (ECGM MV)</td><td>PromoCell</td><td>C-22120</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G-2500</td><td>Prepare 1% gelatin in PBS, incubate 5 ml in a T75 for 15 min and remove before culturing cells.</td></tr><tr><td>TAT ELISA</td><td>Enzyme Research Lab.</td><td>TAT-EIA-C</td><td></td></tr><tr><td>Mouse anti-human CD16</td><td>DAKO</td><td>F7011</td><td>Dilution 1:100</td></tr><tr><td>Goat anti-mouse Alexa 488</td><td>Molecular Probes</td><td>A11001</td><td>Dilution 1:500</td></tr><tr><td>Phalloidin-Texas Red</td><td>Molecular Probes</td><td>A22287</td><td>Dilution 1:200</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>Dilution 10 &amp;mu;g/ml</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E-6758</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco Life Tecnologies</td><td>25200-056</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco Life Technologies</td><td>10437</td><td></td></tr><tr><td>1 well cell culture slides</td><td>BD Falcon</td><td>354101</td><td></td></tr><tr><td>Blood Chambers</td><td>NA</td><td>NA</td><td>Blood Chamber may be manufactured in PMMA or any other plastic able to bind CHS</td></tr><tr><td>Clamps</td><td>Office Depot</td><td>2052204</td><td></td></tr><tr><td>Corline Heparin Surface (CHS)</td><td>Corline Systems AB</td><td>945-00</td><td></td></tr><tr><td>Hypodermic needle</td><td>Terumo</td><td>NN-1850R</td><td>Size: 18 G x 5 mm</td></tr><tr><td>Unfractionated Heparin (UFH)</td><td>Leo Pharma</td><td>585679</td><td></td></tr><tr><td>K&lt;sub&gt;3&lt;/sub&gt;EDTA</td><td>Alfa Aesar</td><td>1709958</td><td>Prepare a 0.34 M stock solution in milliQ H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PFA</td><td>Sigma-Aldrich</td><td>P-6148</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>80i</td><td></td></tr><tr><td>Light microscope</td><td>Nikon</td><td>TS100</td><td></td></tr><tr><td>Image analysis software</td><td>Broad Institute</td><td>CellProfiler</td><td>Available for free at www.cellprofiler.org</td></tr></tbody></table>

a novel in vitro model for studying the interactions between human whole blood and endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

The Necessity of CHS Coating
In order to measure reactions in whole blood specific to those elicited by the endothelial cells, all materials used for the blood endothelial cell chamber must be furnished with a CHS coating prior to use. Figure 2A (left) shows an uncoated chamber after 30 min of blood contact with a clearly visible clot present. Treating the chamber with CHS (Figure 2A, right) on the other hand protects the blood from uncontrolled activation, ensuring that the results are due to the endothelial cell-blood interactions.
The Effect of the Blood Volume in the Chamber
Activation of coagulation is more likely in stagnant blood as the probability of interaction between activated coagulation factors is increased. In the blood endothelial cell chamber model, blood is kept in motion due to the circulation of an air bubble inside the chamber. In order to determine the effect of changes in the blood volume, and thus also air bubble size, a CHS coated chamber was connected to a CHS coated glass slide that was tested with 1.5 ml or 1.75 ml whole blood for 30 min. The volume of blood without any movement by the air bubble is 2.5 times larger when 1.75 ml of blood is added to the chamber compared to when 1.5 ml of blood is used (Figure 2B). The measured TAT-values suggested increased TAT-formation with increased blood volume (Figure 2D). When HUVEC (Figure 2C) were incubated with either 1.5 or 1.75 ml of whole blood, no difference was noted between the two groups, suggesting that endothelial cells may modulate the activation of coagulation (Figure 2D).
The Effect of TNF&#945; on Blood Cell Recruitment and Activation of Coagulation
In order to study the effect of TNF&#945; on leukocyte recruitment, HUVEC were treated with 20 ng/ml TNF&#945; 4 hr prior to blood contact. Blood contact - with either 1.5 ml or 1.75 ml blood - was continued for 30 min after which the culture slides were stained for CD16 (Figure 2F), imaged and the number of CD16+ cells was quantified. The recruitment of CD16+ cells increased significantly with TNF&#945; treatment (Figure 2E) from 100 to 256 CD16+ cells/mm2 (p &#60; 0.0001) with 1.5 ml blood and from 172 to 378 (P &#60; 0.0001) with 1.75 ml blood. The formation of TAT complexes was measured in the corresponding plasma samples of the blood incubated with either TNF&#945; treated or untreated cells (Figure 2G). TNF&#945; stimulated cells induced an approximate doubling of TAT complexes as compared to untreated cells.
<img alt="Figure 2" src="/files/ftp_upload/52112/52112fig2.jpg" width="600" /> 
Figure 2: Functionality of the blood endothelial cell chamber model. (A) Chambers with or without CHS were incubated with whole blood. Without CHS, a clot was formed after 30 min. Measurement of thrombin-antithrombin (TAT) complexes, an indirect marker of coagulation, in blood incubated without CHS was &#62; 6,000 &#956;g/ml. (B) The volume of blood not kept moving by the rotating air bubble will vary with different blood volumes. With the addition of 1.5 ml of blood, this volume will be 0.3 ml, whereas it will increase to 0.74 ml with the addition of 1.75 ml. (C) HUVEC imaged with phase contrast microscopy show typical endothelial cell morphology of a confluent monolayer prior to blood contact. (D) TAT values for completely CHS coated chambers show a slight difference when different blood volumes are added. The addition of 1.5 ml blood resulted in 35 &#177; 11 &#956;g/ml (n = 7) in comparison to 1.75 ml, which resulted in 85 &#177; 70 &#956;g/ml TAT (n = 8). This difference is no longer present when HUVEC are incubated with the same blood volumes; 66 &#177; 55 &#956;g/ml for 1.5 ml (n = 5) and 66 &#177; 29 &#956;g/ml for 1.75 ml (n = 7). (E) Quantification of the number of CD16+ cells present on either unstimulated or TNF&#945; stimulated HUVECs after blood contact. The number of CD16+ cells is significantly increased with TNF&#945; stimulated cells incubated either with 1.5 ml (basal: 100 &#177; 40 cells/mm; TNF&#945;: 256 &#177; 121 cells/mm) or 1.75 ml (basal: 172 &#177; 67 cells/mm; TNF&#945;: 378 &#177; 185 cells/mm) of whole blood. (F) Representative images of unstimulated and TNF&#945; stimulated HUVEC stained for phalloidin (red), CD16 (green) and DAPI (blue). Scale bar = 250 &#956;m. (G) The formation of TAT complexes is roughly doubled by TNF&#945; stimulated cells as compared to untreated cells incubated with blood (1.5 ml: 2.1 &#177; 0.1 times more TAT, n = 3; 1.75 ml: 1.7 &#177; 1.0 times more TAT, n = 4). All values are presented as mean &#177; standard deviation; p values were calculated by unpaired t tests. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52112/52112fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium at JoVE.com

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

a-novel-vitro-model-for-studying-interactions-between-human-whole

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.4K Views.  Uppsala University. The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

Video: A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Bioengineering

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

Developmental Biology

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Medicine

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

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