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 border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:91px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:272px;">Comments/Description</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Human Umbilican Vein Endothelial Cells (HUVEC)</td>
			<td style="width:91px;">PromoCell</td>
			<td style="width:119px;">C-12200</td>
			<td style="width:272px;">Any other type of primary human endothelial cell may be used.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Endothelial Cell Growth Medium MV (ECGM MV)</td>
			<td style="width:91px;">PromoCell</td>
			<td style="width:119px;">C-22120</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Gelatin</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:119px;">G-2500</td>
			<td style="width:272px;">Prepare 1% gelatin in PBS, incubate 5 ml in a T75 for 15 min and remove before culturing cells.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">TAT ELISA</td>
			<td style="width:91px;">Enzyme Research Lab.</td>
			<td style="width:119px;">TAT-EIA-C</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse anti-human CD16</td>
			<td style="width:91px;">DAKO</td>
			<td style="width:119px;">F7011</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse Alexa 488</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">A11001</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Phalloidin-Texas Red</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">A22287</td>
			<td>Dilution 1:200</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">DAPI</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">D1306</td>
			<td>Dilution 10 &#956;g/ml</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">EDTA</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">E-6758</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:91px;">Gibco Life Tecnologies</td>
			<td style="width:119px;">25200-056</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:91px;">Gibco Life Technologies</td>
			<td style="width:119px;">10437</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1 well cell culture slides</td>
			<td style="width:91px;">BD Falcon</td>
			<td style="width:119px;">354101</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Blood Chambers</td>
			<td style="width:91px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:272px;">Blood Chamber may be manufactured in PMMA or any other plastic able to bind CHS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Clamps</td>
			<td style="width:91px;">Office Depot</td>
			<td style="width:119px;">2052204</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Corline Heparin Surface (CHS)</td>
			<td style="width:91px;">Corline Systems AB</td>
			<td style="width:119px;">945-00</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypodermic needle</td>
			<td style="width:91px;">Terumo</td>
			<td style="width:119px;">NN-1850R</td>
			<td style="width:272px;">Size: 18 G x 5 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Unfractionated Heparin (UFH)</td>
			<td style="width:91px;">Leo Pharma</td>
			<td style="width:119px;">585679</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">K3EDTA</td>
			<td style="width:91px;">Alfa Aesar</td>
			<td style="width:119px;">1709958</td>
			<td style="width:272px;">Prepare a 0.34 M stock solution in milliQ H20</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">PFA</td>
			<td style="width:91px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-6148</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescence microscope</td>
			<td style="width:91px;">Nikon</td>
			<td style="width:119px;">80i</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Light microscope</td>
			<td style="width:91px;">Nikon</td>
			<td style="width:119px;">TS100</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Image analysis software</td>
			<td style="width:91px;">Broad Institute</td>
			<td style="width:119px;">CellProfiler</td>
			<td style="width:272px;">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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52112/52112fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.3K 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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

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

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Chapters in this video

Introducing Shear Stress in the Study of Bacterial Adhesion

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes