Name: assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood
Uploaded: 2017-09-05T13:00:00
Description: Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

This study was supported by the Swiss National Science Foundation (SNSF, Grant No. 320030_156193). The authors thank Dr. Beno&#238;t Werlen for providing the Biosilon microcarrier beads. We also thank Prof. Hans Peter Kohler and Prof. Andr&#233; Haeberli for help with setting up the microcarrier bead model.

German Landrace pigs (wild type bred in a local farmhouse and transgenic bred at the Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany), weighing between 30 kg to 40 kg, were used in this study. All animals were housed under standard conditions with water and food ad lib. All animal experiments were performed in accordance with the U.K. Animals Act (scientific procedures) and the NIH Guide for the Care and Use of Laboratory Animals, as well as the Swiss anima...

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	<li>Rajendran, P., 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=The+vascular+endothelium+and+human+diseases.">The vascular endothelium and human diseases.</a> Int. J. Biol. Sci. 9, 1057-1069 (2013).</li><li>Rajwal, S., Richards, M., O'Meara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+recalcified+citrated+whole+blood+-+a+pragmatic+approach+for+thromboelastography+in+children.">The use of recalcified citrated whole blood - a pragmatic approach for thromboelastography in children.</a> Pediatric Anesthesia. 14, 656-660 (2004).</li><li>Kohler, H. P., Muller, M., Mombeli, M., Mtraub, M. M., Maeberli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Suppression+of+the+Coagulation+of+Nonanticoagulated+Whole-Blood+in-Vitro+by+Human+Umbilical+Endothelial-Cells+Cultivated+on+Microcarriers+Is+Not+Dependent+on+Protein-C+Activation.">The Suppression of the Coagulation of Nonanticoagulated Whole-Blood in-Vitro by Human Umbilical Endothelial-Cells Cultivated on Microcarriers Is Not Dependent on Protein-C Activation.</a> Thromb. Haemost. 73, 719-724 (1995).</li><li>Biedermann, B., Rosenmund, A., Muller, M., Kohler, H. P., Haeberli, A., Straub, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cells+suppress+prothrombin+activation+in+nonanticoagulated+whole+blood+in+vitro.">Human endothelial cells suppress prothrombin activation in nonanticoagulated whole blood in vitro.</a> J. Lab. Clin. Med. 124, 339-347 (1994).</li><li>Banz, Y., Cung, T., Korchagina, E. Y., Bovin, N. V., Haeberli, A., Rieben, 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+cell+protection+and+complement+inhibition+in+xenotransplantation:+a+novel+in+vitro+model+using+whole+blood.">Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood.</a> Xenotransplantation. 12, 434-443 (2005).</li><li>Cowan, P. J., d'Apice, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+activation+and+coagulation+in+xenotransplantation.">Complement activation and coagulation in xenotransplantation.</a> Immunology and Cell Biology. 87, 203-208 (2009).</li><li>Reitsma, S., Slaaf, D. W., Vink, H., van Zandvoort, M. A. M. J., oude Egbrink, M. G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx:+composition,+functions,+and+visualization.">The endothelial glycocalyx: composition, functions, and visualization.</a> Pflugers Arch. 454, 345-359 (2007).</li><li>Wiegner, R., Chakraborty, S., Huber-Lang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement-coagulation+crosstalk+on+cellular+and+artificial+surfaces.">Complement-coagulation crosstalk on cellular and artificial surfaces.</a> Immunobiology. 221, 1073-1079 (2016).</li><li>Bongoni, A. K., 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=Transgenic+Expression+of+Human+CD46+on+Porcine+Endothelium:+Effect+on+Coagulation+and+Fibrinolytic+Cascades+During+Ex+Vivo+Human-to-Pig+Limb+Xenoperfusions.">Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions.</a> Transplantation. 99, 2061-2069 (2015).</li><li>Miyazaki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+shear+stress+can+initiate+both+platelet+aggregation+and+shedding+of+procoagulant+containing+microparticles.">High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles.</a> Blood. 88, 3456-3464 (1996).</li><li>Wuensch, A., 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=Regulatory+Sequences+of+the+Porcine+THBD+Gene+Facilitate+Endothelial-Specific+Expression+of+Bioactive+Human+Thrombomodulin+in+Single-and+Multitransgenic+Pigs.">Regulatory Sequences of the Porcine THBD Gene Facilitate Endothelial-Specific Expression of Bioactive Human Thrombomodulin in Single-and Multitransgenic Pigs.</a> Transplantation. 97, 138-147 (2014).</li></ol>

The model presented here is suitable for coagulation related studies allowing the analysis of different aspects of coagulation and its interaction with EC.8 In xenotransplantation research, it is a useful system to test the anticoagulant properties of different genetically modified porcine ECs after incubation with human blood 9.
The most critical steps of the protocol are those ensuring complete cell coverage (confluency) of the microbeads befor...

The vascular endothelium consists of a monolayer of endothelial cells (EC) which line the lumen of blood vessels. In a physiological state, quiescent EC are responsible for the maintenance of an anticoagulant and anti-inflammatory environment.1 This is mediated by the expression of anticoagulant and anti-inflammatory proteins on the EC surface. For example, EC activation caused by ischemia/reperfusion injury or vascular rejection of (xeno-)transplanted organs results in a change of the endothelial surface from an anticoagulant and anti-inflammatory state to a pro-coagulant and pro-inflammatory state.1
To study the fascinating and complex interaction between the endothelium and coagulation factors, in vitro models which mimic as closely as possible the in vivo situation are highly desirable. A common limitation which characterizes conventional in vitro coagulation assays is the use of anticoagulated blood which makes the analysis of coagulation-mediated effects arduous and even recalcification of citrated whole blood cannot reproduce results obtainable with fresh non-anticoagulated blood.2 Besides, in traditional flat-bed cell culture systems it is impossible to exploit the anticoagulant properties of the endothelium as a sufficient endothelial cell surface per blood volume cannot be reached. The model presented here overcomes these limitations by culturing EC on the surface of spherical microcarrier beads, so that an EC surface-to-blood ratio of &#62;16 cm2/mL can be reached, which is similar to the situation in small arterioles or veins, and which was described to be sufficient to allow "natural" anticoagulation of the blood by the EC surface.3,4 Whole blood can be used without added anticoagulants in this setting. Blood samples can be collected during the experiment and cytokines, coagulation factors and soluble complement activation markers can be detected and quantified. Furthermore, EC-coated microcarrier beads may be analyzed for complement and immunoglobulin deposition as well as the expression of EC activation markers by confocal microscopy. Another interesting application includes the testing of drugs which are supposed to prevent endothelial cell activation and, thus, coagulation.5 Although this model cannot completely replace animal experimentation, it offers a method to test specific functional hypotheses ex vivo using cells and thus reduce the number of animals used in basic research on ischemia/reperfusion injury or (xeno)transplantation.
The described model was used to mimic a xenotransplantation setting in which porcine aortic endothelial cells (PAEC) are grown on the microcarrier beads and incubated with whole, non-anticoagulated human blood. Different transgenic PAEC, carrying several human genes such as CD46 for the regulation of the complement system and/or thrombomodulin (hTBM) for the regulation of the coagulation system, were analyzed for their anticoagulant properties. Endothelial cell activation, complement, and coagulation systems are tightly controlled and interconnected.6 It is therefore important to understand how the different transgenic cells behave after exposure to human blood with regard to adhesion molecule expression and cytokine release, shedding of the glycocalyx and loss of anticoagulant proteins.7

<table>
	<tbody>
		<tr>
			<td>PAEC</td>
			<td></td>
			<td></td>
			<td>Isolated from pig aorta in our Lab</td>
		</tr>
		<tr>
			<td>Microcarrier beads (Biosilon)</td>
			<td>Thermo Fisher Scientific</td>
			<td>160-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, Bovine</td>
			<td>Gibco</td>
			<td>A10644-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma</td>
			<td>M7528</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>32404-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral tubes</td>
			<td>Sarstedt</td>
			<td>02.1726.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>Simport</td>
			<td>T406-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirrer flasks</td>
			<td>Tecnomara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological stirrer</td>
			<td>Brouwer</td>
			<td>MCS-104S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti CD31</td>
			<td>R&#38;D Systems</td>
			<td>MAB33871</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG-Cy3</td>
			<td>Jackson Immuno Research</td>
			<td>112-166-003</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Goat anti VE-cadherin</td>
			<td>Santa Cruz</td>
			<td>sc-6458</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti vWF</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>Dilution 1:100</td>
		</tr>
		<tr>
			<td>Dk anti Gt IgG &#8211; Alexa 488</td>
			<td>Molecular Probes</td>
			<td>A11055</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Goat anti Rabbit - FITC</td>
			<td>Southern Biotech</td>
			<td>4050-02</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>32670-25MG-F</td>
			<td>Dilution 1 &#181;g/mL</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Concentration: 10% in cell culture medium</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton Dickinson</td>
			<td>367286</td>
			<td>Size: 0.8x19 mm (21&#610; 3/4&#34;)</td>
		</tr>
		<tr>
			<td>Adapter</td>
			<td>Sarstedt</td>
			<td>14.1205</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 710</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Image J</td>
			<td></td>
			<td>Available for free at: https://imagej.net</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium SupplementMix</td>
			<td>PromoCell</td>
			<td>C-39216</td>
			<td>2mL in 500mL of DMEM</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Concentration: 1% in cell culture medium</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td>Concentration: 1% in cell culture medium</td>
		</tr>
		<tr>
			<td>Heparinun natricum</td>
			<td>Drossa Pharm AG</td>
			<td></td>
			<td>Stock concentration: 5000 U.I./ mL</td>
		</tr>
		<tr>
			<td>Chamberslides</td>
			<td>Bioswisstec AG</td>
			<td>30108</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 x 2H2O</td>
			<td>Merck</td>
			<td>2382.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x6 H2O</td>
			<td>Merck</td>
			<td>1.5833.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Axon Lab AG</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycergel mounting medium</td>
			<td>Dako</td>
			<td>C0563</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After 7 - 10 days of culture in the spinner flask (Figure 1) the cells were confluent, covering the whole surface of the microcarrier beads (Figure 2). Verification of the confluency state is an important step because a non-confluent monolayer of EC on the microbeads will lead to a marked decrease in the clotting time, given the microcarrier beads&#39; surface is strongly pro-coagulant (clotting time: 4&#160;&#177; 1 min) (<stron...

Watch this Scientific Journal Video about Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood at JoVE.com

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

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 ...

The overall goal of this assay is to assess the anticoagulant properties of endothelial cells, using whole non-anticoagulated blood. This method can be used to answer research questions which are related to cardiovascular research, transportation and surgery related research. Essentially, can be used for all questions which have to do with anti-immunity and the activation of endothelial cells.The main advantage of this technique is that whole blood, without anticoagulants can be used as anticoagulation is guaranteed by endothelial cells. To begin the protocol, mix seven milliliters of microcarrier beads with 42 milliliters of coagulant solution in a 50 milliliter tube and incubate the beads for one hour at room temperature. After incubation, wash the beads with 25 milliliters of phosphate buffered saline or PBS and rinse the beads by pipetting up and down.Wait until the beads settle at the bottom of the tube, before discarding the supernatant and repeating. Next, wash the beads one time with 25 milliliters of Dulbecco's Modified Eagle's Medium, DMEM. Then, cover the beads with 10 milliliters of medium 199 and allow them to equilibrate for 10 minutes before further use.Remove the cell culture media from the T175 flask containing PAEC and add five milliliters of PBS. Then, remove PBS from the T175 flask and replace it with five milliliters of trypsin with 0.05%EDTA. Incubate the cells for three to four minutes at 37 degree celsius.Collect the cells by rinsing the flask with 15 milliliters of cell culture media and transfer the suspension to a 50 milliliter tube. Centrifuge the cells, after centrifugation, remove excess media and re-suspend the pellet in five milliliters of cell culture media. Add 20 milliliters of cell culture media to the cell suspension and re-suspend the cells.Add 20 milliliters of cell culture media without cells into a 500 milliliter magnetic stirrer flask. Add the cells to the washed micro carrier beads and mix the solution carefully with a 25 milliliter serological pipette. Transfer the bead cell mixture into the magnetic spinner flask.Rinse the tube with 10 milliliters of cell culture media to collect the remaining cells. Add an additional 85 milliliters of cell culture media into the spinner flask. Place the flask into the incubator overnight, at 37 degree celsius on a shaker.Add 50 milliliters of cell culture media for a total volume of 200 milliliters. Continue stirring for an additional 24 hours on a shaker. Add colorless rockwell park memorial institute or RPMI media, until 320 milliliters of the total volume is reached.Remove 100 milliliters of the old media and add 100 milliliters of fresh supplemented colorless RPMI, every 48 hours. Culture the cells for five to seven days depending on the confluent state of the cell coated beads. Using a 10 milliliter serological pipette, remove the cell coated beads from the magnetic stirrer flask and transfer them into 12 milliliter round bottom polypropylene tubes.After one to two minutes, when the beads have settled down, remove the excess media. Add more beads to the tubes until each tube contains exactly two millimeters of beads. Next, add five milliliters of clear RPMI to each tube and mix the solution carefully with a 10 milliliter serological pipette.Let the beads settle down and remove excess media. Repeat the washing procedure one more time with RPMI and remove all excess medium. Carefully and slowly draw blood from a healthy volunteer and collect it in a nine milliliter neutral polypropylene tube, with no anticoagulant.Slowly transfer eight milliliters of blood into each of the polypropylene tubes containing two milliliters of cell coated beads, to make a total volume of 10 milliliters. Be sure to carefully handle the blood and the beads to avoid premature EC activation. Carefully tilt the blood bead mixture to ensure equal mixing and seal the cap with paraffin film.Place the tubes on a horizontal tilting table with gentle settings, inside a 37 degree celsius incubator and record the clotting times. At set time intervals, remove at least 1.5 to two milliliters of the blood bead mixture for serum or plasma analysis. For serum collection, leave the blood to coagulate.Finally, centrifuge the tubes at 2, 500 G for 10 minutes at four degree celsius. Store the serum or plasma at 80 degree celsius until use. Microcarrier beads stained for CD31 and nuclei clearly show confluency.Visual observation determined that prolongation of the clotting time could be observed if a monolayer of EC was present on the surface of the microcarrier beads. A significant increase in clotting time was observed when porcine aortic endothelial cells, PAEC GalTKO/hCD46/hTBM were present on microcarrier beads, suggesting a successful modulation of both the complement and coagulation systems. Once mastered, the incubation of endothelial cell coated beads with non-anticoagulated human blood can be done in about six hours.The actual duration of the experiment depends mainly on the clotting time, which is seen in the experiment. While attempting this procedure, it's important to remember to ensure complete coverage of the micro beads with endothelial cells and prevent pre-mature activation of human blood.

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