Name: assessment of the anticoagulant and anti-inflammatory properties of endothelial cells using 3d cell culture and non-anticoagulated whole blood
Uploaded: 2017-09-05T13:00:00.000+00:00
Duration: 7 min 8 s
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 animal protection law. All animal studies complied with the ARRIVE guidelines. The animal experimentation committee of the cantonal veterinary service (Canton of Bern, Switzerland) approved all animal procedures, permission no. BE70/14. Experimental protocols were refined according to the 3R principles and state-of-the-art anesthesia and pain management were used to minimize the number of animals and reduce the exposure of the animals to stress and pain during the experiments.
Blood was drawn from healthy individuals by closed system venipuncture in accordance with Swiss jurisdiction and ethics guidelines of the Bern University Hospital. The phrase &#34;non-anticoagulated blood&#34; means that the blood has not been treated with any anticoagulant.
The following steps are performed under sterile conditions. Familiarity with basic cell culture sterile technique is required.
1. Isolation of PAEC
NOTE: Thoracic aorta segments of 6 to 10 cm were obtained from euthanized German Landrace pigs of 3 to 6 months of age (used for other in vivo experiments) and immediately transferred into a 500-mL glass bottle containing transport medium (DMEM + 1% penicillin/streptomycin).
<ol>
	<li>Pre-coat a 6-well plate with fibronectin 12.5 &#181;g/mL in PBS 1x and place it in an incubator at 37 &#176;C for 1 h.</li>
	<li>Pre-warm sterile PBS 1x and cell culture medium (DMEM).</li>
	<li>Take out the porcine aorta from transport medium.</li>
	<li>Place the aorta on a polystyrene plate.</li>
	<li>Flush with warm PBS gently beforehand.</li>
	<li>Cut the aorta longitudinally and fix it with needles.</li>
	<li>Add warm cell culture medium on the inner vessel surface.</li>
	<li>Aspirate fibronectin-cell culture medium and add fresh cell culture medium (DMEM supplemented with 10% FBS, 1% penicillin/streptomycin and 0.4% of Endothelial Cell Growth Medium Supplement Mix).</li>
	<li>Soak one cotton swab in the cell culture medium. Swab the cotton wool bud on the very top of the inner vessel surface gently and slowly in the same direction.</li>
	<li>Rub the cells in one well of 6-well plate round by round.</li>
	<li>Do the same for the rest of the wells.</li>
	<li>Check cells under the microscope and place the 6-well plate in incubator at 37 &#176;C, 5% CO2.</li>
	<li>Change the medium on the second day and change it again every 2 - 3 days.</li>
	<li>When cells are going to be confluent, trypsinize cells and seed them into a T75 flask (PAEC P1).</li>
</ol>
2. PAEC Characterization
<ol>
	<li>Pre-coat an 8-well chamberslide with fibronectin 12.5 &#181;g/mL in PBS 1x and place it in an incubator at 37 &#176;C for 1 h.</li>
	<li>Seed 5 x 104 cells/well and incubate overnight in the incubator at 37 &#176;C.</li>
	<li>Wash the cells twice with PBS++(PBS supplemented with CaCl2 and MgCl2), 300 &#956;L/well.</li>
	<li>Fix cells with 3.7% paraformaldehyde for 10 min at room temperature, 200 &#956;L/well.</li>
	<li>Wash cells 3 times with PBS++, 300 &#956;L/well.</li>
	<li>Add 300 &#956;L of PBS 1x-3% BSA (blocking buffer) and leave 30 min at room temperature.</li>
	<li>Apply primary antibodies (anti-VE-cadherin, anti-CD31, anti-vWF) diluted in PBS 1x-1%BSA-0.05% detergent, 160 &#956;L/well and incubate for 1 h at room temperature.</li>
	<li>Wash 3 times with PBS++ (200 &#956;L/well).</li>
	<li>Apply secondary antibodies and DAPI diluted in PBS 1x-1%BSA-0.05% detergent, 160 &#956;L/well, and incubate for 1 h at room temperature.</li>
	<li>Wash 3 times with PBS++ (200 &#956;L/well).</li>
	<li>Mount slides with glycerol based mounting medium and verify endothelial cell markers expression under a fluorescence microscope. 
	NOTE: Culture porcine aortic endothelial cells in a T175 flask (DMEM low glucose medium + 10% FBS, 1% penicillin/streptomycin and 0.4% of Endothelial Cell Growth Medium Supplement Mix) until 90% confluence is reached. (seeding density 1 x 106 cells, 90% confluence correspond roughly to 5 x 106 cells).</li>
</ol>
3. Coating of Microcarrier Beads
<ol>
	<li>Mix 7 mL of microcarrier beads with 42 mL of collagen solution in a 50-mL tube (100 &#956;g/mL, diluted in a 0.2% acetic acid solution) and incubate for 1 h at room temperature.</li>
	<li>Wash beads two times with 25 mL of PBS pH 7.4 (add 25 mL of PBS, mix well with the pipet and wait until the beads are settled down then discard the supernatant and repeat) and one time with 25 mL of DMEM medium.</li>
	<li>Cover the beads in the 50-mL tube with 10mL of medium 199 supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-Glutamine, 0.4% of Endothelial Cell Growth Medium Supplement Mix and 25 &#956;L of heparin (5000 IU/mL) and allow equilibration for 10 min before further use.</li>
</ol>
4. Collecting Cells
<ol>
	<li>Remove the cell culture medium from the T175 flask containing PAEC and add 5 mL of PBS pH 7.4.</li>
	<li>Remove PBS from the T175 flask.</li>
	<li>Add 5 mL of Trypsin-0.05% EDTA and incubate for 3 - 4 min at 37 &#176;C.</li>
	<li>Collect the cells by rinsing the flask with 15 mL of cell culture medium and transfer the suspension in a 50-mL tube.</li>
	<li>Centrifuge cells at 1,200 x g for 8 min at room temperature, remove excess medium and resuspend the pellet in 5 mL of cell culture medium.</li>
</ol>
5. Seeding Cells into the Stirrer Flask
<ol>
	<li>Add 20 mL of cell culture medium to the cell suspension and resuspend.</li>
	<li>Add 20 mL cell culture medium (w/o cells) into the 500-mL magnetic stirrer flask.</li>
	<li>Add the cells to the washed microcarrier beads from step 3.3 and mix carefully with a 25-mL serological pipette.</li>
	<li>Transfer the beads/cell mixture into the magnetic spinner flask.</li>
	<li>Rinse the 50-mL tube with 10 mL of cell culture medium to collect the remaining cells.</li>
	<li>Add an additional 85 mL of cell culture medium into the spinner flask and place it into the incubator overnight at 37 &#176;C on a shaker (100 x g, mixing interval: 3 min every 45 min).</li>
	<li>Add 50 mL of cell culture medium (total volume 200 mL) and continue stirring for additional 24 h at 37 &#176;C on a shaker (100 x g, mixing interval: 3 min every 45 min).</li>
	<li>Add colorless RPMI medium (supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-Glutamine, 0.4% of Endothelial Cell Growth Medium Supplement Mix and 25 &#956;L of Heparin) until 320 mL of total volume is reached.</li>
	<li>Replace the medium every 48 h: Remove 100 mL of old medium and add 100 mL of fresh supplemented colorless RPMI.</li>
	<li>Culture the cells for 5 to 7 days. The time depends on the confluence state of the cell-coated beads.</li>
</ol>
6. Confluence Verification
<ol>
	<li>Collect 200 &#956;L of cell-coated beads using a pipette and transfer them into a polypropylene tube.</li>
	<li>Wash the beads 3 times with 600 &#956;L of PBS 1x (add PBS, tilt the tube and mix gently to avoid detachment of the cells, wait the beads to settle down, discard the PBS and repeat).</li>
	<li>Fix the beads for 10 min by adding 200 &#956;L of parapicric acid.</li>
	<li>Wash 3 times with 600 &#956;L of PBS 1x.</li>
	<li>Add DAPI diluted in PBS 1x and incubate for 10 min.</li>
	<li>Transfer the beads on a glass slide and apply a coverslip using glycerol based mounting medium.</li>
	<li>Visualize the beads under a confocal microscope.</li>
</ol>
7. Experimental Procedure
<ol>
	<li>Remove the cell-coated beads from the magnetic stirrer flask (procedures do not have to be done under sterile conditions) with a 10-mL serological pipette and transfer them into 12 mL round-bottom polypropylene tubes.</li>
	<li>Let the beads settle down (around 1 - 2 min) and remove excess medium.</li>
	<li>Add more beads to the tubes until every tube contains exactly 2 mL of beads.</li>
	<li>Add 5-mL clear RPMI to each tube and mix carefully using a 10-mL serological pipette.</li>
	<li>Let the beads settle down and remove excess medium.</li>
	<li>Repeat the washing procedure one more time with RPMI and remove all excess medium.</li>
</ol>
8. Incubation with Non-anticoagulated Blood
<ol>
	<li>Carefully and slowly (using neither jet nor vacutainers) draw blood from a healthy volunteer and collect it in 9 mL neutral polypropylene tubes (no anticoagulant).</li>
	<li>Slowly transfer 8 mL of blood with a 10-mL serological pipette into each of the polypropylene tubes containing 2 mL of cell-coated beads (the total volume will be 10 mL). Always avoid rough handling of blood or beads to avoid premature EC activation. The procedure takes 1 - 2 min.</li>
	<li>Carefully tilt the blood/bead mixture to ensure equal mixing and seal the cap with paraffin film.</li>
	<li>Place the tubes on a horizontal tilting table (with gentle tilting settings only) inside a 37 &#176;C incubator and record clotting times.
	<ol>
		<li>At set time intervals, e.g. after 10, 20, 30, 50, 70, 90 min, remove at least 1.5 - 2 mL of blood-bead mixture for serum or plasma analysis. 
		NOTE: for 6 time points we suggest having more than 3 replicates within one group of cells, as the blood sampling will be done in different tubes.</li>
		<li>For collection of serum, leave the blood to coagulate. To collect the plasma, add EDTA or citrate to 2 mL tubes before adding blood samples.</li>
		<li>Store the tubes on ice, centrifuge at 2,500 x g for 10 min at 4 &#176;C and store serum/plasma at 80 &#176;C until use. 
		NOTE: Details on materials are provided in Table of Materials</li>
	</ol>
	</li>
</ol>

<ol>
	<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 authors have nothing to disclose.

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 before starting the experiment and those pertaining to the careful collection and handling of the non-anticoagulated whole blood to avoid premature platelet activation due to high shear stress, which may occur if vacutainers are used to collect the blood.10 Nevertheless, this system has some limitations, including the absence of a recirculating closed system and the absence of physiological shear stress which would better represent the in vivo conditions.
Despite these limitations, the described model offers significant advantages over currently existing models. Due to the use of microcarrier beads, the EC surface to blood volume ratio is increased, allowing the establishment of the anti-coagulant and anti-inflammatory environment and use of non-anticoagulated whole blood. In current flat-bed cell culture models, this is not possible and mechanisms which involve blood clotting due to EC activation therefore often require the use of animal experimentation. In part, this can be avoided using the described microcarrier bead model.
Furthermore, this system allows for a broad spectrum of applications. Its versatility resides in the possibility of testing different drugs or compounds which are not only related to (xeno-) transplantation but also to human diseases. The effects of the drugs on the endothelium and on the coagulation system can be easily assessed by immunofluorescence, ELISA and multiplex suspension array analysis. This was previously done in a study on the effect of transgenic expression of human thrombomodulin in a xenotransplantation setting.11
A possible modification of the described method could be the collection of non-anticoagulated whole blood directly into tubes which are filled previously with cell-coated beads in order to reduce air-contact and blood activation by using the pipette. The use of human aortic endothelial cells (HAEC) may be an interesting control and is already incorporated into future plans. Argon topping of the tubes might be used to reduce the contact of non-anticoagulated blood with air, which is known to lead to contact activation of the clotting cascade. If the blood is drawn too quickly, for example by using vacuumed tubes with rubber stopcocks and introducing blood into the tube in a fine jet, then platelets become activated and coagulation will occur sooner. To avoid platelet activation, use large diameter hypodermic needles (21&#610; - 16&#610;).

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&amp;amp;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 &amp;ndash; 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 &amp;micro;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>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>2 mL in 500 mL 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>CaCl&lt;sub&gt;2&lt;/sub&gt; x 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>2382.0500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;x6 H&lt;sub&gt;2&lt;/sub&gt;O</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) (Figure 3).
Another important point which needs attention is the speed of the tilting plate. A high tilting speed will enhance blood clotting. A prolongation of the clotting time could be observed if a monolayer of EC was present on the surface of the microcarrier beads. The use of GalTKO/hCD46/hTBM transgenic PAEC showed a significant increase in the clotting time compared to WT PAEC (Figure 3). The absence of the Gal-&#945;-1,3-Gal xenoantigen on the PAEC (GalTKO) showed an increase in clotting time (25 &#177; 8 min for PAEC WT and 68 &#177; 30 min for PAEC GalTKO). Another strong increase in clotting time was observed when PAEC GalTKO/hCD46/hTBM were present on microcarrier beads (205 &#177; 32 min), which suggests a successful modulation of both the complement (hCD46) and coagulation systems (hTBM). The end of the experiment is defined when a visible clot is formed.The variability within samples of the same of group is due both to inter assay-variability and the blood donor. Every experiment had 3 replicates and each time a different blood donor was used to increase the reliability of the data. For each donor, a blood analysis (platelet count, WBC, RBC, HCT and other parameters) was performed by external healthcare laboratories. The results shown in Figure 3 are obtained from different experiments performed with different blood donors.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56227/56227fig1.jpg" /> 
Figure 1: Schematic Representation of the Microcarrier-based Assay. (A) EC are expanded in T175 flasks and (B) transferred into spinner flasks together with collagen-coated microcarrier beads. (C) Fresh non-anticoagulated blood is collected from a healthy volunteer, (D) mixed with EC-coated microcarrier beads, and incubated at 37 &#176;C. The phrase &#34;non-anticoagulated blood&#34; means that the blood has not been treated with any anticoagulant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56227/56227fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56227/56227fig2.jpg" /> 
Figure 2: Immunofluorescence Staining on EC-coated Microcarrier Beads. Microcarrier beads were retrieved and stained for CD31 (PECAM-1) to assess the confluency. The nuclei were stained in blue (DAPI) and CD31 was stained in red (Cy3). Scale bar: 100 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56227/56227fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56227/56227fig3.jpg" /> 
Figure 3: Clotting Times of Different EC. Clotting times were determined visually and the end of the experiment was defined when a visible clot was observed. A strong procoagulant effect was observed when non-EC-coated microcarrier beads were exposed to whole non-anticoagulated human blood. The absence of the Gal-&#945;-1,3-Gal xenoantigen on the PAEC (GalTKO) showed an increase in clotting time (25&#160;&#177; 8 min for PAEC WT and 68 &#177; 30 min for PAEC GalTKO). A further increase in clotting time was observed when PAEC GalTKO/hCD46/hTBM were present on microcarrier beads, which suggests a successful modulation of both the complement (hCD46) and coagulation systems (hTBM). Data are shown as mean&#160;&#177; standard deviation. Statistical analysis has been done using ANOVA for multiple comparisons with Bonferroni correction. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56227/56227fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

assessment-anticoagulant-anti-inflammatory-properties-endothelial

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K Views.  University of Bern. We presenteren een in vitro model waarmee de studie en analyse van coagulatie in hele, niet-vertonen bloed. Antistolling in het systeem is afhankelijk van de natuurlijke anticoagulation effect van gezonde endotheliale cellen en endothelial cel activatie zal resulteren in de bloedstolling.

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

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

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Developmental Biology

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Immunology and Infection

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

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 ...

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 ...

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

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