We thank Jan Voorberg from the Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, Academic Medical Center, Amsterdam, The Netherlands, for his input in this manuscript. This work was supported by the Dutch CardioVascular Alliance (DCVA) [2012-08, 2014-11] awarded to the Phaedra and the Reconnect consortium as well as the Impulse grant 2018 awarded to the Phaedra IMPACT consortium. These grants include collective funding by the Dutch Heart Foundation, Dutch Federation of University Medical Centers, The Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences. Furthermore, this work was funded by the European Research Council under the Advanced Grant &#8216;VESCEL&#8217; program (Grant number: 669768). XDM is funded by a research grant of the Institute for CardioVascular Research (ICaR-VU) at the VU University Medical Center, Amsterdam, the Netherlands.

<ol>
	<li>Yau, J. W., Teoh, H., Verma, S. <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+control+of+thrombosis.">Endothelial cell control of thrombosis.</a> BMC Cardiovascular Disorders. 15, 130 (2015).</li><li>Pearson, J. D. <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+function+and+thrombosis.">Endothelial cell function and thrombosis.</a> Best Practice &amp; Research: Clinical Haematology. 12 (3), 329-341 (1999).</li><li>Bochenek, M. L., Schafer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Endothelial+Cells+in+Acute+and+Chronic+Thrombosis.">Role of Endothelial Cells in Acute and Chronic Thrombosis.</a> Hamostaseologie. 39 (2), 128-139 (2019).</li><li>Swieringa, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+Control+of+Fibrin+Distribution+and+Microelasticity+in+Thrombus+Formation+Under+Flow.">Platelet Control of Fibrin Distribution and Microelasticity in Thrombus Formation Under Flow.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 36 (4), 692-699 (2016).</li><li>Macfarlane, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Enzyme+Cascade+in+the+Blood+Clotting+Mechanism,+and+Its+Function+as+a+Biochemical+Amplifier.">An Enzyme Cascade in the Blood Clotting Mechanism, and Its Function as a Biochemical Amplifier.</a> Nature. 202, 498-499 (1964).</li><li>Versteeg, H. H., Heemskerk, J. W., Levi, M., Reitsma, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fundamentals+in+hemostasis.">New fundamentals in hemostasis.</a> Physiological Reviews. 93 (1), 327-358 (2013).</li><li>Opneja, A., Kapoor, S., Stavrou, E. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+platelets,+the+coagulation+and+fibrinolytic+systems+to+cutaneous+wound+healing.">Contribution of platelets, the coagulation and fibrinolytic systems to cutaneous wound healing.</a> Thrombosis Research. 179, 56-63 (2019).</li><li>Tabas, I., Garcia-Cardena, G., Owens, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+the+cellular+biology+of+atherosclerosis.">Recent insights into the cellular biology of atherosclerosis.</a> Journal of Cell Biology. 209 (1), 13-22 (2015).</li><li>Karino, T., Motomiya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+through+a+venous+valve+and+its+implication+for+thrombus+formation.">Flow through a venous valve and its implication for thrombus formation.</a> Thrombosis Research. 36 (3), 245-257 (1984).</li><li>ISTH Steering Committee for World Thrombosis Day. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis:+a+major+contributor+to+global+disease+burden.">Thrombosis: a major contributor to global disease burden.</a> Thrombosis Research. 134 (5), 931-938 (2014).</li><li>Witkin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+pulmonary+embolism:+the+role+of+the+pulmonary+embolism+response+team.">Acute and chronic pulmonary embolism: the role of the pulmonary embolism response team.</a> Current Opinion in Cardiology. 32 (6), 672-678 (2017).</li><li>Kim, N. H., 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=Chronic+thromboembolic+pulmonary+hypertension.">Chronic thromboembolic pulmonary hypertension.</a> European Respiratory Journal. 53 (1), 1801915 (2019).</li><li>Zhang, M., Zhang, Y., Pang, W., Zhai, Z., Wang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+biomarkers+in+chronic+thromboembolic+pulmonary+hypertension.">Circulating biomarkers in chronic thromboembolic pulmonary hypertension.</a> Pulmonary Circulation. 9 (2), 2045894019844480 (2019).</li><li>Giri, 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=Interventional+Therapies+for+Acute+Pulmonary+Embolism:+Current+Status+and+Principles+for+the+Development+of+Novel+Evidence:+A+Scientific+Statement+From+the+American+Heart+Association.">Interventional Therapies for Acute Pulmonary Embolism: Current Status and Principles for the Development of Novel Evidence: A Scientific Statement From the American Heart Association.</a> Circulation. 140 (20), 774-801 (2019).</li><li>Sharma, S., Lang, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+the+pathophysiology+of+chronic+thromboembolic+pulmonary+hypertension.">Current understanding of the pathophysiology of chronic thromboembolic pulmonary hypertension.</a> Thrombosis Research. 164, 136-144 (2018).</li><li>Eisenberg, P. R., 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=Fibrinopeptide+A+levels+indicative+of+pulmonary+vascular+thrombosis+in+patients+with+primary+pulmonary+hypertension.">Fibrinopeptide A levels indicative of pulmonary vascular thrombosis in patients with primary pulmonary hypertension.</a> Circulation. 82 (3), 841-847 (1990).</li><li>Bochenek, M. L., 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=From+thrombosis+to+fibrosis+in+chronic+thromboembolic+pulmonary+hypertension.">From thrombosis to fibrosis in chronic thromboembolic pulmonary hypertension.</a> Thrombosis and Haemostasis. 117 (4), 769-783 (2017).</li><li>Nagy, M., Heemskerk, J. W. M., Swieringa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+microfluidics+to+assess+the+platelet-based+control+of+coagulation.">Use of microfluidics to assess the platelet-based control of coagulation.</a> Platelets. 28 (5), 441-448 (2017).</li><li>Barendrecht, A. D., 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=Live-cell+Imaging+of+Platelet+Degranulation+and+Secretion+Under+Flow.">Live-cell Imaging of Platelet Degranulation and Secretion Under Flow.</a> Journal of Visualized Experiments. (125), e55658 (2017).</li><li>Vajen, T., 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=Laminar+Flow-based+Assays+to+Investigate+Leukocyte+Recruitment+on+Cultured+Vascular+Cells+and+Adherent+Platelets.">Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets.</a> Journal of Visualized Experiments. (134), e57009 (2018).</li><li>Michels, A., Swystun, L. L., Mewburn, J., Albanez, S., Lillicrap, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+von+Willebrand+Factor+Pathophysiology+Using+a+Flow+Chamber+Model+of+von+Willebrand+Factor-platelet+String+Formation.">Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation.</a> Journal of Visualized Experiments. (126), e55917 (2017).</li><li>Smeets, M. W. J., Mourik, M. J., Niessen, H. W. M., Hordijk, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stasis+Promotes+Erythrocyte+Adhesion+to+von+Willebrand+Factor.">Stasis Promotes Erythrocyte Adhesion to von Willebrand Factor.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (9), 1618-1627 (2017).</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=Endothelial+cell+heterogeneity.">Endothelial cell heterogeneity.</a> Cold Spring Harbor Perspectives in Medicine. 2 (1), 006429 (2012).</li><li>Aitsebaomo, J., Portbury, A. L., Schisler, J. C., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brothers+and+sisters:+molecular+insights+into+arterial-venous+heterogeneity.">Brothers and sisters: molecular insights into arterial-venous heterogeneity.</a> Circulation Research. 103 (9), 929-939 (2008).</li><li>Szulcek, R., 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=Delayed+Microvascular+Shear+Adaptation+in+Pulmonary+Arterial+Hypertension.+Role+of+Platelet+Endothelial+Cell+Adhesion+Molecule-1+Cleavage.">Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage.</a> American Journal of Respiratory and Critical Care Medicine. 193 (12), 1410-1420 (2016).</li><li>Casa, L. D., Deaton, D. H., Ku, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+high+shear+rate+in+thrombosis.">Role of high shear rate in thrombosis.</a> Journal of Vascular Surgery. 61 (4), 1068-1080 (2015).</li><li>Jain, 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=Assessment+of+whole+blood+thrombosis+in+a+microfluidic+device+lined+by+fixed+human+endothelium.">Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium.</a> Biomedical Microdevices. 18 (4), 73 (2016).</li><li>van Nieuw Amerongen, G. P., Draijer, R., Vermeer, M. A., van Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+prolonged+increase+in+endothelial+permeability+induced+by+histamine+and+thrombin:+role+of+protein+kinases,+calcium,+and+RhoA.">Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: role of protein kinases, calcium, and RhoA.</a> Circulation Research. 83 (11), 1115-1123 (1998).</li><li>Smits, 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=Blood+Outgrowth+and+Proliferation+of+Endothelial+Colony+Forming+Cells+are+Related+to+Markers+of+Disease+Severity+in+Patients+with+Pulmonary+Arterial+Hypertension.">Blood Outgrowth and Proliferation of Endothelial Colony Forming Cells are Related to Markers of Disease Severity in Patients with Pulmonary Arterial Hypertension.</a> International Journal of Molecular Sciences. 19 (12), 3763 (2018).</li><li>Tobias, M. D., Wambold, D., Pilla, M. A., Greer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+serial+hemodilution+with+hydroxyethyl+starch,+albumin,+and+0.9%+saline+on+whole+blood+coagulation.">Differential effects of serial hemodilution with hydroxyethyl starch, albumin, and 0.9% saline on whole blood coagulation.</a> Journal of Clinical Anesthesia. 10 (5), 366-371 (1998).</li><li>Jain, 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=Primary+Human+Lung+Alveolus-on-a-chip+Model+of+Intravascular+Thrombosis+for+Assessment+of+Therapeutics.">Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.</a> Clinical Pharmacology &amp; Therapeutics. 103 (2), 332-340 (2018).</li><li>Veiseh, M., Veiseh, O., Martin, M. C., Asphahani, F., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+peptides+enhance+single+cell+adhesion+and+viability+on+microarrays.">Short peptides enhance single cell adhesion and viability on microarrays.</a> Langmuir-ACS. 23 (8), 4472-4479 (2007).</li><li>Kuo, C. W., Chueh, D. Y., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+size-dependent+cell+adhesion+on+nanostructured+interfaces.">Investigation of size-dependent cell adhesion on nanostructured interfaces.</a> Journal of Nanobiotechnology. 12, 54 (2014).</li><li>McCormack, J. J., Lopes da Silva, M., Ferraro, F., Patella, F., Cutler, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weibel-Palade+bodies+at+a+glance.">Weibel-Palade bodies at a glance.</a> Journal of Cell Science. 130 (21), 3611-3617 (2017).</li><li>Guilarte, M., Sala-Cunill, A., Luengo, O., Labrador-Horrillo, M., Cardona, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mast+Cell,+Contact,+and+Coagulation+System+Connection+in+Anaphylaxis.">The Mast Cell, Contact, and Coagulation System Connection in Anaphylaxis.</a> Frontiers in Immunology. 8, 846 (2017).</li><li>Westein, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerotic+geometries+exacerbate+pathological+thrombus+formation+poststenosis+in+a+von+Willebrand+factor-dependent+manner.">Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner.</a> Proceedings of the National Academy of Sciences. 110 (4), 1357-1362 (2013).</li><li>Huveneers, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vinculin+associates+with+endothelial+VE-cadherin+junctions+to+control+force-dependent+remodeling.">Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling.</a> Journal of Cell Biology. 196 (5), 641-652 (2012).</li><li>Szulcek, R., Bogaard, H. J., van Nieuw Amerongen, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+cell-substrate+impedance+sensing+for+the+quantification+of+endothelial+proliferation,+barrier+function,+and+motility.">Electric cell-substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility.</a> Journal of Visualized Experiments. (85), e51300 (2014).</li><li>Yamasaki, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+arterial+stiffness+and+platelet+activation.">Association between arterial stiffness and platelet activation.</a> Journal of Human Hypertension. 19 (7), 527-533 (2005).</li><li>Tsai, M., 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=In+vitro+modeling+of+the+microvascular+occlusion+and+thrombosis+that+occur+in+hematologic+diseases+using+microfluidic+technology.">In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.</a> Journal of Clinical Investigation. 122 (1), 408-418 (2012).</li><li>Zheng, 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=In+vitro+microvessels+for+the+study+of+angiogenesis+and+thrombosis.">In vitro microvessels for the study of angiogenesis and thrombosis.</a> Proceedings of the National Academy of Sciences. 109 (24), 9342-9347 (2012).</li><li>Myers, D. R., 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=Endothelialized+microfluidics+for+studying+microvascular+interactions+in+hematologic+diseases.">Endothelialized microfluidics for studying microvascular interactions in hematologic diseases.</a> Journal of Visualized Experiments. (64), e3958 (2012).</li></ol>

The authors declare no conflict of interest.

Coagulation is a result of the complex and temporally controlled interplay between the endothelium and blood components. This in vitro assay presents a method to investigate thrombogenic properties of endothelial cells in real-time. Various types of primary human endothelial cells can be used, facilitating in situ thrombosis in an organ and patient-specific&#160;manner. In this study, we illustrated the use of this protocol comparing thrombogenic properties of PAECs isolated from healthy donors versus CTEPH patients. Live thrombus formation was studied by perfusion of whole blood from healthy subjects over&#160;histamine-activated endothelium, while the effect of a DOAC was tested as an antithrombotic agent.
Besides the use of commercially available microchannels, as shown in the representative results, the introduction of the custom-made microfluidic channels enables the study of the influence of vascular geometry changes on thrombus formation. For example, flow decreases at a branch point or stenosis results in an increase in shear stress and more platelet activation36. However, a significant limitation of these custom-made microfluidic channels is the requirement of high cell numbers to form a stable monolayer of ECs as described in step 2.3.2. This may present&#160;a limiting factor when patient-derived cells are scarce. The strengths of the commercially available microslides are that surface areas are modified for cell culture and growth areas are bigger, thereby allowing ECs to form a confluent and stable monolayer. On the other hand, a bigger surface area will result in a bigger lumen. According to Equation 1, this requires higher blood volumes to reach similar flow rates as in the custom-made microfluidics.
An improvement to&#160;this protocol could be to investigate live EC&#160;loss or changes in barrier integrity. EC&#160;damage in this protocol is only measured at the end of the experiment. For live tracking, ECs can be tagged with mCherry VE-cadherin, for example37. However, as this would need a highly optimized protocol with efficient virus transfection, Electric Cell-substrate Impedance Sensing (ECIS) could be used as an alternative to study endothelial integrity and barrier function38. Perfusion over special ECIS flow channels allows longitudinal monitoring of endothelial barrier integrity under flow. These specific ECIS features allow for parallel measurements of endothelial barrier properties and thrombus formation. Alternative ways for parallel EC barrier measurements, especially in the custom-made arrays include the use of fluorescent dextrans in the perfusate, which diffuse out of the lumen, depending on the EC barrier properties.
A limitation of the described protocol is that ECs are removed from the human body and cultured on tissue culture plastic, which is a stiff, artificial substrate. Cells adapt to their biophysical environment. This could possibly affect endothelial response to platelet activation, as there is an association between platelet activation and wall stiffness39. Despite these adaptations to culture plastics, cells do&#160;keep disease-specific characteristics that can be identified in direct comparison with ECs derived from healthy donors as shown with&#160;control, CTEPH-PAEC, and HUVEC that exerted&#160;different patterns of platelet adhesion and fibrin deposition after 5 min of blood perfusion.
In contrast to other protocols, this system uses whole blood while others&#160;study EC interaction with a single blood component such as platelets and leukocytes19,20,21. There have been more advanced microfluidic models developed that allow the study of endothelial function in a vascular model with a round vessel geometry and soft extracellular matrix. However, these are optimized with HUVECs40,41,42. The novelty of the described protocol is the use of primary ECs combined with whole blood&#160;bringing the modelling of in situ thrombosis one step closer to in vivo conditions. Having optimized the protocol for the use of patient-derived ECs and patient-derived blood further optimizes disease modelling in vitro, allowing the assessment of personalized thrombus formation and drug treatment.
The described protocol can be applied to study the effect of anticoagulation therapies on patient-derived cells. While we used dabigatran to inhibit&#160;thrombus formation, it is possible to use other&#160;anticoagulants, such as rivaroxaban, which directly inhibits factor Xa in the coagulation cascade. Direct-platelet inhibitors like clopidogrel or aspirin can be studied as well, as these act on the primary phase of hemostasis where platelets bind to ECs. Ultimately, the thrombogenic capacities of patient-specific ECs in interaction with the patient&#39;s own blood can be used to predict the personal effect of anticoagulation therapy on the individual patient. Furthermore, a knockdown of specific proteins can provide additional functional information.
There are some steps in the described protocol that are critical for a successful perfusion experiment. First, during the isolation of primary cells, it is necessary to obtain a highly pure EC culture. Second, it is important that the ECs form a stable confluent monolayer. If this is not the case, a slight change in shear stress can cause endothelial damage and activation of the coagulation cascade, or platelets can start binding to the basement membrane, which will provide false positive results. Third, it is essential to prevent air bubbles, as those can damage the endothelium and thereby influence the results.
After recalcification of the citrated blood with calcium chloride and magnesium chloride, it is important to immediately start the perfusion experiment. Recalcification induces a rapid response in platelet activation and thrombus formation, resulting in fast clotting in the sample.
In conclusion, we describe a highly versatile protocol to study whole blood-endothelial cell interactions during thrombosis.&#160;

The endothelium forms the inner cellular layer of blood vessels and separates blood from the surrounding tissue. It has been described as a dynamic organ that actively regulates its micro-environment and responds to external stimuli1. Because of its direct contact with flowing blood, the endothelium is pivotal in the control of hemostasis and thrombosis and is the primary source of many of the major regulatory molecules that control platelet aggregation, coagulation, and fibrinolysis2. Healthy, nonactivated endothelial cells (EC) produce several molecules that counteract platelet activation and prevent coagulation and thrombus formation to maintain blood flow, such as prostacyclin, thrombomodulin, or tissue factor pathway inhibitor (TFPI)2,3. This prevents the adhesion of platelets, platelet aggregation, and thrombus formation. Injury or activation of the vessel wall results in a procoagulant endothelial phenotype that initiates localized platelet adhesion and clot formation2,4. Upon endothelial activation platelets&#160;adhere to von Willebrand Factor (VWF), a multimeric protein released from ECs, or to exposed binding sites of the underlying subendothelial matrix. Subsequently, molecular changes in platelets and the exposure to tissue factor (TF) initiate the activation of the coagulation system, which induces thrombus formation by fibrin polymerization5,6. Together, the resulting clot provides the basis for wound closure by re-endothelialization7. Perturbations of the coagulation system may result in bleeding disorders, such as von Willebrand disease, hemophilia, or thrombosis, that often result from&#160;a dysregulated pro- and antithrombotic balance of the endothelial hemostatic pathway2,3.
The process of hemostasis occurs in both arterial and venous circulation. However, the mechanisms underlying arterial and venous thrombosis are fundamentally different. While arterial thrombosis, as seen in ischemic heart disease, is mostly driven by the rupture of an atherosclerotic plaque under conditions of high shear stress, venous thrombosis mostly develops in the absence of endothelial injury in a condition of stasis8,9,10. A deep vein thrombus may embolize and travel towards the pulmonary arteries, where it causes a pulmonary embolism. This can result in chronic vascular obstructions leading to significant impaired functional capacities&#160;that might foster the development of chonic lung diseases&#160;including the development of chronic thromboembolic pulmonary hypertension (CTEPH)11,12,13,14. CTEPH is characterized by elevated pulmonary pressure due to obstructions of the pulmonary arteries by thromboembolic material following at least three&#160;months of anticoagulation therapy15. In addition to lung emboli, it is postulated that the pulmonary endothelium provides a prothrombotic environment in CTEPH that facilitates&#160;in situ thrombosis and chronic obstructions of the pulmonary arteries, causing the increase in blood pressure that ultimately can result in heart failure, if untreated16,17.
Over the past years, various studies have led to the development of assays to examine thrombus formation by measuring platelet function and coagulation18. However, most of them either study the interaction of whole blood with single extracellular matrix components like collagens or fibrins, or endothelial function in interaction with single blood components, such as endothelial-platelet or endothelial-leukocyte interaction19,20,21,22. These assays are most commonly performed with human umbilical vein&#160;endothelial cells (HUVEC), as these cells are easily obtained. However, hemostatic genes are differentially expressed across the vascular tree, vessel types, and organ systems23,24, which makes the use of HUVECs to represent endothelial cells involved in arterial thrombosis or pulmonary embolisms problematic23.
In addition to EC plasticity, disease-specific hemodynamic alterations and changes in vascular morphology can promote thrombus formation at the normal endothelium25. Higher shear rates, due to local vasoconstriction or changes in vessel geometry, for example, may result in acute thrombus formation, causing a stenosis that accelerates the cessation of blood flow26. The use of custom-made microengineered flow channels allows to specifically design vascular geometries that are representative of the (patho)biology. In this way, it is possible to study the effect of local biomechanical forces on healthy or diseased EC27.
There are anticoagulation therapies available for targeting different phases and molecules in the coagulation cascade, which all pose&#160;particular risks and benefits that can be specific to certain disorders. The approach of disease modeling described in this paper is especially suited to test the effects of various anticoagulation and antiplatelet therapies on thrombus dynamics.
The aim is to present a model of thrombosis that includes primary ECs, yielding a versatile model suitable for the analysis of various forms of thrombosis depending on the type of primary ECs&#160;used. As an illustration, we used pulmonary artery endothelial cells from CTEPH patients in interaction with whole human blood containing all components involved in thrombus formation (platelets, leukocytes, erythrocytes, clotting proteins, and cofactors). This approach can be applied in commercial parallel flow channels or in custom-made microfluidic flow channels with a specific vascular design. As such, the model can eventually be used in the study of thrombus formation and resolution, for the assessment of inflammatory responses in disease modeling, for antiplatelet or anticoagulation therapy, and ultimately for personalized medicine.
This study describes the isolation of primary human pulmonary artery endothelial cells. For the isolation of other primary human endothelial cell types, we refer to previously published methods, including pulmonary microvascular endothelial cells25, human umbilical vein endothelial cells28, and blood circulating endothelial colony forming cells Figure 1A29.

<table border="0" cellpadding="0" cellspacing="0" style="width:895px;" width="894">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">20 mL syringe</td>
			<td style="width:200px;">BD Plastipak</td>
			<td style="width:161px;">300613</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">20X objective</td>
			<td style="width:200px;">Olympus</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2-Propanol (IPA)</td>
			<td style="width:200px;">Boom</td>
			<td style="width:161px;">76051455 . 5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Aladdin Syringe Pump</td>
			<td style="width:200px;">Word Precision Instruments</td>
			<td style="width:161px;">AL-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa488-Fibrinogen</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">F13191</td>
			<td>15 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Alexa647 Goat anti Rabbit</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">A21245</td>
			<td align="right">0.180555556</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Biopsy punch 1 mm diameter, Integra Miltex</td>
			<td style="width:200px;">Ted Pella</td>
			<td style="width:161px;">15110-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">CaCl2</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">21115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Calcein AM-Red</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">C3099</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">CD42b-APC</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-100-208</td>
			<td>1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Citric Acid</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Collagen Typ I</td>
			<td style="width:200px;">Corning</td>
			<td>354249</td>
			<td>0,1 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Corning CellBIND Surface 60 mm Culture Dish</td>
			<td style="width:200px;">Corning</td>
			<td style="width:161px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cross-flow hood</td>
			<td style="width:200px;">Basan</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Desiccator</td>
			<td style="width:200px;">Duran</td>
			<td>24 782 69</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">D-Glucose</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">14431-43-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">EDTA</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">15575-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Endothelial Cell Medium (ECM)</td>
			<td style="width:200px;">ScienCell</td>
			<td style="width:161px;">1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Fibronectin</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Flow tubings</td>
			<td style="width:200px;">ibidi</td>
			<td>10831, 10841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Gelatin</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">104070</td>
			<td>0.1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">HEPES</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Hoechst 33342</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:161px;">H1399</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ibidi &#181;-Slide VI 0.4 flow chambers</td>
			<td style="width:200px;">ibidi</td>
			<td style="width:161px;">80606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ImageJ</td>
			<td style="width:200px;">ImageJ</td>
			<td style="width:161px;">v1.49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">KCl</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">7447-40-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Lab oven</td>
			<td style="width:200px;">Quincy</td>
			<td>10GC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">LS720 Fluorescent microscope</td>
			<td style="width:200px;">etaluma</td>
			<td style="width:161px;">LS720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Luer connector</td>
			<td style="width:200px;">ibidi</td>
			<td style="width:161px;">10802, 10825</td>
			<td>Male elbow connectors, and Female tube connectors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">MACS magnetic beads (anti-CD144)</td>
			<td style="width:200px;">Miltenyi Biotec</td>
			<td style="width:161px;">130-097-857</td>
			<td>1:5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">MgCl2</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:161px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microscopy slides, 76 x 26 mm</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:161px;">AAAA000001##12E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Negative photoresist, SU-8</td>
			<td>Microchem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Non-Essential Amino Acids (NEAA)</td>
			<td style="width:200px;">Lonza</td>
			<td style="width:161px;">13-114E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Paraformaldehyde (PFA)</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">818715</td>
			<td>4% PFA in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Penicillin/streptomycin (P/S)</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:161px;">15140-122</td>
			<td>1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:161px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Plasma chamber, CUTE</td>
			<td style="width:200px;">Femto Science</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Polydimethylsiloxane (PDMS), Sylgard 184</td>
			<td style="width:200px;">Dow</td>
			<td>101697</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">sodium citrate blood collection tubes</td>
			<td style="width:200px;">BD Vacutainer</td>
			<td style="width:161px;">363048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">trisodium citrate</td>
			<td style="width:200px;">Merck</td>
			<td style="width:161px;">6448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:200px;">Gibco</td>
			<td style="width:161px;">25300-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">VE-Cadherin (D87F2)-XP</td>
			<td style="width:200px;">Cell Signaling</td>
			<td style="width:161px;">2500</td>
			<td>1:300</td>
		</tr>
	</tbody>
</table>

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.

The representative results can be divided into three parts, each representing the respective steps of experimental setup, data collection, and data analysis. Depending on the research question, parameters for each step can be changed. The presented data are applied to study the influence of the&#160;endothelium on thrombus formation.
Experimental Setup 
It is well-established that endothelial cells are highly heterogeneous in structure and function, depending on location and time, during health and disease23. Various sources of endothelial cells can be used to study endothelial-blood interaction, which in this case were commercially available HUVECs and PAECs&#160;(Figure 1A). HUVECs have been the most commonly used in laboratory models, while PAECs are patient-derived isolated cells from the pulmonary arteries. Furthermore, there are&#160;well-established protocols available to isolate microvascular endothelial cells (MVEC) from the pulmonary circulation or blood circulating endothelial colony forming cells (ECFC)25,28,29.
After isolation, endothelial cells were characterized by VE-cadherin, CD31, and TIE2 staining to confirm an endothelial phenotype. Characterization for the presence of &#945;SMA and cytokeratin indicated the absence of a fibroblast or epithelial-like phenotype (Figure 1B). After obtaining a highly pure population of ECs, passage 3&#8211;5 cells were used to seed either a commercial microslide or custom-made microfluidic flow channels (Figure 1C). While the commercially available microslides are primarily parallel flow chambers, or Y-shaped channels with specifically defined parameters in height or bifurcation angle, the use of microfluidic slides makes it possible to adapt the experimental parameters to in vivo vessel geometry and blood flow dynamics31. However, custom-made microfluidic sizes are smaller&#160;and&#160;tend to limit&#160;cell spread and induce more cell death32,33. Using a surplus of cells compensates for the fact that only a small percentage of cells will adhere. This was observed when a stenosis was introduced, where endothelial cells showed a more elongated phenotype compared to a parallel microfluidic channel (Figure 1D). Commercial flow chambers have a bigger&#160;surface area that cells can bind to. This requires fewer cell numbers for seeding.
To study endothelial cell-blood interaction, whole blood was perfused over an endothelial monolayer. Citrated blood was collected on the day of the experiment and immediately before perfusion recalcified. Cells were stimulated with histamine to induce VWF release and platelet adhesion 30 min prior to perfusion (Figure 1E)34,35. Because of the small dimensions, the custom-made microfluidic channels allowed use of smaller blood volumes.
Data collection 
To investigate thrombus formation on PAECs, Calcein AM-Red fluorescently labeled blood cells and Alexa488-conjugated fibrin were perfused for 5 min at 2.5 dyne/cm2 (Figure 2A&#8211;C). Adherent blood cells and deposited fibrin were quantified. Images were acquired every 30 s and quantified with ImageJ. It was important to subtract the background to eliminate the autofluorescence of nonadhered platelets. The triangle algorithm for thresholding has been used to define minimal background. It allowed for measurement of small platelet aggregates (Figure 2D).
Expectedly, under nonstimulated conditions, there was no binding of platelets and fibrin to the endothelium. To promote VWF release and platelet binding, PAECs were stimulated with histamine, which resulted in an immediate increase of platelet adhesion reaching a plateau after 2.5 min. At this time, platelets started to secrete autocrine factors that induced platelet aggregation and fibrinogen cleavage into fibrin. Fibrin was deposited after 3 min and formed a stable aggregate with platelets after 4 min (Figure 2E).
To investigate whether this effect could be inhibited by a direct oral anticoagulant (DOAC), blood was treated with 10 nM dabigatran. Dabigatran was added to the blood dilution, where it inhibits&#160;Factor IIa in the coagulation pathway, and prevented the cleavage of fibrinogen to form fibrin fibers. When dabigatran-treated blood was perfused over stimulated PAECs, clot formation could be directly inhibited mainly by delaying fibrin deposition (Figure 2F).
Data analysis 
To study the influence of various endothelial sources on thrombus formation, the cellular changes upon 5 min&#160;blood perfusion were analyzed. The endothelium was fixed and adherent platelets were labeled with CD42b before imaging under a confocal microscope. This provided a&#160;detailed analysis for colocalization of platelets and fibrin that could indicate dysfunctional coagulation factors in the blood. The influence of EC&#160;in clot formation was determined by standard immunofluorescent staining. Endothelial cell-cell contacts were maintained, as confirmed by VE-cadherin staining,indicating that the blood clots formed on top of the&#160;endothelial monolayer rather than on the underlying matrix in between endothelial gaps (Figure 3). Furthermore, the use of different cell sources resulted in different patterns of thrombi&#160;forming&#160;on the endothelium. HUVECs are venous endothelial cells and showed limited&#160;platelet adhesion and fibrin deposition, while diseased primary PAEC from CTEPH patients showed abundant&#160;platelet adhesion and more fibrin deposition upon histamine stimulation compared to healthy PAEC. This suggests that the endothelium of CTEPH patients show higher responsiveness to vasoactivation that results in increased thrombus formation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61068/61068fig1.jpg" /> 
Figure 1: Schematic overview of the protocol. (A) Various sources and different types of endothelial cells were isolated and cultured for usage in a microfluidic flow channel for perfusion experiments. Representative brightfield images of different types of endothelial cells. Scale bar = 50 &#181;m. (B) Isolated cells were characterized by immunofluorescent staining to confirm an endothelial phenotype. Scale bar = 50 &#181;m. (C) Cells can be seeded in either commercial flow slides or custom-made microfluidic channel. (D) Representative brightfield images of HUVEC grown in different channel geometries. Scale bar = 50 &#181;m. (E) Experimental setup of blood perfusion experiments. Citrated blood was collected, diluted with saline buffer, and perfused over endothelial cells with a syringe pump. The lung and umbilical vein in this figure were modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic Licence. http://smart.servier.com/ <a href="https://www.jove.com/files/ftp_upload/61068/61068fig1large.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/61068/61068fig2.jpg" /> 
Figure 2: Image acquisition and quantification of thrombus formation. (A) Representative time-lapse imaging of adhered Calcein AM-Red labeled platelets and deposited Alexa488-conjugated fibrin at 1, 3, and 5 min after whole blood perfusion over unstimulated PAECs (B) and over histamine stimulated PAECs. (C) Representative time-lapse images of platelet adhesion and fibrin deposition at 1, 3, and 5 min of perfusion with whole blood incubated with dabigatran perfused over histamine-stimulated PAECs. Scale bar = 50 &#181;m. (D) Schematic overview of image quantification in ImageJ. (E) Quantification of platelet adhesion and fibrin deposition for every 30 s on an unstimulated and histamine stimulated endothelium. (F) Quantification of the effect of dabigatran on thrombus formation quantified by platelet adhesion and fibrin deposition. Data are represented as mean &#177; SD, n = 3. <a href="https://www.jove.com/files/ftp_upload/61068/61068fig2large.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/61068/61068fig3.jpg" /> 
Figure 3: Representative confocal images of flow experiments to characterize thrombus formation in platelet adhesion and fibrin deposition on endothelial cells. Endothelial cell-cell contacts were characterized by VE-cadherin and measured in control PAECs, patient-derived CTEPH PAECs, and HUVEC. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61068/61068fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time at JoVE.com

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

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 ...

in-vitro-microfluidic-disease-model-to-study-whole-blood-endothelial

Research

JoVE Journal

Immunology and Infection

8.6K Views.  VU University Medical Center, Amsterdam Cardiovascular Sciences (ACS). 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.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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 ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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.

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).

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

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

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

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

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

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

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.