Name: in vitro microfluidic disease model to study whole blood-endothelial interactions and blood clot dynamics in real-time
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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><tbody><tr><td>NaCl</td><td>Merck</td><td>106404</td><td></td></tr><tr><td>20 mL syringe</td><td>BD</td><td>300613</td><td></td></tr><tr><td>20X objective</td><td>Olympus</td><td>N1492900</td><td>LUCPLFLN20XPH/0.45</td></tr><tr><td>2-Propanol (IPA)</td><td>Boom</td><td>760514555000</td><td></td></tr><tr><td>Aladdin Syringe Pump</td><td>Word Precision Instruments</td><td>AL-4000</td><td></td></tr><tr><td>Alexa488-Fibrinogen</td><td>Invitrogen</td><td>F13191</td><td>15 ug/mL</td></tr><tr><td>Alexa647-Goat anti Rabbit</td><td>Invitrogen</td><td>A21245</td><td>1:100</td></tr><tr><td>Biopsy punch 1 mm diameter</td><td>Ted Pella</td><td>15110-10</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>21115</td><td></td></tr><tr><td>Calcein AM-Red</td><td>Invitrogen</td><td>C3099</td><td>1:1000</td></tr><tr><td>CD42b-APC</td><td>Miltenyi Biotec</td><td>130-100-208</td><td>1:100</td></tr><tr><td>Citric Acid</td><td>Merck</td><td>100244</td><td></td></tr><tr><td>Collagen Type I, rat tail</td><td>Corning</td><td>354249</td><td>0.1 mg/mL</td></tr><tr><td>Corning CellBIND Surface 60 mm Culture Dish</td><td>Corning</td><td>3295</td><td></td></tr><tr><td>Cross-flow hood</td><td>Basan</td><td></td><td></td></tr><tr><td>Desiccator</td><td>Duran</td><td>2478269</td><td></td></tr><tr><td>D-Glucose</td><td>Merck</td><td>14431-43-7</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>Endothelial Cell Medium (ECM)</td><td>ScienCell</td><td>1001</td><td></td></tr><tr><td>Fibronectin Human Plasma</td><td>Sigma-Aldrich</td><td>F0895</td><td></td></tr><tr><td>Flow tubing</td><td>ibidi</td><td>10831, 10841</td><td></td></tr><tr><td>Gelatin</td><td>Merck</td><td>104070</td><td>0.1%</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H1399</td><td>1:1000</td></tr><tr><td>micron-Slide VI 0.4 flow chambers</td><td>ibidi</td><td>80606</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td>v1.49</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>7447-40-7</td><td></td></tr><tr><td>Lab oven</td><td>Quincy</td><td>10GC</td><td></td></tr><tr><td>LS720 Fluorescent microscope</td><td>Etaluma</td><td>LS720</td><td></td></tr><tr><td>Luer connector</td><td>ibidi</td><td>10802, 10825</td><td>Male elbow connectors, Female tube connectors</td></tr><tr><td>MACS magnetic beads (anti-CD144)</td><td>Miltenyi Biotec</td><td>130-097-857</td><td>1:5</td></tr><tr><td>MgCl2</td><td>Sigma-Aldrich</td><td>M1028</td><td></td></tr><tr><td>Microscopy slides, 76 x 26 mm</td><td>Thermo Scientific</td><td>AAAA000001##12E</td><td></td></tr><tr><td>Negative photoresist, SU-8</td><td>Microchem</td><td></td><td></td></tr><tr><td>Non-Essential Amino Acids (NEAA)</td><td>Lonza</td><td>13-114E</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Merck</td><td>818715</td><td>4% PFA in PBS</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Gibco</td><td>15140-122</td><td>1%</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190-094</td><td>no calcium or magnesium</td></tr><tr><td>Plasma chamber, CUTE</td><td>Femto Science</td><td></td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS), Sylgard 184</td><td>Dow</td><td>101697</td><td></td></tr><tr><td>sodium citrate blood collection tubes</td><td>BD</td><td>363048</td><td></td></tr><tr><td>trisodium citrate</td><td>Merck</td><td>106448</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300-045</td><td></td></tr><tr><td>VE-Cadherin (D87F2)-XP</td><td>Cell Signaling</td><td>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

8.8K Views.  VU University Medical Center, Amsterdam Cardiovascular Sciences (ACS). Dit In Vitro Disease Model, kan worden gebruikt om de interactie van volbloed en patiënt afgeleide cellen, met name de endotheel, te bestuderen om trombogeniciteit en de effectiviteit van antistollingsmiddelsmiddelen te evalueren. Dit is endotheel cellen kunnen worden gebruikt op verschillende omstandigheden en kan worden gewijzigd door de invoering van op maat gemaakte microfluidics of door het aanpassen van de bloedstroom parameters. Bovendien kan dit model worden gebruikt om de trombusdyn...

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

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

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

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

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

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

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

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 ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

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

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

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

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

Medicine

Combining the Chandler Loop and RT-FluFF for Ex Vivo Clot Lysis Monitoring

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

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

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

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

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Biology

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

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

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