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Perspective Article
portion of the circuit. The pump chosen needs to support the desired flow rate (macrovascular: ≤70 mL/min, microvascular: ≤50 μL/min). Syringe pumps deliver con- sistent continuous flow rates. Peristaltic pumps can deliv- er continuous and pulsatile flow patterns. Due to the rotor mechanism of peristaltic pumps, the continuous flow setting may deliver a “dampened” pulsatile flow. Pumps which deliver inconsistent flow rates may lead to bubble formation in the circuit. The flow rate can be adjusted to simulate the desired environment (e.g. arter- ies, capillaries, veins) and shear stress. When calculating the shear rate, it is important to note that blood is a non- Newtonian fluid meaning that its viscosity and the shear stress that it exerts on the endothelium are dependent on the amount of pressure exerted on it. Lastly, the entire cir- cuit can be placed in an incubator or the pump computer can be left outside the incubator (if there are ports avail- able for the passage of cables).
Whole blood, blood product, whole blood-blood prod- uct mixtures and diluted blood can be perfused through the system to simulate blood flow. Undiluted blood best recapitulates the intravascular milieu by maintaining blood concentration and viscosity. However, undiluted anticoagulated whole blood still carries increased risks of thrombosis (particularly in microvascular channels) and is opaque – making it optically difficult to assess blood cell adhesion to endothelium. In these situations, blood can be diluted to maintain circulation and enable in situ micro- scopic analysis. Mixing whole blood with packed RBCs at a ratio of 9:1 simulates a one unit transfusion of packed RBCs. The whole blood should be diluted such that the hematocrit is below the transfusion threshold of 7 g/dL to prevent post-transfusion hyperviscosity. While this dilu- tion simulates the hemodilution observed after crystalloid resuscitation, it is unlikely to approximate the blood vis- cosity observed in patients with chronic anemia. Notably, all blood and blood products circulated through in vitro models are anticoagulated as this treatment is necessary for ex vivo blood storage prior to experimentation. Sodium citrate, ethylenediamine-tetraacetic acid (EDTA) and heparin anticoagulation are non-toxic and suitable for use. Sodium citrate and EDTA work by chelating calcium ions
and their anticoagulant effect can be weakened by adding calcium. Sodium citrate is required for coagulation analy- ses. Furthermore, high levels of sodium citrate can alter the pH of blood, EDTA can activate endothelium and both chelators can interfere with microparticle formation. Heparin provides enduring anticoagulation, but it inter- feres with coagulation analyses and immune cell function. It follows that the requirement for anticoagulated blood represents a shortcoming of in vitro vascular models. This pitfall can be dealt with to some extent by using in vivo models to corroborate in vitro results.
Various outcome measures can be recorded from these in vitro vascular models. Real-time video microscopy can be used to visualize blood cell adhesion to the endotheli- um. The perfusant can be analyzed for soluble factors (e.g. cytokines, microparticles, biological response media- tors) and cell surface markers. After blood circulation, the endothelial cells can be imaged in situ or removed from the scaffold with trypsin for further analysis. In our labo- ratory, a vascular model with a 3 mm, full-length channel seeded with human umbilical vein endothelial cells is pri- marily used to simulate transfusion reactions by circulat- ing mixtures of recipient whole blood and donor blood products (Online Supplementary Material). Whole blood circulation leads to increased formation of annexin V-pos- itive microparticles and erythrocyte microparticles com- pared to statically held whole blood (2.13 x 1010 versus 6.08 x 109 microparticles/mL, P<0.0001 (Online Supplementary Material).
Conclusion
In vitro vascular models combine blood, endothelial and flow components into a single system. In this way, endothelialized flow models simulate the blood product- recipient blood and blood-endothelial interactions that occur under flow conditions after blood product transfu- sion. Five key factors of the experimental set-up (channel geometry, scaffold molding, endothelial cell seeding, cir- cuit construction, and perfusate) can be manipulated depending on the desired organ system, transfusion sce- nario and outcome measures.
References
1 Jy W, Mao WW, Horstman L, Tao J, Ahn YS. Platelet microparticles bind, activate and aggregate neutrophils in vitro. Blood Cells Mol Dis. 1995;21(3):217-231.
2. Straat M, Boing AN, Tuip-De Boer A, Nieuwland R, Juffermans NP. Extracellular vesicles from red blood cell products induce a strong pro-inflammatory host response, dependent on both numbers and storage duration. Transfus Med Hemother. 2016;43(4):302-305.
3. Sadallah S, Eken C, Schifferli JA. Erythrocyte-derived ectosomes have immunosuppressive properties. J Leukoc Biol. 2008;84(5):1316-1325.
4. Karsten E, Herbert B. Red blood cells: the immune system's hidden regulator. Annual scientific meeting of Haematology Society
of Australia and New Zealand, the Australian & New Zealand Society of Blood Transfusion and the Thrombosis and Haemostasis Society of Australia and New Zealand. Sydney, Australia, 2017.
5. Rayner SG, Zheng Y. Engineered microves- sels for the study of human disease. J Biomech Eng. 2016;138(11).
6. Li YS, Haga JH, Chien S. Molecular basis of the effects of shear stress on vascular endothelial cells. J Biomech. 2005;38(10):1949-1971.
7. Granger D, Senchenkova E. Leukocyte– Endothelial Cell Adhesion. Inflammation and the Microcirculation. San Rafael (CA): Morgan & Claypool Life Sciences, 2010.
microengineered model of RBC transfusion- induced pulmonary vascular injury. Sci Rep. 2017;7(1):3413.
10. Hathcock JJ. Flow effects on coagulation and thrombosis. Arterioscler Thromb Vasc Biol. 2006;26(8):1729-1737.
11. Sakariassen KS, Orning L, Turitto VT. The impact of blood shear rate on arterial throm- bus formation. Future Sci OA. 2015;1(4):FSO30.
12. Eckstein EC, Bilsker DL, Waters CM, Kippenhan JS, Tilles AW. Transport of platelets in flowing blood. Ann N Y Acad Sci. 1987;516:442-452.
13. Xu C, Wootton DM. Platelet near-wall excess in porcine whole blood in artery- sized tubes under steady and pulsatile flow conditions. Biorheology. 2004;41(2):113-
8. Hack CE, Zeerleder S. The endothelium in
sepsis: source of and a target for inflamma-
tion. Crit Care Med. 2001;29(7 Suppl):S21- 125.
27.
9. Seo J, Conegliano D, Farrell M, et al. A
14. Dimmeler S, Haendeler J, Nehls M, Zeiher AM. Suppression of apoptosis by nitric
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