A vascular microphysiological system with a defined injury and coagulation site

Introduction:: The expanding use of vascular microdevices demands engineering improvements to complement our

understanding of vascular biology. Our understanding of vascular physiology explains normal coagulation as a

response to vascular injury or damage. Disruption of vascular endothelium reveals collagen that provides

platelet binding and coagulation factor activation sites which can initiate the coagulation cascade. Vascular

damage also promote tissue factor expression, leading to extrinsic coagulation. In otherwise healthy, undamaged

blood vessels, coagulation is limited by the anticoagulant behavior of endothelial cells stimulated by fluid shear.

However, in many vascularized microfluidics, it is common to elicit coagulation during perfusion of blood

plasma. Herein, we present a vascular microfluidic for the regulation of injury-specific coagulation. Our results

confirm the importance of fluid shear in mimicking the anticoagulant behavior of blood vessel endothelium in

vitro. The presented device also represents a novel tool for testing therapeutics that augment hemostasis.


Materials and Methods:: Microfluidic molds were 3D printed with a Boston Micro Fabrication (BMF) microArch S240 and coated with

3µm of parylene C to improve demolding of poly(dimethylsiloxane) (PDMS). Soft PDMS (25:1 elastomer-to-

crosslinker) was cured on molds for 20min prior to adding standard 10:1 PDMS and curing for at least 2hr at

80ºC. PDMS microchannels were bonded to microscope slides after plasma treatment. Soft PDMS facilitated

the pressurization and lateral deformation of microchannel walls adjacent to a 5mm x 200µm x100µm (l x w x

h) endothelialized microchannel. Prior to injury, cells were perfused with cell culture media for 18hr at either 0

or 5dyne/cm 2 . Lateral PDMS actuators were pressurized for 30sec to compress and “injure” an 800µm length of

endothelium. Injured endothelial cells were identified by annexinV-TRITC staining for phosphatidylserine, a

phospholipid revealed during cell death and a stimulant of coagulation. After injury, endothelial microchannels

were perfused with blood plasma spike with fluorescent fibrinogen and the accumulation of fibrin(ogen)

(coagulation) was monitored via confocal microscopy. Change in fluorescent fibrin(ogen) intensity and area

were monitored over 30min. Relative fibrin(ogen) fluorescence was measured upstream of, at, and downstream

of injury sites. Additional tests compared the coagulation of adult and umbilical cord-derived plasmas, as well

as plasma supplemented with synthetic platelet-like particles (PLPs).


Results, Conclusions, and Discussions:: The fluorescent intensity and area of fibrin(ogen) were found to increase over time, with significantly greater

coagulation in “injured” devices, and greater coagulation of adult plasma compared to umbilical cord plasma.

Pretreating cells with fluid shear also enhanced the injury site-specificity for coagulation. Fluorescent plot

profiles reveal a greater degree of overlap between fibrin(ogen) with phosphatidylserine in vascular channels

pre-treated with shear.

Shear treatment improved the colocalization of coagulation and injury site, confirming that fluid shear promotes

anticoagulant behavior of blood vessel endothelial cells, and endothelial damage promotes coagulation. For a

given length of an endothelial microfluidic, this means we can achieve greater injury site-specificity for

coagulation. For those who use vascular microfluidics, or are interested in connecting organ chips with

vascularized channels, our work provides a greater understanding of how to turn coagulation on and off in these

systems.

Coagulation rates in endothelial channels treated with and without fluid shear highlight the importance of fluid

flow in recapitulating the anticoagulant behavior of vascular endothelial cells. For vascular microdevice

engineers interested in perfusing blood or blood plasma, this means committing to the use of pumps or rockers

to induce fluid flow within the vasculature, prior to the addition of blood. Coagulation in otherwise healthy

vascular endothelium is not a faithful recapitulation of normal physiology. Instead, coagulation should be a

response to injury (hemostasis) or the addition of exogenous clotting factors or procoagulants. Ultimately, we

should be able to transfer blood or blood plasma to vascularized in vitro systems and circulate the blood without

clotting. Continuous blood circulation in multi-organ chip networks would represent a significant step in the

accurate recapitulation of physiology that makes organ chips exciting pharmaceutical tools.


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