The authors have no acknowledgements.

This protocol follows the institutional ethical guidelines for research on human samples and informed consent was obtained from all donors involved. Approval for the experiments described here was obtained from the institutional review board of the Antwerp University Hospital.
Note: Temperature indications are always room temperature, unless specified.
1. Preparation Flow Chamber Setup
<ol>
	<li>Preparing Lanes, Tubing and Pins
	<ol>
		<li>Vortex the collagen suspension vigorously and dilute 1/20 in the isotonic glucose solution supplied by the provider to a final concentration of 50 &#181;g/ml. 
		Note: We use equine tendon collagen, mainly made up of type I fibrils. The equine collagen type I is often referred to as &#34;Horm&#34; collagen and is the golden standard for this type of assay9 for both historical as well as biological reasons. Human type III collagen can also be used, but the fibrils coat less well and the platelet response is not as strong. Other coating surfaces can also be used, for example von Willebrand Factor (VWF), fibrinogen, fibronectin, laminin, vitronectin, thrombospondin-1 or combinations of these16.</li>
		<li>Take a new disposable biochip from the provider&#39;s container. The dimensions of the biochips used here are 0.4W x 0.1H x 20L in mm3.</li>
		<li>Pipet 0.8 &#181;l into the lane(s) of the microfluidic biochip on one end of the chip and mark as outlet. Make sure that the lane is filled 5/6th with the collagen containing coating solution prepared in 1.1.1. Ensure that there are no air bubbles. 
		Note: Channels are partially coated to avoid accumulation of collagen fibers at the entrance of the channel (see discussion).</li>
		<li>Incubate at 4 &#176;C for 4 hr or overnight in a humidified and closed container.</li>
		<li>Block the coated channels by pipetting blocking buffer (1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS; 0.9% (w/v) NaCl, pH 7.4) at the other end and mark as inlet. Make sure that the lane is completely filled with the blocking buffer avoiding air bubbles.</li>
		<li>Cut tubing at equal length (12 cm). Use one per lane and connect each tubing with a pin. For example, an experiment comparing two conditions, run in duplicate will require four tubing stretches and four pins to be prepared.</li>
		<li>Rinse the tubing with distilled water using a syringe and a 26 G needle or the accompanying connectors.</li>
		<li>Saturate the tubing with blocking buffer. Store in a closed and humidified container for a minimum of 1 hr.</li>
	</ol>
	</li>
	<li>Preparing Pump and Manifold
	<ol>
		<li>Rinse the pump and manifold with distilled water, remove air bubbles.</li>
		<li>Aspirate the blocking buffer out of the biochip lane(s) using the fixed 10 &#181;l tip at the outlet. Clean the surface of the biochip with a precision dust free wipe and denaturated alcohol to remove prints and dust.</li>
		<li>Fix the biochip on an automated microscope stage. If more than one lane is used simultaneously in one run, connect the eight-lane manifold splitter to the biochip outlet. 
		Note: The eight-lane manifold splitter is a piece of hardware (Figure S1) connected to the pump and the biochip. It allows operation of all available (eight) lanes on a biochip or a combination of lanes which can be operator-defined in the accompanying software.</li>
		<li>Use the pins in the tubing to fix them in the biochip inlet. Place the other end of the tubing (without pin) in a 1.5 ml conical test tube filled with HBS. Rinse all tubing and their connected lanes with 1 ml HBS using the pump, so as to remove remainder blocking buffer and poorly adhering collagen.</li>
	</ol>
	</li>
</ol>
2. Preparation of Blood Samples
<ol>
	<li>Collection and separation of fresh whole blood from a healthy volunteer.17
	<ol>
		<li>Collect the first milliliters of blood in an evacuated tube containing Ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and exclusively use this sample for complete blood count (CBC) with an automated hematology analyzer.</li>
		<li>Collect a volume of blood in a suitable anticoagulant using evacuated tubes. Standard anticoagulants for flow chamber experiments are heparin or hirudin when fibrin formation is not part of the study protocol and sodium citrate when it is. 
		Note: Heparin was used as an anticoagulant for all the experiments described in the results. 
		Note: The amount of blood depends on the number of experiments to be performed. Approximately 1 tube (7 ml) for 3 lanes.</li>
		<li>Place the tubes on a rotator pending blood reconstitution. 
		Note: The assay should be completed within 3 hr of phlebotomy.</li>
		<li>Centrifuge for 15 min at 250 g to prepare platelet rich plasma (PRP). Do not use the centrifuge break to prevent disturbance of the loosely packed pellet.
		<ol>
			<li>When more than one tube was collected, pool the blood in a single conical centrifugation tube. 
			Note: Centrifugation can be done more slowly or less long, depending on the PRP yield and differential cell &#34;contamination&#34; preferred.</li>
		</ol>
		</li>
		<li>Remove and discard the PRP and buffy coat yielding packed red blood cells with few platelets. 
		Note: The platelet count in the packed red cell fraction is 13 &#177;5 x 103 per &#181;l (mean &#177;SD, n = 12) on average in our hands.</li>
	</ol>
	</li>
	<li>Blood Reconstitution
	<ol>
		<li>Thaw blood group AB (Rhesus D negative) plasma at 37 &#176;C for 5 min and 20 sec per 4 ml.</li>
		<li>Determine the hematocrit of the packed red blood cells prepared in 2.1.5 using an automated hematology analyzer.</li>
		<li>Determine the platelet concentration in the blood bank prepared platelet concentrate that will be used to reconstitute the red cell fraction above.</li>
		<li>Calculate the volume of packed red blood cells and platelet concentrate that will yield 40% hematocrit and 250 x 10&#179; platelets/&#181;l in a 1 ml sample. 
		Note: Other target titers of cells can be set arbitrarily, depending on the study protocol.</li>
		<li>Transfer packed red blood cells and plasma into a fresh tube using a clipped pipet tip and add platelet concentrate until a sample volume of 1 ml is reached. 
		Note: Depending on the variables studied, the plasma fraction should be equal in all reconstituted samples because plasma has a significant influence on thrombus formation rate. For instance, repeated freeze-thawing or plasma taken from different donors or on different anticoagulants may influence the result.</li>
		<li>Mix the reconstituted blood gently by inverting and perform a CBC.</li>
		<li>Prepare a &#34;blank&#34; control sample in which the volume of platelet fraction is replaced by the same volume of 0.9% (m/v) sodium chloride in water to determine the concentration of endogenous platelets (i.e. non-blood bank platelets) in the reconstituted blood using a CBC.</li>
	</ol>
	</li>
	<li>Labeling
	<ol>
		<li>Pipet 1 ml reconstituted blood in a test tube containing 1 &#181;l 5 mM Calcein AM (5 &#181;M final concentration). 
		Note: Other cell dyes can be used14.</li>
		<li>Mix gently by inverting.</li>
		<li>Incubate for 5 min at 37 &#176;C prior to use.</li>
	</ol>
	</li>
</ol>
3. Perfusion Assay
<ol>
	<li>Focus the objective on the collagen fibers adhered at the bottom of the lanes. Ideally, use phase-contrast or differential interference contrast (DIC) settings for this focusing strategy. Select &#8216;Set current Z for selected tile regions&#8217; in the experiment software to digitally fix the selected Z-positions.</li>
	<li>Select a region of interest (ROI) in the lane (xy) in the experiment software of the microscope that will be recorded during the experiment. 
	Note: The ROI can be any surface area arbitrarily chosen within a perfusion lane. It is advisable not to analyze thrombus formation close to the in- and outlet of a lane so to avoid side effects of the variable flow profile in that region, even though this is relatively small. The ROI surface area should contain a significant number of platelets or thrombi to allow leveling of the signal. In this protocol the ROI is a digitally stitched aggregate of three equally sized side-by-side images resulting in 0.62 mm2 in the middle of the 2 cm long lane.</li>
	<li>Mix the samples gently by inverting and position these next to the biochip on the automated stage.</li>
	<li>Place the tubing that is connected with the inlet of the biochip in the test tubes containing the reconstituted blood samples.</li>
	<li>Launch the pump at 50 dyne/cm2 (or other shear stresses as desired) for those channels linked to test tubes containing the reconstituted blood samples using the software of the pump. 
	Note: Other shear stresses can be used.</li>
	<li>Record images every 15 sec for 5 min in real-time using the acquisition and experiment software of the microscope. 
	Note: Other time series can be used depending on the experimental set-up. 
	Note: We generally use a 100X magnification (10X objective and 10X lenses), but higher (or lower) magnification can easily be used as an alternative.</li>
</ol>
4. Wash Out
<ol>
	<li>Wash out all tubing attached to the outlet and connected to the multichannel manifold or pump using distilled water, followed by sodium hypochlorite (bleach) 0.5% (v/v) and finally 0.1 M NaOH in water. Discard the tubing pinned to the biochip inlet as hazardous waste.</li>
</ol>
5. Data Analysis
<ol>
	<li>Determine thrombus growth kinetics with the image analysis software. The following commands are specific for ZEN2012.
	<ol>
		<li>Open the plugin Image Analysis to determine the surface coverage of the platelets.</li>
		<li>Set the fluorescence threshold in the Analyze Interactive tab to define the pixel intensity that correlates with a positive signal, i.e. an adhered platelet or adhering platelets.</li>
		<li>Use Create Tables to automatically generate a spreadsheet that will contain the separate surface areas (in &#181;m2) of those &#34;objects&#34; containing pixels with a signal between the selected thresholds. This is performed for each time point. 
		Note: Once fluorescence thresholds have been chose, the analysis software automatically detects &#39;objects&#39; in the view field that fulfill the criteria. These objects are thrombi, small platelet aggregates or single platelets and cover a number of pixels. Every object is listed in the spreadsheet separately.</li>
		<li>Save these spreadsheets in xml format and open them in a spreadsheet program for further calculations.</li>
		<li>Total the surface areas of the selected objects by summation and divide the result by the total area of the measurement field (&#181;m2). This will yield the relative surface coverage (%). Do so for every time point.</li>
		<li>Plot these surface coverages in function of the perfusion time and calculate the slope by linear regression, yielding the thrombus growth kinetic of that particular condition.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Microfluidic flow chamber experiments are an excellent tool to investigate platelet function in flowing blood and are used to evaluate hemostasis in vitro in varying experimental contexts. Despite poor interlaboratory standardization9, we demonstrate that within our laboratory the experimental variation is acceptable. This allows to reliably compare (paired) samples within a given study. This was validated using the well documented phenomenon of platelet storage lesion, which is a detrimental conseque...

Hemostasis requires the combined and regulated activity of cells, proteins, ions and tissues in a restricted spatiotemporal context1. Uncontrolled activity may lead to hemorrhage or thrombosis and morbidity or mortality in a spectrum of disorders related to blood coagulation. A microfluidic flow chamber experiment is a challenging technique that mimics hemostasis in vitro. This approach allows investigation of the complex interplay of processes that take part in hemostasis with a leading role for blood platelets.
Following vascular injury, platelets adhere to the exposed subendothelial matrix (glyco)proteins to prevent blood loss. Following adhesion, platelets activate and aggregate in response to auto- and paracrine signaling which finally leads to the formation of a platelet network, stabilized by fibrin and resulting in a firm, wound sealing thrombus2. Unlike most other platelet function tests, experiments with flow chambers take into account the physical parameter of blood flow and therefore the influence of rheology on the participating cells and biomolecules3,4.
Flow chamber experiments have generated landmark insights in hemostasis and thrombosis by varying key parameters that influence hemostatic (sub)processes including the adhesive matrix, rheology and flow profiles, cellular composition, presence of toxins or drugs, ionic strength and many more. In the past two decades, low throughput flow chamber experiments requiring large sample volumes (10-100 ml) have evolved to microfluidic chambers often consisting of small parallel-plate chambers and including modern technology for perfusing whole blood at controlled wall shear conditions5. Microscaling has significantly increased assay throughput mostly because the hardware setup has simplified and less (blood) volume is required, rendering the experiment more accessible and versatile. For instance, blood from small laboratory animals can now be used without the need to sacrifice animals. Blood samples of genetically modified mice have thus aided in the identification of key molecules promoting or inhibiting hemostasis and in novel basic insights6.
Specialized research laboratories often still use custom made flow chambers for instance from polydimethylsiloxane (PDMS)7 that polymerizes on lithographed molds which can be blueprinted by software. The resulting chamber is inexpensive, disposable and can be easily disassembled for post hoc analysis. Furthermore, basically any design of vessels, including bifurcations or sharp turns can be built on command. This advantage is also its downside since standardization was already the primary problem with flow chamber experiments, and PDMS custom made chambers have not aided this. On top of this particular issue, coating (conditions), fluorescent probes, anticoagulant, temperature and time between sampling and analysis are all poorly standardized8. Standardization of these variables is challenging, but nonetheless required to permit comparison of results between laboratories. This topic is the major subject of the International Society on Thrombosis and Haemostasis in Scientific and Standardization subcommittee on Biorheology9,10.
Platelet concentrates (PC) are transfused in patients suffering from various diseases that cause thrombocytopenia and/or bleeding. But platelets in PC are known to desensitize, especially in function of storage time11, a deterioration process linked to ageing and commonly referred to as platelet storage lesion. It is sometimes claimed that such platelets restore in circulation once transfused12, but evidence for this is scarce. Furthermore, the functionality of platelets making up a PC is not routinely tested because the relationship between such assays and therapeutic or prophylactic efficacy is unclear13. Microfluidic flow chambers offer a means to investigate platelet function in PC to optimize the chain of manipulations between collection and issuing. It is a powerful research tool for direct (paired) comparisons of PC as we have previously published14,15 and is described here.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368856</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Heparin</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368480</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">366575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hirudin Blood tube</td>
			<td style="width:220px;">Roche</td>
			<td style="width:268px;">6675751 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">739280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:268px;">A08928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tube 5mL</td>
			<td style="width:220px;">Simport</td>
			<td style="width:268px;">11691380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">1888271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:268px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:268px;">5511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">188V8CF-400-100-02P10</td>
			<td>Named in figure S1 A as &#39;Biochip&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td>Named in figure S1 B as &#39;Disposable tubing&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">SS-P-B1IC-B1OC-PACK200</td>
			<td>Named in figure S1 B as &#39;Pin&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Connectors for single inlet cables of biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiflow8 connect</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">MF8-CONNECT-BIC3-N-THROMBOSIS</td>
			<td>Named in figure S1 B as &#39;Reusable tubing&#39; and &#39;Splitter&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microfluidic pump</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">N/A</td>
			<td>Venaflux Assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:268px;">1130630</td>
			<td style="width:409px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:268px;">C1430</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach 10%</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1M NaOH</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:268px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8. Named in figure S1 A as &#39;Pump&#39; and &#39;Manifold&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z1</td>
			<td>equipped with a colibri-LED and high resolution CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:268px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Platelet incubater</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:268px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:220px;">VWR</td>
			<td style="width:268px;">58816-121</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To demonstrate intra-assay variation, three identical reconstituted whole blood samples were perfused simultaneously over collagen coated surfaces (Figure 1). This resulted in a coefficient of variation of 8.7%. This statistic suggests acceptable intra-assay and intralaboratory variation permitting reliable comparison between related samples.
The inlet of the commercial flow chamber we describe here is perpendic...

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

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.

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 ...

microfluidic-flow-chambers-using-reconstituted-blood-to-model

Research

JoVE Journal

Bioengineering

9.3K Views.  Belgium Red Cross-Flanders. 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. 

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 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 ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.