Name: assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay
Uploaded: 2014-12-30T15:00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay at JoVE.com

Research reported in this publication was supported by National Eye Institute of the National Institutes of Health under award numbers: R01EY022084/S1 (KMC), T32EY007145 (HS) and P30EY014104. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support was provided by the Massachusetts Lions Eye Research Fund (K.M.C.) and a Special Scholar Award from Research to Prevent Blindness (to K.M.C.).

All experiments on animals were handled in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the guidelines and regulations set forth by the Massachusetts Eye and Ear Infirmary Animal Care Committee.
The day before the experiment:
1. Preparation of Tubing for Flow Chamber
<ol>
	<li>To prepare the jugular side, connect 1 cm PE10 polyethylene tubing to 6 cm silicon tubing followed by 5 cm polyethylene tubing. For the carotid side, prepare similarly to the jugular side with the addition of a T-tube within the 6 cm silicon tubing about 1.5 cm away from the 1 cm polyethylene tubing (Figure 1A, B). In order to insert the T-tube, widen the silicon tube by using fine forceps if necessary.</li>
	<li>Connect one end of 10 cm of silicone tubing to the pressure transducer then insert the T end of the carotid side T-tube into the other end. Ensure the connection sites are tight and leak free. Apply a small rectangular piece of parafilm over where the tubing fits together, if necessary, to prevent leakage (Figure 1C).</li>
</ol>
2. Coating the Chamber
<ol>
	<li>Prepare the endothelial surface coating protein (e.g., P selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1)) in 0.1% BSA. As a guideline, 1 &#956;g of protein in 200 &#956;l BSA is enough to coat 4-5 chambers. Using a 1 ml syringe connected to a 25 G needle, infuse each chamber (0.04 x 0.4 x 50 mm) with endothelial surface coating protein. Store the coated chambers in a 15 ml tube at 4 &#176;C overnight.</li>
</ol>
The day of the experiment:
3. Preparing the Chamber
<ol>
	<li>Remove the coated chambers from 4 &#176;C and connect the jugular and carotid tubing to each side. Seal the connections by carefully stretching a small rectangular piece of parafilm over where the tubing meets the chamber. Using a syringe, fill the chamber and tubing with 0.1% BSA (check for leakage). Incubate for 45 min.</li>
	<li>Prepare a 35 mm Petri dish to hold the chamber by cutting notches on each side of the dish. Stabilize the chamber in the notches; then use a small drop of silicone to affix the chamber to the dish at the notched site. Make sure the chamber is level by looking under the microscope. Allow the silicone to dry for at least 15 min.</li>
	<li>Fill a 60 ml locking syringe with heparin and connect to the pressure transducer. Connect the pressure transducer to the carotid tubing in the following order: Transducer, silicon tubing, and T-tube.
	<ol>
		<li>Use a 1 cm PE50 between the silicon tubing connected to the transducer and the T-tube to secure the connection. Pass the carotid artery silicon tubing through the pressure clamp (Figure 1D). Run 100USP heparin through the tubing and chamber until there are no air bubbles.</li>
	</ol>
	</li>
</ol>
4. Setting up the Software
<ol>
	<li>To prepare the software, turn on the computer, microscope, and open lab device. Double click on the Leica application suite (LAS) (or equivalent) and prepare a folder to save the video: browse &#8594; acquire &#8594; movie &#8594; define sequence of clips (clips = 15, recording time = 30 sec, and intervals = 15 sec) then click the &#8220;+&#8221; sign to confirm.</li>
	<li>To record the flow pressure open the LabChart (or equivalent) file and coordinate the file name to the recording folder in step 4.1, above.</li>
</ol>
5. Surgical Procedure
<ol>
	<li>Anesthetize an 8-10 week-old mouse using a combination of ketamine (60 mg/kg) and xylazine (6 mg/kg) injected intra-peritoneally (IP). Confirm that deep sedation is achieved within 5-10 min by loss of pedal withdrawal reflex. If necessary, isoflurane delivered via face mask can be given as needed.</li>
	<li>While under anesthesia, place a warming pad under the cage and set to 37 &#176;C to maintain body temperature. Once proper anesthesia is confirmed, place the mouse in a ventral position and coat the eyes with bacitracin ointment to prevent ocular dryness.</li>
	<li>Thereafter, shave the hair at the incision site using a razor blade and thoroughly clean the site with alcohol.</li>
	<li>Once fully anesthetized secure the animal with tape keeping the mouse in a ventral position. Expose the neck area with scissors and remove the thyroid gland to expose the trachea and vessels (Figure 2A).</li>
	<li>Clean the carotid artery until it is fully exposed taking care not to damage the vagus nerve (Figure 2B). Fold a piece of suture and pass the bend underneath the carotid artery then cut at the bend so there are now 2 pieces of suture underneath the carotid. Loop each suture into knots (do not tighten the knots).</li>
	<li>Repeat the same procedure to expose the left jugular vein (Figure 2C), preparing the sutures in the same manner (Figure 2D).</li>
	<li>Connect the tubing to the mouse vasculature.
	<ol>
		<li>Heparinize the mouse by injecting 1 ml of heparin IP.</li>
		<li>Tighten the upper suture on the carotid artery. Place the vessel clamp as low as it can go on the carotid artery (Figure 3A). Use forceps to hold the artery by the upper suture and make a small incision, about 1/8 of the circumference of the artery, using the microscissors (Figure 3B).</li>
		<li>Pass the carotid artery tubing through the second knot in the upper suture and into the incision made in the artery. Tie off the lower suture so that the artery is sealed around the tubing (several knots should be made to ensure a tight seal; Figure 3C).</li>
		<li>Repeat the same procedure for inserting the jugular vein tubing (the vessel clamp is not needed for the jugular side; Figure 3D).</li>
		<li>Test the flow by releasing the vessel clamp. If there are no leaks move the mouse to the microscope and connect the carotid tubing and jugular vein to the chamber tubing (see attached video for demonstration of surgical procedure).</li>
	</ol>
	</li>
</ol>
6. Recording Rolling Velocity
<ol>
	<li>Open the carotid clamp to fill the chamber, making a circuit with the mouse circulatory system. Allow the blood to circulate for 3-5 min so that the flow conditions stabilize. During the waiting period of 3-5 min, adjust the microscope stage so that the chamber is level from one end to the other with the microscope objective and its edge is parallel with the video recording window on the screen.</li>
	<li>Fill the modified Petri dish containing the glass chamber with water then lower the 10X water immersion objective into the dish so that the cells flowing through the chamber are visible. The light intensity may have to be adjusted for adequate viewing of the cells. Generally a lower intensity is better.</li>
	<li>Adjust the clamp on the tubing so that the transducer reads a steady 30 mm Hg, correlating to a medium physiological flow pressure, or as desired.</li>
	<li>Press &#8220;start&#8221; to initiate recording both the video and the pressure with the first time lapse close to the jugular end and moving against the flow toward the carotid end until completed. 
	NOTE: Several coating conditions using different adhesion molecules can be studied in the same mouse by using multiple chambers. Only one chamber is recorded at a time.
	<ol>
		<li>When recording is finished, close the valve at the input site, stopping the blood flow. Remove the chamber and fit a new chamber (coated with a different adhesion molecule) to the circuit using fine forceps in one hand and grooved forceps in the other hand. Resume recording for the new chamber as outlined in the steps 6.1-6.4.</li>
	</ol>
	</li>
	<li>Euthanize all mice by CO2 asphyxiation followed by a spinal cord dislocation at the end of the experiment. The procedure is terminal, therefore, the mice are euthanized by a spinal cord dislocation at the end of the experiment.</li>
</ol>
7. Interpreting the Results
<ol>
	<li>To analyze the recorded data use the &#8220;M TrackJ&#8221; plugin for ImageJ software23.</li>
	<li>Convert the file to grayscale by Select file &#8594; import &#8594; AVI &#8594; convert to grayscale.</li>
	<li>Standardize the frames by Select &#8594; image &#8594; property &#8594; enter the frame intervals (i) &#8594; global. Calculate the frame intervals by dividing the total video duration (usually 30 sec) by the number of frames in one video (shown in the upper corner of the Image J video window).</li>
	<li>Measure the flow of each leukocyte by Select plugin &#8594; M TrackJ &#8594; Add tracks &#8594; select a leukocyte &#8594; Track it with each frame for 15 - 20 sec &#8594; Merge.</li>
	<li>Repeat step 7.4 for all the leukocytes in view.</li>
	<li>Determine the mean rolling velocity of the leukocytes by Select measure &#8594; two columns appear &#8594; Copy all of the M TrakJ:Tracks into a spreadsheet. The column labeled &#8220;mean V&#8221; is the leukocyte rolling measured in pixels/second. Convert into &#956;m/sec by dividing by 2/3.</li>
	<li>To analyze the pressure data highlight and export into a spreadsheet and perform the following calculation: &#916;P (mm Hg) and t (dyne/cm2) for a comparison between each experimental group. Once calculated, copy the data into prism software for graphing.</li>
</ol>

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The authors (L.M., H.S., and K.M.C.) have no competing financial interests to disclose.

The process of leukocyte recruitment is a crucial step in the inflammatory response; it involves the migration of leukocytes from the circulatory system towards target tissues, where they are able to exert their effector function. Leukocyte recruitment is integral in a variety of inflammatory conditions such as atherosclerotic plaques, myocardial infarction, ischemia/reperfusion, and transplant surgery1, as well as multiple CNS related neuro-inflammatory conditions10-12,20,24,25. Considering the diversity of the disease conditions that leukocyte recruitment spans, the autoperfused microflow chamber provides an indispensible tool allowing the investigator the ability to study leukocyte migration dynamics.
Over the past several decades, a variety of in vitro assays have been developed to study the dynamics of leukocyte cell adhesion26. Unfortunately, all of these assays require the extraction of the leukocytes from the blood, introducing mechanical activation. To get closer to the in vivo environment, adaptations were made to collect whole blood samples and control the hemodynamic flow conditions16,17. Here, we extend the previous advances in the field by linking a coated chamber to the mouse circulatory system. We are able to regulate the flow of the blood to a physiologic range and study the dynamics of leukocyte rolling. The coated chamber gives us the ability to study leukocyte interactions with specific adhesion molecules. Since the system is functioning as a unit operated by a live mouse, it more closely mimics the natural environment, allowing us to study leukocyte-endothelial interactions in a variety of immunologic mouse models. Additionally, this system allows us to take advantage of the multiple genetic mouse models available. While the system does not completely replicate the in vivo environment, it provides a platform to study specific elements of the leukocytes under physiologic conditions, a feat that has not previously been possible. Even though this takes us a step closer to a more physiologic environment there are limitations to the system. It does not completely replicate the complex 3D matrix of the vasculature so we are only able to assess certain elements of leukocyte interactions that are restricted to the chamber coating. In addition, great care should be taken to ensure a closed system to the mouse circulation by following all the steps mentioned in the protocol. The introduction of air bubbles will greatly affect the accuracy and reproducibility of the experiments.
While we describe the use of the Autoperfused Microflow chamber for assessing leukocyte rolling dynamics the procedure has the potential to be personalized by investigators to study a variety of diseases. For example, cancer cells metastasize by expressing many of the common integrins shared by leukocytes. Studying their rolling dynamics in a setting that more closely mimics an in vivo environment could help in our knowledge, and possibly the prediction, of the invasive nature of certain cancer cells. One possible approach could be to combine a labeling technique for the cancer cells, such as GFP27, along with the flow chamber assay to track rolling dynamics of the cancer cells expressing GFP. Given the flexibility of coating the chamber with a variety of substances and connecting multiple chambers to the same mouse it will be interesting to see how this procedure is modified for use in other labs in combination with genetically modified mice and disease models. The technique we describe here just touches on a much broader application potential that is only limited by the creativity of the investigator.

Inflammation is the body&#8217;s universal response to injury and is a crucial step in both innate and adaptive immune system function. In response to injury and/or inflammatory stimuli, endothelial cells upregulate specific adhesion molecules; this leads to leukocyte extravasation through the microvascular endothelium, primarily in post capillary venules. This process starts with tethering of the free-flowing leukocytes in the bloodstream on the endothelium. Stable rolling and firm adhesion of leukocytes, which in turn leads to transmigration and the secretion of cytotoxic agents, follow this tethering1,2. Selectins are known to mediate the early steps of the cascade3-5; integrins are responsible for the later steps of firm adhesion and transmigration1,6-8.
A growing body of evidence suggests leukocytes and endothelial adhesion molecules have vital roles in animal models of ischemia reperfusion injury, asthma, psoriasis, multiple sclerosis, and age-related macular degeneration9-12. Under these conditions, the inflammatory response is misdirected to attack one&#8217;s own body, resulting in the breakdown of healthy tissue. Existing anti-inflammatory agents (such as non-steroidal anti-inflammatory drugs, corticosteroids, or other chemotherapeutic agents) carry the risk of severe side effects with long-term use13. Therefore, it is of great interest to have the appropriate tools with the ability to identify disease-specific molecules, which can ultimately be targeted to have the desired anti-inflammatory effect while remaining non-toxic14.
Existing in vitro methods such as the static leukocyte adhesion assay were used as early as 197615. The parallel flow chamber was first used in vitro in 1987 to study leukocyte-endothelial interactions under flow conditions. In these experiments, stimulated human polymorphonuclear leukocytes (PMNs) from venous blood were perfused over a monolayer of primary human umbilical vein endothelial cells (HUVECs). To control for the hemodynamic flow conditions, the Harvard Apparatus syringe pump was employed16. Alternatively, to avoid artificial leukocytes isolation, whole blood was used in combination with the glass chamber coated with immobilized adhesion molecules17.
To avoid leukocyte stimulation and to mechanistically study their interaction with the adhesion molecules under approximate physiologic conditions, an ex vivo autoperfused, extracorporal, arteriovenous circuit was developed16. In this circuit, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing interaction of native PBLs within a glass microflow chamber coated with single or co-immobilized adhesion molecules. A major advantage to this system is the ability to employ genetically engineered mice, in which inflammatory pathways are directly or indirectly manipulated. Additionally, there is the ability to pinpoint the isolated contribution of leukocyte adhesion molecules to inflammation, free of external activation, under flow conditions. The application of a flow regulator at the input point of the flow chamber provides a wide range of experimental variations of shear forces to mimic either arterial or venous systems18-22. Here we describe in great detail a protocol concerning the preparation and performance of the ex vivo autoperfused microflow chamber assay.

<table border="0" cellpadding="0" cellspacing="0" width="792">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">Material</td>
			<td style="width:220px;">Vendor</td>
			<td style="width:237px;">Part number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro glass chamber 0.4x0.04x50mm</td>
			<td>VitroCom</td>
			<td>2540-050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing PE 10</td>
			<td>Fisher Scientific</td>
			<td>427400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing PE 60</td>
			<td>Fisher Scientific</td>
			<td>427416</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Silicone tubing 002</td>
			<td>Fisher Scientific</td>
			<td>11-189-15A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y tube</td>
			<td>Value Plastics</td>
			<td>Y210-6</td>
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		<tr height="21">
			<td height="21" style="height:21px;">T tube</td>
			<td>value plastics</td>
			<td>T410-6</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Silicone gel</td>
			<td>Hardware store - Home Depo</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">35mm petri dish</td>
			<td>Corning</td>
			<td>430165</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>Pechiney Plastic Packaging</td>
			<td>PM996</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine forceps</td>
			<td>FST</td>
			<td>11253-25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>FST</td>
			<td>15000-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Tube holder</td>
			<td>FST</td>
			<td>00608-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Clamp applicator</td>
			<td>FST</td>
			<td>18057-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Vascular clamp</td>
			<td>FST</td>
			<td>18055-04</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-0 silk sutures</td>
			<td>George Tiemann &#38; Co</td>
			<td>160-1215-6/0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25x1G&#160; needles</td>
			<td>BD</td>
			<td>305125</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30x1/2G needles</td>
			<td>BD</td>
			<td>305106</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin 100 USP units/ml</td>
			<td>Hospital pharmacy</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

During each recording only interacting leukocytes are evident on the chamber. Results from a representative experiment can be seen in Figure 4. The white arrows point to individual leukocytes that were captured by the adhesion molecule of interest and are rolling along the chamber amongst other free-flowing ones within the blood flow. A common artifact outside the chamber can be seen with the large dark spheres in all the images. The dark spheres help the reader appreciate the leukocyte movement in relation to a fixed point. Leukocytes advancement over a period of 22 sec can be seen in relation to the spheres. The graph in the same figure shows a final plot of the data where Tx1 decreases the rolling velocity (a shift to the left) and Tx2 increases the velocity (a shift to the right) compared to the control.
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig1.jpg" width="700" /> 
Figure 1: Preparation of tubing for the flow chamber. (A) Tools required for tubing and chamber assembly (micro glass chamber 0.4 x 0.04 x 50 mm, polyethylene tubing PE10, polyethylene tubing PE60, silicon tubing 002, Y tube, T tube, 35 mm Petri dish, fine forceps, fine scissors, tube holder, vascular clamp, 7-0 silk suture. (B) Preparation of the carotid artery and jugular vein tubing for re-directing the mouse blood flow through the coated chamber. (C) Connecting the carotid artery tubing to the pressure transducer. (D) Pass the tubing from the transducer through the clamp to adjust the rate of the blood flow. <a href="https://www.jove.com/files/ftp_upload/52130/52130fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig2.jpg" width="700" /> 
Figure 2: Exposing the carotid artery and jugular vein. (A) Carefully cut the neck area to expose the trachea. (B) Clean the right carotid artery so that a piece of suture can be passed underneath the vessel (dotted yellow line outlines the carotid artery). (C) Clean the left jugular vein so that a piece of suture can be passed under the vessel (dotted yellow line outlines the jugular vein). (D) Arrange loosely tied sutures at the upper and lower regions of the carotid artery and jugular vein (arrows indicated the upper and lower regions of the vessels). <a href="https://www.jove.com/files/ftp_upload/52130/52130fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig3.jpg" width="700" /> 
Figure 3: Connecting the tubing to the carotid artery and jugular vein. (A) Tighten the upper knot on the carotid and place the clamp below the lower knot. (B) Using the microscissors, make a small incision in the carotid, about 1/8 the circumference (dotted yellow circle indicates the incision). (C) Insert the tubing into the carotid and secure with at least two sutures (green dotted line shows the tubing and the yellow dotted line the carotid artery). (D) Similarly insert tubing in the jugular (green dotted line shows the tubing and the yellow dotted line the jugular vein). <a href="https://www.jove.com/files/ftp_upload/52130/52130fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52130/52130fig4.jpg" width="700" /> 
Figure 4: Representative time course of leukocytes rolling through the flow chamber (Scale bar = 100 &#956;m) (white arrowheads point out rolling leukocytes). Graph depicts representative results after exporting into a graphing program (Tx1 and Tx2 are experimental treatment groups and CTR is the control group). Tx1 represents a treatment in which leukocytes slow down leading to a decrease in leukocytes rolling velocity and a left shift of the graph compared to the untreated group (CTR), while Tx2 leads to a right shift reflecting an increase in leukocytes rolling velocity. <a href="https://www.jove.com/files/ftp_upload/52130/52130fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay at JoVE.com

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

assessing-leukocyte-endothelial-interactions-under-flow-conditions-an

Research

JoVE Journal

Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

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 ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by ...