CHO-P cells were a kind gift from Dr. Barbara Furie (Beth Israel Deaconess Medical Center, Harvard Medical School). This work was supported by National Institute of Health grant HL095722 to J.M.K. This work was also supported in part by a Movember-Prostate Cancer Foundation Challenge Award to J.M.K.

1. Cell Culture
<ol>
	<li>Human promyelocytic leukemia (HL-60) cells
	<ol>
		<li>Culture HL-60 cells in 75 cm2 flasks with 15 ml of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM), supplemented with 20% (v/v) fetal bovine serum (FBS), 1% (v/v) L-Glutamine and 1% (v/v) Penicillin-Streptomycin.</li>
		<li>Change media every 3 days by aspirating half of the cell suspension volume and replacing it with complete IMDM media.</li>
		<li>For carboxyfluorescein diacetate, succinimidyl ester (CFSE) staining, centrifuge HL-60 cell suspension (400 x g, 5 min), resuspend in a 1 &#956;M CFSE solution (prepared in prewarmed PBS) and incubate for 15 min at 37 &#176;C. Then centrifuge cells, aspirate supernatant and resuspend cells in fresh prewarmed medium for 30 min. Wash cells in PBS and then use for rolling experiments (see Figure 1B for representative image of CFSE-stained HL-60 cells on P-sel-coated surface).</li>
	</ol>
	</li>
</ol>
Note: CFSE staining is optional, and is presented here to demonstrate the rolling phenomenon in the microfluidic channel. Analysis of rolling parameters presented in this manuscript was performed on unstained cells using standard brightfield imaging.
<ol start="2">
	<li>Lung microvascular endothelial cells (LMVECs)
	<ol>
		<li>Coat 100 mm Petri dishes with 0.1% gelatin solution (v/v in PBS) and incubate at 37 &#176;C for at least 30 min.</li>
		<li>Culture LMVECs on gelatin-coated 100 mm Petri dishes in complete endothelial growth medium (endothelial basal medium-2 (EBM-2)), supplemented with a specific growth supplement kit, see REAGENTS). Change media every other day and sub-culture cells upon reaching 80-90% confluence.</li>
		<li>For sub-culture, wash cells with PBS and then detach cells with 4 ml of 1x&#160;Trypsin-EDTA for 3 min at 37 &#176;C and neutralize in an equal volume of complete EBM-2 media. Transfer the cell suspension to a 15 ml tube and centrifuge (400 x g, 5 min). Following centrifugation, resuspend the pellet in 1 ml of complete endothelial media and count cells with a hemocytometer. Do not over-passage the cells, as this affects their morphology and function- use only cells under passage 7 for all experiments.</li>
	</ol>
	</li>
</ol>
<ol start="3">
	<li>Chinese Hamster Ovary-P-selectin (CHO-P) Cells
	<ol>
		<li>CHO-P cells, which are CHO cells stably transfected to express human P-sel, were provided by collaborators (Beth Israel Deaconess Medical Center, Harvard Medical School)11,12.</li>
		<li>Culture CHO-P cells in T175 cm2 flasks in 25 ml of F-12 media.</li>
		<li>For passaging, wash cells with 10 ml of PBS for 4-5 sec and then trypsinize in 10 ml of 1x Trypsin-EDTA for 3 min at 37 &#176;C, followed by neutralization in full media.</li>
		<li>Centrifuge the cell suspension (400 x g, 5 min), carefully aspirate the supernatant, resuspend the cell pellet in 1 ml of full media and count the cells with a hemocytometer.</li>
	</ol>
	</li>
</ol>
2. Operation of the Integrated Multi-well Plate Microfluidic System
<ol>
	<li>Make sure all the equipment is properly connected and turn on the different modules: computer, controller, inverted microscope, and CCD camera.</li>
	<li>Open the imaging software; make sure the multi-well plate module and the imaging module are properly presented on the screen.</li>
	<li>Connect the tubes to the vapor trap (connected to the controller) and also connect them to the Pressure Interface.</li>
	<li>Place the multi-well plate in the plate heater/adaptor. Add reagents to wells (described below) and attach interface on top of the plate. Place plate for imaging on automated stage.</li>
	<li>The interface attaches to the top of the plate and applies a pneumatic pressure from the controller to the top of the wells, driving the fluid through the microfluidic channels at the defined flow rate, easily controlled using the multi-well plate module screen under Manual mode.</li>
	<li>Reagents in the channel flow across an observation area, located between the wells. Microfluidic channel dimensions are 350 &#956;m wide x&#160;70 &#956;m tall. The length of the linear channel is 1 mm and the bottom of the channels comprises a 180 &#956;m coverslip glass, which is compatible with brightfield, phase, fluorescence and confocal microscopy.</li>
	<li>Acquire videos using a CCD camera (stream acquisition, 11 frames/sec) and analyze via compatible software.</li>
</ol>
3. Coating of Microfluidic Channels with a Protein Substrate or a Cell Monolayer
<ol>
	<li>Coating microfluidic channel with fibronectin or P-/E-selectin
	<ol>
		<li>Prepare 1 ml of 20 &#956;g/ml fibronectin solution in PBS. Alter volume based on the number of channels to be coated (use 25-50 &#956;l of fibronectin per channel).</li>
		<li>Add 25-50 &#956;l of fibronectin solution to each inlet well. Apply shear force of 2 dyn/cm2 for 5 min to perfuse the channel. Please note the bead of liquid appearing in the outlet well. Incubate for 30-45 min at R.T.</li>
		<li>Aspirate the solution from wells (do not aspirate directly from the middle circle that feeds the channel)1,13. Add 200-500 &#956;l of PBS into outlet well and wash channel with PBS by applying shear flow of 2 dyn/cm2 for 5 min. The channel is now properly coated with fibronectin and ready to be used.</li>
		<li>To coat with P- or E-sel, prepare a 5 &#956;g/ml solution of the desired human recombinant protein in PBS, and coat the channels as described above, with 1 hr incubation at 37 &#176;C to allow surface coating.</li>
	</ol>
	</li>
	<li>Creation of CHO-P or LMVEC monolayer inside the microfluidic channel
	<ol>
		<li>Gently trypsinize cells from culture dishes for 3 min, quench using a 2-fold volume of full media and centrifuge (5 min at 400 x g). Resuspend cells with 10 ml of full media and centrifuge (5 min at 400 x g) again.</li>
		<li>Count the cells to determine cell concentration in the suspension. To ensure the formation of a confluent LMVEC monolayer inside the channel, bring cell concentration to 15-20 million cells/ml. For a confluent CHO-P cell monolayer, use 50-60 million cells/ml. Use 25-50 &#956;l of cell suspension for each channel - determine initial cell number used for the experiment accordingly.</li>
		<li>Add 25-50 &#956;l of cell suspension in the appropriate concentration to the inlet well. Place the plate on the microscope stage and introduce cells into the channel (2 dyn/cm2) until cells are observed on the screen filling the entire channels, and then stop the flow.</li>
		<li>Fill both outlet and inlet with 200 &#956;l of either full LMVEC or CHO media. Let the cells settle and adhere for 3 hr in the incubator (37 &#176;C, 5% CO2).</li>
		<li>Following the 3 hr incubation, wash the channel with full media (2 dyn/cm2, 10-15 min) to remove unattached cells. Cells should now appear completely confluent and the channel is now ready for use. Depending on initial cell seeding density, additional 2-3 hr of settling time may be required to ensure complete coverage of the surface with the cells.</li>
	</ol>
	</li>
</ol>
4. LMVEC Pro-inflammatory Activation and Antibody Blocking of P-/E-selectin
<ol>
	<li>Prepare a TNF-&#945; solution (10 ng/ml) in LMVEC basal media.</li>
	<li>To induce inflammatory activation of LMVEC in the channels, add 100 &#956;l of the TNF-&#945; solution to the inlet well and introduce the solution into the channel by applying shear flow of 2 dyn/cm2 for 5 min. For control channels (nonactivated ECs), add 100 &#956;l of LMVEC basal media to the inlet well and introduce into the channel (2 dyn/cm2 for 5 min). Channel is now ready for a rolling assay.</li>
	<li>To block P-sel and E-sel on LMVECs and CHO-P cells, introduce neutralizing P-sel (clone AK4, 5 &#956;g/ml in basal media) or E-sel (clone P2H3, 5 &#956;g/ml in basal media) antibodies into the channel and incubate for 1 hr at 37 &#176;C. Next, wash channels with basal media (2 dyn/cm2 for 5 min). Channels are now ready for a rolling assay.</li>
</ol>
5. HL-60 Rolling Assay on Substrate/Cell Monolayer-Coated Microfluidic Channels
<ol>
	<li>Carefully examine the channels under the microscope to confirm that channels are properly coated (in the case of coating with cells, a fully confluent cell monolayer should be observed).</li>
	<li>To prepare HL-60 cell suspension for the rolling experiments, centrifuge HL-60 cell suspension (5 min at 400 x g) and wash once with basal media. Count the cells and resuspend in IMDM (basal media, containing Ca2+ and Mg2+) to create a HL-60 cell suspension with 5 million cells/ml. Use 25-50 &#956;l of cell suspension for each channel to perform the rolling assay.</li>
	<li>Add 25-50 &#956;l of the cell suspension to outlet well, place plate inside the temperature-controlled plate holder (37 &#176;C) and place on the microscope stage. Next, introduce cells into the channel by applying shear force of 2 dyn/cm2 (cells should be observed within 10-15 sec flowing from outlet to inlet).</li>
	<li>To examine the rolling response as a function of shear stress, reduce shear to 0.25 dyn/cm2 and acquire 20-30 sec videos (using &#34;stream acquisition&#34; function) in each desired shear (increase shear gradually from 0.25 up to 5 dyn/cm2. It is also possible to use higher shears).</li>
	<li>Acquire videos using a CCD camera (stream acquisition, 11 frames/sec) and analyze rolling paths and rolling velocities via compatible software.</li>
</ol>
6. Flow Cytometry to Detect Expression Of Surface Molecules
<ol>
	<li>Following trypsinization, prepare a cell suspension (using 1-2 x&#160;105 cells/sample) of desired cell type (HL-60, CHO-P or LMVECs) in PBS (-/-), supplemented with 2% FBS. Wash cells twice and bring sample volume to 50 &#956;l (using the same buffer).</li>
	<li>Incubate each sample with the desired fluorophore-conjugated Ab (see attached table for detailed information) at 4 &#176;C for 20 min (cover with aluminum foil).</li>
	<li>Wash the cells twice (same buffer) and bring final volume of stained cell suspension to 200 &#956;l. Analyze samples using a flow cytometer to detect expression of surface molecules.</li>
</ol>

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Authors declare no conflict of interests.

One of the major challenges in successful translation of exogenous cell-based therapy is the inability to efficiently deliver cells to sites of injury and inflammation with high engraftment efficiency3. Cell rolling represents a critical step in the process of cell homing, facilitating the deceleration of cells on the walls of blood vessels, eventually leading to their firm adhesion and transmigration through the endothelium into the tissue5. Better understanding of the rolling process for candidate...

One of the major challenges in the successful clinical translation of cell-based therapy is the inefficient delivery or targeting of systemically infused cells to desired sites3,4. Consequently, there is a constant search for approaches to improve cell homing, and specifically cell rolling, as a strategy to improve cell therapy. Cell rolling on blood vessels is a key step in the cell homing cascade, classically defined for leukocytes that are recruited to disease sites5. This step is governed by specific interactions between endothelial selectins, i.e. P-and E-selectin (P-and E-sel), and their counter ligands on the surface of leukocytes5,6. Better understanding and improved efficiency of cell homing, and specifically the rolling step, are of great importance in the quest for new platforms to improve cell-based therapy. To date this has been achieved by using parallel plate flow chambers (PPFCs), comprising two flat plates with a gasket between them, with an inflow and outflow port located on the upper plate, through which a cell suspension is perfused by using a syringe pump7,8 ,9. The surface of the bottom plate can be coated with a relevant cell monolayer/substrates and the interaction between perfused cells and the surface under shear flow is then explored7. However, PPFC is a low throughput, reagent-consuming, and fairly tedious method, with bubble formation, leakage, and poorly controlled flow presenting major drawbacks.
An alternative technique to the traditional PPFC is a multi-well plate microfluidic system, permitting higher throughput performance of cellular assays (up to 10 times higher than PPFCs) under accurate, computer-controlled shear flow, with low reagent consumption1,10. Cell rolling experiments are performed inside the microfluidic channels, which can be coated with cell monolayers or engineered substrates and imaged using a microscope, with rolling properties readily analyzed using a suitable software. In this study, we demonstrate the capabilities of this multi-well plate microfluidic system by studying the rolling properties of human promyelocytic leukemia (HL-60) cells on different surfaces. HL-60 rolling on substrates like P-and E-sel, as well as on cell monolayers expressing different rolling receptors, was analyzed. In addition, antibody (Ab) blocking was used to demonstrate direct involvement of specific selectins in mediating the rolling movement of HL-60 on those surfaces. Rolling experiments were performed with increased throughput, under stable shear flow, with minimal reagent/cell consumption, allowing efficient analysis of key rolling parameters such as rolling velocity, number of rolling cells, and rolling path properties.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Human Lung Microvascular Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2527</td>
			<td></td>
		</tr>
		<tr>
			<td>P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P)</td>
			<td>Kind gift by Dr. Barbara Furie11,12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 Cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture Reagents</td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 Media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium Supplements</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 MV SingleQuots</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM - Iscove&#39;s Modified Dulbecco&#39;s Medium 1x</td>
			<td>Gibco</td>
			<td>12440</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12 (1x) Nutrient Mixture (Ham)</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (L/G) 200 mM</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>Sa550</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>BD Falcon</td>
			<td>BD-353003</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml polypropylene conical tubes)</td>
			<td>MedSupply Partners</td>
			<td>TC1500</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Solution (2%)</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS (without calcium chloride and magnesium chloride)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution (10x)</td>
			<td>Sigma</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Anti-hE-Selectin/CD62E</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-hP-Selectin</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG&#173;1 &#954; Isotype Control</td>
			<td>BD Bioscience</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SLeX /CD15s Ab, Clone: 5F18</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgM-FITC Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC2859</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD162, Clone: KPL-1</td>
			<td>BD Pharmingen</td>
			<td>556055</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG1 k Isotype Control</td>
			<td>BD Pharmingen</td>
			<td>550617</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-P-Selectin Ab (AK4)</td>
			<td>Santa Cruz</td>
			<td>SC19996</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E-Selectin Ab, Clone P2H3</td>
			<td>Millipore</td>
			<td>MAB2150</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC3877</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Human P-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>137-PS-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human E-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>724-ES-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Human, Plasma</td>
			<td>Invitrogen</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Bioflux 1000</td>
			<td>Fluxion Biosciences</td>
			<td>Bioflux Montage was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFlux 48-well plates</td>
			<td>Fluxion Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow Cytometer</td>
			<td>BD Bioscience</td>
			<td>CFlow Plus was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-S</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSnap HQ2 CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

HL-60 cells roll on P- and E-selectin surfaces, but not on fibronectin
HL-60 cells are considered gold standard &#34;rollers&#34; as they express a variety of homing ligands, including the rolling ligands P-sel glycoprotein ligand-1 (PSGL-1) and Sialyl-Lewis X (SLeX)5,14 (Figure 1A). The surface protein PSGL-1 acts as a scaffold for the tetra-saccharide SLeX, mediating specific interaction with P- and E-sel, which are up-regulated on the endothelium during inflammation...

Watch this Scientific Journal Video about Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System at JoVE.com

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.

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 ...

systematic-analysis-vitro-cell-rolling-using-multi-well-plate

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Bioengineering

11.9K Views.  Brigham and Women's Hospital. 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.

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

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 ...

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

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 ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

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 ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...