Umbilical cords were collected with the assistance of the Birmingham Women's Health Care NHS Trust. HMM was supported by an Arthritis Research UK Career Development Fellowship (19899) and Systems Science for Health, University of Birmingham (5212).

<ol>
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N., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+the+beginning+programs+the+end.">Resolution of inflammation: the beginning programs the end.</a> Nature immunology. 6 (12), 1191-1197 (2005).</li><li>Schmidt, S., Moser, M., Sperandio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+leukocyte+recruitment+and+its+deficiencies.">The molecular basis of leukocyte recruitment and its deficiencies.</a> Molecular immunology. 55, 49-58 (2013).</li><li>Luu, N. T., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Ability+of+Exogenous+Chemotactic+Agents+to+Disrupt+Transendothelial+Migration+of+Flowing+Neutrophils.">Differential Ability of Exogenous Chemotactic Agents to Disrupt Transendothelial Migration of Flowing Neutrophils.</a> The Journal of Immunology. 164 (11), 5961-5969 (2000).</li><li>Smith, C. W., Rothlein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+an+endothelial+determinant+for+CD+18-dependent+human+neutrophil+adherence+and+transendothelial+migration.">Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration.</a> The Journal of clinical investigation. 82 (5), 1745-1756 (1988).</li><li>Luscinskas, F. W., Brock, A. F., Arnaout, M. A., Gimbrone, M. 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The authors declare that they have no conflicts of interest.

Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils by inflamed endothelium. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We have previously observed a dose-dependent increase in neutrophil adhesion to and transmigration through TNF&#945;-stimulated EC9,29. We also describe how both models can be adapted to study the effects of stromal ce...

Inflammation is a protective response to microbial infection or tissue injury that requires tight regulation of leukocyte entry into and exit from the inflamed tissue to allow resolution1,2. Cross-talk between endothelial cells (EC) that line blood vessels, circulating leukocytes and tissue-resident stromal cells is essential for coordinating this process3. However, uncontrolled recruitment of leukocytes and their ineffective clearance underpin the development of chronic inflammatory diseases4. Our current understanding of leukocyte recruitment in health and disease is incomplete and more robust models are needed to analyze this process.
The mechanisms supporting the recruitment of leukocytes from blood through vascular EC in post-capillary venules have been well described1,2,5. Circulating leukocytes are captured by specialized receptors (e.g., VCAM-1, E-selectin, P-selectin) which are up-regulated on inflamed endothelium. These transient interactions allow leukocytes to interact with surface bound chemokines and lipid-derived mediators (either endothelial or stromal in origin) that activate integrins expressed by leukocytes6-11. This in turn stabilizes adhesion and drives migration across the endothelium and into the tissue12-15. Within tissue, recruited leukocytes are subjected to stromal-derived agents that influence their motility, function and survival16,17. Growing evidence strongly suggests that signals received at each stage of the recruitment process conditions leukocytes for the next. However, our understanding of leukocyte recruitment remains incomplete and very little is known about the components shaping leukocyte movement within tissue.
In Birmingham we have developed several in vitro &#8220;vascular&#8221; models to study the recruitment of leukocytes from flow9,18,19. We now understand that vascular EC act as immediate regulators of leukocyte recruitment responding to changes in their local microenvironment. Specifically, tissue-resident stromal cells can actively regulate the inflammatory response, in part by conversing with neighboring vascular EC to influence their role in recruitment3. We have previously shown that various stromal cells modulate the ability of EC to support adhesion and migration of leukocytes in a tissue-specific manner, and that these effects become altered in chronic diseases13,16,20,21. Thus, stromal cells establish tissue &#8216;address-codes&#8217; that define the context of each inflammatory response22. More recently, we have demonstrated that bone-marrow derived MSC (BMMSC) potently down-regulate the response of EC to cytokines, leading to a reduction in the recruitment of both neutrophils and lymphocytes23.
The mechanisms governing recruitment elucidated in vitro have largely used assays incorporating a single cell type (e.g., EC) or protein in isolation. However, these studies do not take into consideration the effects of the local tissue environment (i.e., the presence of stromal cells) on recruitment of leukocytes and their subsequent migration into the tissue. Here we describe two flow-based methods in which stromal cells, specifically mesenchymal stem cells (MSC), are co-cultured with EC23. Such models allow us to examine the effect of stromal cell on endothelial responses, in particular their ability to support leukocyte recruitment from flow.

<table><tbody><tr><td>Collagenase Type Ia</td><td>Sigma</td><td>C2674</td><td>Dilute in 10 ml PBS to get a final concentration of 10 mg/ml. Store at -20 &amp;deg;C in 1 ml aliquots.</td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma</td><td>D8662</td><td>With calcium and magnesium chloride. Keep sterile and store at RT.</td></tr><tr><td>1X Medium M199</td><td>Gibco</td><td>31150-022</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Gentamicin sulphate</td><td>Sigma</td><td>G1397</td><td>Store at 4 &amp;deg;C. Add to M199 500 ml bottle.</td></tr><tr><td>Human epidermal growth factor</td><td>Sigma</td><td>E9644</td><td>Store at -20 &amp;deg;C in 10 &amp;micro;l aliquots.</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Sigma</td><td>F9665</td><td>FCS must be batch tested to ensure the growth and viability of isolated EC. Heat inactivate at 56 &amp;deg;C. Store in 10 ml aliquots at -20 &amp;deg;C.</td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290-026</td><td>Potent and becomes toxic within a week so fresh complete HUVEC medium must be made up every week. Store at -20 &amp;deg;C in 1 ml aliquots.</td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0135</td><td>Stock is in ethanol. Store at -20 &amp;deg;C in 10 &amp;micro;l aliquots.</td></tr><tr><td>Collagenase Type II</td><td>Sigma</td><td>C6885</td><td>Dilute stock in PBS to a final concentration of 100 mg/ml. Store at -20 &amp;deg;C in 100 &amp;micro;l aliquots.</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H3631</td><td>Dilute stock in PBS to a final concentration of 20,000 U/ml. Store at -20 &amp;deg;C in 100 &amp;micro;l aliquots.</td></tr><tr><td>100 &amp;micro;m cell strainer for 50 ml centifuge tube</td><td>Scientific Lab Supplies (SLS)</td><td>352360</td><td>Other commercially available cell strainers (e.g. Greiner bio-one) can also be used.</td></tr><tr><td>DMEM low glucose</td><td>Biosera</td><td>LM-D1102/500</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Penicillin/Streptomycin mix</td><td>Sigma</td><td>P4333</td><td>Store at -20 &amp;deg;C in 1 ml aliquots.</td></tr><tr><td>25 cc tissue culture flask</td><td>SLS</td><td>353109</td><td></td></tr><tr><td>75 cc tissue culture flask</td><td>SLS</td><td>353136</td><td></td></tr><tr><td>Bone marrow mesenchymal stem cells vial</td><td>Lonza</td><td>PT-2501</td><td>Store in liquid nitrogen upon arrival. Cells are at passage 2 upon arrival but are designated passage 0. Exapand to passage 3 and store in liquid nitrogen for later use.</td></tr><tr><td>Mesenchymal stem cell growth medium (MSCGM)</td><td>Lonza</td><td>PT-3001</td><td>Warm in 37 &amp;deg;C water bath before use. For Cell Tracker Green staining use medium without FCS.</td></tr><tr><td>EDTA (0.02%) solution</td><td>Sigma</td><td>E8008</td><td>Store at 4 &amp;deg;C. Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Trypsin solution</td><td>Sigma</td><td>T4424</td><td>Store at -20 &amp;deg;C in 2 ml aliquots. Thaw at RT and use immediately.</td></tr><tr><td>Cryovials</td><td>Greiner bio-one</td><td>2019-02</td><td>Keep on ice before adding before adding cell suspension.</td></tr><tr><td>Mr. Frosty Freezing Container</td><td>Nalgene</td><td>5100-0001</td><td>Store at RT. When adding cryovials with cells store at -80 &amp;deg;C for 24 hr before transfering cells to liquid nitrogen.</td></tr><tr><td>Ibidi u-Slide VI (0.4), T/C treated, sterile</td><td>Ibidi</td><td>IB-80606</td><td>Alternative models include glass capillaries, Cellix Biochips (www.cellixltd.com), BioFlux Plates (www.fluxionbio.com/bioflux/) and GlycoTech parallel plate flow chambers (http://www.glycotech.com/apparatus/parallel.html).</td></tr><tr><td>Cell tracker green dye</td><td>Life technologies</td><td>C2925</td><td>Store in 5 &amp;micro;l aliquots at -20 &amp;deg;C. Dilute in 5 ml prewarmed (at 37 &amp;deg;C) MSCGM.</td></tr><tr><td>Cell counting chambers</td><td>Nexcelom</td><td>SD-100</td><td>Alternatively a haemocytometer can be used.</td></tr><tr><td>Cellometer auto T4 cell counter</td><td>Nexcelom</td><td>Auto T4-203-0238</td><td></td></tr><tr><td>Tumor necrosis factor &amp;alpha; (TNF&amp;alpha;)</td><td>R&amp;amp;D Systems</td><td>210-TA-100</td><td>Dilute stock in PBS to a final concentration of 100,000 U/ml. Store at -80 &amp;deg;C in 10 &amp;micro;l aliquots.</td></tr><tr><td>6-well, 0.4 &amp;micro;m PET Transwell filters</td><td>SLS</td><td>353090</td><td></td></tr><tr><td>K2-EDTA in 10ml tubes</td><td>Sarstedt</td><td></td><td>Store at RT.</td></tr><tr><td>Histopaque 1119</td><td>Sigma</td><td>11191</td><td>Store at 4 &amp;deg;C. Warm to RT before use.</td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>10771</td><td>Store at 4 &amp;deg;C. Warm to RT before use.</td></tr><tr><td>10 ml round bottomed tube</td><td>Appleton Woods</td><td>SC211 142 AS</td><td></td></tr><tr><td>7.5% BSA Fraction V solution</td><td>Life technologies</td><td>15260-037</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>20 ml Plastipak syringes</td><td>BD falcon</td><td>300613</td><td></td></tr><tr><td>5 ml Plastipak syringes</td><td>BD falcon</td><td>302187</td><td></td></tr><tr><td>2 ml Plastipak syringes</td><td>BD falcon</td><td>300185</td><td></td></tr><tr><td>3M hypo-allergenic surgical tape 9 m x 2.5 cm</td><td>Micropore</td><td>1530-1</td><td>Use to secure the syringe tap onto the wall of the perspex chamber.</td></tr><tr><td>Silicon rubber tubing, internal diameter/external diameter (ID/OD) of 1/3 mm (thin tubing)</td><td>Fisher Scientific</td><td>FB68854</td><td>Cut silicon tubing to the appropriate size. All tubing leading directly to the electronic microvalve must be thin.</td></tr><tr><td>Silicon rubber tubing ID/OD of 2/4 mm (thick tubing)</td><td>Fisher Scientific</td><td>FB68855</td><td></td></tr><tr><td>Portex Blue Line Manometer tubing</td><td>Smiths</td><td>200/495/200</td><td>Tubing leading to the syringe pump.</td></tr><tr><td>3-way stopcock</td><td>BOC Ohmeda AB</td><td></td><td></td></tr><tr><td>Glass 50 ml syringe for pump</td><td>Popper Micromate</td><td>550962</td><td>Must be primed prior to use by removing any air bubbles.</td></tr><tr><td>Glass coverslip</td><td>Raymond A Lamb</td><td></td><td>26 x 76 mm coverslips made to order. Lot number 2440980.</td></tr><tr><td>Parafilm gasket</td><td>American National Can Company</td><td></td><td>Cut a 26 x 76 mm piece of parafilm using an aluminium template and cut a 20 x 4 mm slot into it using a scalpel 10a. Gasket thickness is approximately 133 &amp;micro;m.</td></tr><tr><td>Two perspex parallel plates</td><td>Wolfson Applied Technology Laboratory</td><td></td><td>Specially designed chamber consisting of parallel plates held together by 8 screws. The lower plate has a viewing slot cut out in the middle and a shallow recess milled to allow space for the coverslip, filter and gasket. The upper perspex plate has an inlet and outlet hole positioned over the flow channel.</td></tr><tr><td>Electronic 3-way microvalve with min. dead space</td><td>Lee Products Ltd.</td><td>LFYA1226032H</td><td>Electronically connected to a 12 volt DC power supply.</td></tr><tr><td>Syringe pump for infusion/withdrawal (PHD2000)</td><td>Harvard Apparatus</td><td>70-2001</td><td>Set the diameter to 29 mm and refill (flow) rate.</td></tr><tr><td>L-shaped connector</td><td>Labhut</td><td>LE876</td><td>To attach to the inlet and outlet ports onto the Ibidi microslide channel.</td></tr><tr><td>Video camera</td><td>Qimaging</td><td>01-QIC-F-M-12-C</td><td>Connected to a computer which enables digitall videos to be recorded.</td></tr><tr><td>Image-Pro Plus 7.0</td><td>Media Cybernetics</td><td>41N70000-61592</td><td>For data analysis. Manually tag cells displaying the different behaviors. Track cells for analysis of rolling and migration velocities.</td></tr><tr><td colspan="4">Refer to product datasheets for details on hazards of using the reagents described here.</td></tr></tbody></table>

analyzing the effects of stromal cells on the recruitment of leukocytes from flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

Initially, we analyzed the effect of stimulating EC with TNF&#945; on the recruitment of neutrophils from flow using the Ibidi microslide model (Section 7 - 9). In the absence of TNF&#945;, little if any neutrophils adhered to the endothelial monolayer (Figure 2A). This was expected, as untreated/resting EC do not express the necessary adhesion molecules (selectins) or chemokines to support binding25,26. In contrast, cytokine-stimulation significantly increased neutrophil adhe...

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Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

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Research

JoVE Journal

Immunology and Infection

10.2K Views.  University of Birmingham.  Het vermogen van ontstoken endotheel om leukocyten werven van stroom wordt geregeld door mesenchymale stromale cellen. We beschrijven twee in vitro modellen die primaire humane cellen die kunnen worden gebruikt om neutrofielen werving beoordelen van stroom en onderzoekt de rol van mesenchymale stromale cellen spelen in het reguleren van dit proces. 

Video: Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Developmental Biology

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 ...

Bioengineering

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Medicine

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

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