Research has been supported by the Wallace H. Coulter Foundation Early Career Award in Translational Research. Thanks to Tim Andrews, Pediatric Hematology/Oncology, for help with blood sample collection. Also thanks to Dr. Sam Wellhausen, Flow Cytometry Core, James Graham Brown Cancer Center, for help with running flow cytometry samples.

Part 1. Microfluidic device fabrication
A silicon master mold of the microfluidic lysis device was created using standard photolithographic techniques with the equipment in the University of Louisville cleanroom [1]. AutoCAD (Autodesk, Inc., San Rafael, CA) was used to generate a transparency mask (Fineline Imaging, Colorado Springs, CO) to create negative replicas of the channels. The microfluidic lysis device was fabricated from the silicon master mold using soft lithographic techniques with the elastomer poly(dimethylsiloxane) (PDMS; Dow Corning, Midland, MI). The elastomer with the replicated channels was released, and channel access holes were punched with a 20-gauge needle (Small Parts, Miramar, FL.). The PDMS wafer was irreversibly bonded to a glass slide via oxygen plasma and tested for experimentation. 
Part 2. Protocol for running lysis device
After the microfluidic device has been fabricated and tested, experiments with erythrocyte lysis are ready to begin. The following section details the protocol for the microfluidic device.
2.1 Sample Collection
<ol>
 <li>Collect 4 milliliters of blood sample from median cubital vein, on the anterior forearm vein of patient with heparin as anticoagulant in two 2 ml green top vaccutainers.</li>
 <li>Discard first 2 ml tube as it contains dislodged mature endothelial cells (false positives).</li>
 <li>Resuspend the second tube to mix heparin with blood by turning up and down; save immediately on ice.</li>
 <li>Transfer 1 ml of blood to a 1.5 ml Eppendorf tube. Process the blood sample immediately (within one hour of collection).</li>
</ol>
2.2 Prime microfluidic device with PBS
<ol>
 <li>Obtain a microfluidic device bonded to glass. Press-fit access tubing (Small Parts, Miramar, FL) of slightly larger diameter in the inlets and outlet (Figure 1).</li>
 <li>Attach a 30-gauge syringe needle (Small Parts, Miramar, FL) to the tubing on each of the inlets, providing the macro to micro interface.</li>
 <li>Fill a 1 ml syringe with 1X PBS and make sure to get rid of bubbles by flicking the syringe needle. Connect the PBS-loaded 1 ml syringe to inlet 1 on the microfluidic device. Push the syringe containing the 1X PBS gently by hand until the solution flows out of inlet 2, and then immediately clamp inlet 2 with a standard office binder clip</li>
 <li>Continue pushing the 1X PBS until fluid reaches the outlet port of the microfluidics device. Once the solution flows out of the outlet, clamp the outlet tubing with another binder clip.</li>
 <li>Continue pushing the 1 ml syringe until the 1X PBS flows out of inlet 3, and clamp the inlet 3 port with another office binder clip. The microfluidics device is now fully primed.</li>
</ol>
<img src="/files/ftp_upload/1656/1656fig1.jpg" alt="Figure 1" /> Figure 1. Schematic of device showing inlets for respective solutions and outlet.
2.3 Preparation of syringe pumps and solution
<ol>
 <li>To calibrate the pumps follow these steps:
 <ol class="lalpha">
 <li>For the large Harvard syringe pump (Harvard, PHD 22/2000 Part # 702000): Set the diameter configuration to 22.5 mm, flow rate 600 &mu;l/min. </li>
 <li>For the small Harvard Syringe pump (Harvard, Pico Plus Part # 702213): Set the diameter configuration to 4.61 mm, flow rate 20 &mu;l/min. </li>
 </ol>
 </li>
 <li>Fill one 30 ml syringe with sterile deionized water. This can be accomplished by withdrawing the solution directly from the 50 ml conical tube. (Label the syringe)</li>
 <li>Fill a second 30 ml syringe with 2X phosphate-buffered-saline (PBS), 2% paraformaldehyde (PFA) using the procedure outlined in step 9. (Label the syringe)</li>
 <li>Fill a 1 ml syringe with 1X PBS from the aliquots stored in the 15 ml conical tubes, using the procedure outlined in step 9.</li>
 <li>Connect the 30 ml deionized water syringe to inlet 2 and the 30 ml 2X PBS syringe to inlet 3 (make sure you don t trap bubbles in the tubing or microfluidics device). Remove the clamp from inlets 2 and 3 and the outlet tubing. Connect the 1X PBS 1 ml syringe into inlet 1. Avoid introducing bubbles by filling the syringe needle with liquid, connecting the syringe, and flicking the syringe needle to rid of bubbles.</li>
</ol>
2.4 Erythrocyte lysis
<ol>
 <li>Set all of the syringes on the proper Harvard pumps as outlined below:
 <ol class="lalpha">
 <li>Set the two 30 ml syringes (one containing sterile, de-ionized water and the other with 2X PBS, 2% PFA) together on the large Harvard syringe pump.</li>
 <li>Set the 1 ml syringe (containing 1X PBS) on the small Harvard syringe pump. </li>
 <li>Place the outlet tubing in a waste collector (50 ml conical tube that is labeled waste ). </li>
 </ol>
 </li>
 <li>Turn on the large Harvard syringe pump (with the 30 ml syringes) and let it run for one minute. (Make sure there are no bubbles and no leakage in the device or in the tubing).</li>
 <li>Turn on the small Harvard syringe pump and let it run for an additional minute. (Make sure there are no bubbles and no leakage in the device or in the tubing). Stop the pumps. The microfluidics device is now ready to be used.</li>
 <li>Remove the 1 ml syringe containing 1X PBS from the small Harvard syringe pump.</li>
 <li>After obtaining a blood sample, fill the 1 ml syringe with 0.05 ml of sterile 1X PBS without trapping any bubbles. Next, fill the syringe with 0.5 ml of blood obtained from the Eppendorf tube. (The 1 ml syringe will now contain a final volume of 0.55 ml of the blood and 1X PBS buffer.) Note: Keep the syringe vertical while filling to avoid mixing of the 0.05 ml PBS with the blood. </li>
 <li>Connect the syringe containing the blood to inlet 1 and mount the syringe carefully (avoid pushing the blood through the tubing into the device) on the small Harvard syringe pump (the syringe is mounted vertical into the pump). </li>
 <li>Remove the outlet tubing from the waste tube and put a clean 50 ml centrifuge tube in its place and set it on a bucket of ice. </li>
 <li>Switch on the large Harvard syringe pump first and let it run for 1 minute. Start collecting the sample from the outlet into the 50 ml centrifuge tube. </li>
 <li>Switch on the small Harvard syringe pump.</li>
 <li>Once the blood sample in the 1 ml syringe has completely traveled through the device, stop both pumps. This should take approximately 20 minutes.</li>
 <li>Centrifuge the collected sample for 5 minutes at 350 x g at room temperature with the brake off.</li>
 <li>Remove supernatant by placing pipette tip at the opposite side of the white pellet. Remove as much supernatant as possible, especially red cell debris, without disturbing the white pellet.</li>
 <li>Resuspend the sample in 1 ml of flow buffer (1X PBS, 1% BSA [bovine serum albumin], 2% PFA) by pipetting up and down. Transfer sample to a 1.5 ml Eppendorf tube.</li>
 <li>Microcentrifuge the collected sample for 5 minutes at 4 x g at room temperature. Use same precision as before to remove the supernatant.</li>
 <li>Resuspend the sample in 1 ml of flow buffer by vortexing.</li>
</ol>
2.5 Preparation for flow cytometric analysis
<ol>
 <li>For sample analysis using flow cytometry, add 100 &mu;l of sample to a flow cytometry tube.</li>
 <li>To the 100 &mu;l of sample add specified antibody (ensure correct concentration).</li>
 <li>Allow sample to incubate for 30 minutes at 40 C. </li>
 <li>Wash twice with flow buffer prior to flow cytometry. Wash step includes adding 250 &mu;l of flow buffer, vortexing, centrifuging for 5 minutes at 350 x g, and removing supernatant with one fluid turn of flow cytometry tube. </li>
 <li>Resuspend the pellet in 250 &mu;l of flow buffer. Sample is ready for analysis by flow cytometry.</li>
</ol>
Representative Results
Whole blood has been run through the microfluidic lysis device and post-processing protocol, ridding of erythrocytes and enriching circulating nucleated cells (CNCs). Ideal results will yield a clean flow cytometry scatter plot with separate CNC populations. Displayed below in Figures 2 and 3 are scatter plot comparisons of a whole blood sample without erythrocyte lysis and with erythrocyte lysis by the microfluidic protocol with axes as forward scatter (FSC) versus side scatter (SSC) light. It can be seen that the microfluidic enrichment protocol is necessary to obtain a clean flow cytometry scatter plot. 
<img src="/files/ftp_upload/1656/1656fig2.jpg" alt="Figure 2" /> Figure 2. Flow cytometry scatter plot of whole blood sample without erythrocyte lysis with FSC and SSC axes.
<img src="/files/ftp_upload/1656/1656fig3.jpg" alt="Figure 3" /> Figure 3. Flow cytometry scatter plot of circulating nucleated cells with microfluidic erythrocyte lysis labeled by populations with FSC and SSC axes. Region 1 (red) represents lymphocytes, Region 2 (green) represents monocytes, Region 3 (blue) represents granulocytes, and Region 4 (purple) has yet to be characterized.
Removal of red cell debris by pipette in the protocol is also important when necessary. Below in Figure 4 is a scatter plot showing a surplus of red cell debris. Although not clouding the scatter plot as in Figure 2, one must account for the added events due to erythrocyte debris during data analysis.
<img src="/files/ftp_upload/1656/1656fig4.jpg" alt="Figure 4" /> Figure 4. Flow cytometry scatter plot depicting red cell debris, seen as a cluster of events in Region 5 (cyan).
Staining cells for certain antibody phenotype markers will also generate characteristic results. In the following Figures are antibody phenotype stains for 3 distinct leukocyte populations: lymphocytes, monocytes, and granulocytes. Figure 5 depicts lymphocyte populations plotted with axes CD3 and CD4. Monocytes and granulocytes are shown in Figures 6-7 with axes as FSC versus CD14 and CD66b, respectively.
<img src="/files/ftp_upload/1656/1656fig5.jpg" alt="Figure 5" /> Figure 5. Flow cytometry scatter plot depicting lymphocytes with CD3 and CD4 axes.
<img src="/files/ftp_upload/1656/1656fig6.jpg" alt="Figure 6" /> Figure 6. Flow cytometry scatter plot depicting monocytes with FSC and CD14 axes.
<img src="/files/ftp_upload/1656/1656fig7.jpg" alt="Figure 7" /> Figure 7. Flow cytometry scatter plot depicting granulocytes with FSC and CD66b axes.

<ol>
	<li>White, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+microfluidic+leukocyte+enrichment+protocol+in+mild+phenotype+sickle+cell+disease+(SCD.">Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD.</a> Biomedical Microdevices. 11 (2), 477-483 (2009).</li><li>Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Flow+Microfluidic+Device+for+Rapid+Erythrocyte+Lysis.">Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis.</a> Anal. Chem. 76 (21), 6247-6253 (2004).</li></ol>

An automated microfluidic blood lysis protocol has been reported. Within the protocol are critical steps worth noting. In section P.2.1, it is important to discard the first blood vial collected as the sample contains dislodged mature endothelial cells acting as false positives. Also make sure to run the blood sample within an hour of collection. Priming the microfluidic device in section P.2.2, it is important to initially rid of the bubbles. Additionally, one should avoid introducing bubbles when attaching new syringes to the needles throughout the protocol. When filling the 1 ml syringe with blood in section P.2.4, one must remember to first add 1X PBS and the blood, all while keeping the syringe vertical to avoid mixture of the two solutions. Before starting the syringe pump pushing the blood sample, ensure the large syringe pump has been running so the system is at full speed. After the sample has finished running through the device and been centrifuged, carefully remove as much supernatant as possible, including red cell debris, without disturbing the white pellet. Lastly, in section P.2.5 follow the manufacturer s protocol to assure the correct concentration of antibody is added to the 100 &mu;L sample.
Applications of the microfluidic protocol are immense in clinical research. Interrogating the immune status of an individual in terms of CNCs generates important information on condition of health and may help with diagnosis and understanding of disease pathogenesis. Accessing these CNC populations through blood sample collection is easier and more convenient than human expression analyses involving tumor tissues. To examine these nucleated cells in circulation one must be able to enrich the populations from other blood components, including erythrocytes, the primary function of the reported device. Hence, such studies would benefit greatly from the microfluidic blood lysis protocol.
Significance of the microfluidic protocol emerges in terms of applications. Because nucleated cell enrichment is required in clinical studies of blood, a protocol requiring little expertise and user-mediated steps that produces clear and consistent results is important. The microfluidic protocol ensures consistency through automation and controlled exposure of cells to harsh environments. With uniformity among clinical laboratories data will be directly comparable [2].

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Base</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Curing Agent</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>30-gauge syringe needle</td><td>Small Parts, Inc.</td><td>NE-301PL-25</td><td></td></tr><tr><td>Tygon tubing (.010&quot; ID)</td><td>Small Parts, Inc.</td><td>TGY-010</td><td></td></tr><tr><td>20-gauge syringe needle</td><td>Small Parts, Inc.</td><td>NE-201PL-25</td><td></td></tr><tr><td>Syringe Pump (Harvard PHD 22/2000)</td><td>Harvard Apparatus</td><td>702000</td><td></td></tr><tr><td>Syringe Pump (Harvard Pico Plus)</td><td>Harvard Apparatus</td><td>702213</td><td></td></tr><tr><td>Flow cytometry tubes</td><td>DB Biosciences</td><td>352052</td><td></td></tr><tr><td>Antibody stains</td><td>DB Biosciences</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Fisher Scientific</td><td>05-402-25</td><td></td></tr><tr><td>50 ml tube</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>1 ml syringe</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>30 ml tube</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>10X PBS</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

automated microfluidic blood lysis protocol for enrichment of circulating nucleated cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

Watch this Scientific Journal Video about Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells at JoVE.com

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

automated-microfluidic-blood-lysis-protocol-for-enrichment

Nanyang Technological University

Research

JoVE Journal

Biology

12.3K Views.  University of Louisville. An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

Video: Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Cancer Research

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Immunology and Infection

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

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Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics