This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

Patient informed consent as approved by the institutional review board must be obtained for use of human cells.
1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
<ol>
	<li>Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette.
	<ol>
		<li>Dilute cells with 25 ml of phosphate-buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting the tube, very slowly and carefully add in 25 ml of the diluted cell suspension over the density gradient so that there is no mixing of the whole blood with the density gradient, i.e., no disturbance of the blood-density gradient interface.</li>
	<li>Centrifuge the suspension from step 1.2 at 400 &#215; g for 30 to 40 min in a temperature-controlled swinging-bucket rotor without brake at 20 &#176;C. 
	Note: Three distinct layers should be apparent after centrifugation: the upper layer being plasma; the bottom clear layer being the Ficoll-Paque density gradient; and a thin, middle cellular layer being the PBMCs</li>
	<li>Aspirate and discard the upper layer by suction with a Pasteur pipette, with care not to disturb the interphase layer of mononuclear cells (i.e., lymphocytes, monocytes, and platelets).</li>
	<li>Carefully collect this mononuclear cell layer with a 10 ml pipette to a new 50 ml centrifuge tube.</li>
	<li>Fill the tube with 20 ml of PBS, mix well, and centrifuge at 300 &#215; g for 10 min at 20&#176;C. Remove the supernatant completely after centrifugation.</li>
	<li>Resuspend the cell pellet in 20&#160;ml of PBS and centrifuge at 200 &#215; g for 10-15 min at 20 &#176;C. Remove the supernatant completely after centrifugation. 
	Note: This step removes the platelets-which are unwanted-within the PBMCs; this step can be repeated to ensure complete removal of the platelets.</li>
	<li>If no selection of specific populations is desired, resuspend cell pellet in leukocyte complete medium (10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin in RPMI-1640 medium) for culturing after performing a cell count16 to suspend 1 ml of medium per 107 PBMCs before proceeding to the next step.</li>
</ol>
2. Magnetic Labeling of Leukocyte Populations (Skip to STEP 4 if no Selection of Specific Populations is Required)
<ol>
	<li>Centrifuge PBMC suspension at 300 &#215; g for 10 min and aspirate supernatant completely.</li>
	<li>Resuspend cell pellet in 80 &#181;l of PBS per 107 total cells.</li>
	<li>Add 20 &#181;l of specified magnetic beads (i.e., CD14 magnetic beads for isolation of monocytes, CD4 magnetic beads for isolation of T lymphocytes) per 107 total cells.</li>
	<li>Mix well and incubate for 15 min at 2 to 8 &#176;C in the refrigerator.</li>
	<li>Add 1-2 ml of PBS per 107 cells to wash, and centrifuge at 300 &#215; g for 10 min.</li>
	<li>Aspirate supernatant completely and resuspend up to 108 cells in 500 &#181;l of PBS.</li>
</ol>
3. Magnetic Selection of Leukocyte Subpopulations
Note: A variety of magnetic bead separators and columns are available for isolation of specific population of leukocytes by cell surface marker from a number of manufacturers. Isolation can be by positive selection based on one or a few positive markers or by negative selection after depletion of unwanted populations. A general approach of performing positive selection using one marker (i.e.,&#160;CD4 for T helper lymphocytes, or CD14 for monocytes) is outlined below.
<ol>
	<li>Prepare and prime magnetic separator including separation column according to manufacturer&#39;s instructions.</li>
	<li>Apply tube containing the sample of labeled PBMCs. Provide two 15 ml centrifuge tubes for collection of the positively labeled and unlabeled cell fractions, following manufacturer&#39;s instructions.</li>
	<li>Count cell number15 and resuspend the positively selected leukocytes (i.e.,&#160;CD4+ T lymphocytes or CD14+ cells) in leukocyte complete medium, using 1 ml of medium per 107 cells.</li>
</ol>
4. CFSE Staining of Leukocytes for Assessment of Proliferation
Note: CFSE has been widely used in immunological investigations, for both in vivo and in vitro studies. Our protocol has been optimized for in vitro study of MSC/PBMC (or other leukocyte) interactions. While the general steps involved in conducting CFSE in vitro labeling are similar, there may be differences in the specific dose and timing of the protocol14. This may be due to a number of factors, i.e., the specific cell type used, cell numbers used-which can likely affect the intensity of the CFSE fluorescent signal.
<ol>
	<li>Dilute the CFSE stock solution (10 mM) in PBS to the desired working concentration of 10 &#181;M (CFSE-working solution). Prepare 107 cells (i.e., PBMCs or T cells) for labeling with the CFSE-working solution.</li>
	<li>Centrifuge at 300 &#215; g for 10 min to obtain a cell pellet and aspirate the supernatant.</li>
	<li>Resuspend the cells gently in 1 ml of pre-warmed (37 &#176;C) CFSE-working solution and incubate the cells for 10 min at 37 &#176;C.</li>
	<li>To wash off excess CFSE, dilute the cell suspension with 10x (by volume) of precooled (4 &#176;C) RPMI medium containing 10% FBS. Sediment the cells by centrifugation at 300 x g for 5 min and discard the supernatant. Wash the cell pellet in this manner twice more.</li>
	<li>Re-pellet the cells by centrifugation and count cell number15. Resuspend 107 cells (either PBMCs or T cells) in 1 ml fresh prewarmed leukocyte complete medium.</li>
</ol>
5. Co-culture of MSCs with Leukocytes and Activation of Leukocytes
<ol>
	<li>Pre-warm MSC complete medium (10% FBS (pretested for optimal MSC growth), 1% L-glutamine, and 1% penicillin/streptomycin in DMEM-low glucose medium) to 37 &#176;C for no more than 30 min.</li>
	<li>Seed MSCs at 50,000 cells in 1&#160;ml of MSC complete medium in 24-well plates (MSC density: 25,000 cells/cm2) for attachment O/N in a 37 &#176;C incubator, allowing for the stem cells to reach 80% confluence.</li>
	<li>Aspirate medium, and based on seeded MSC numbers, add CFSE-labeled PBMCs (previously labeled-please refer to protocol Step 4 above) to cultured MSCs (seeded the previous day in 24-well plates) in 1&#160;ml of leukocyte complete medium at a 1:10 (cell ratio) co-culture ratio of MSCs to PBMCs.</li>
	<li>Add the mitogen phytohemagglutinin (PHA), a non-specific leukocyte activator, to a final concentration of 10 &#181;g/ml in a total volume of 1 ml leukocyte complete medium per well.
	<ol>
		<li>Alternatively, to stimulate for the activation of T lymphocytes specifically: use &#945;-CD3/28 microbeads and add to the two-cell co-culture to obtain a bead-to-T lymphocyte ratio of 1:1.</li>
	</ol>
	</li>
	<li>For negative control, plate 500,000 PBMCs/well (or a specific effector leukocyte population; cell density: 250,000 leukocytes/cm2) in a 24-well plate with 1 ml leukocyte complete medium only; for positive control, in addition to plating the same number of PBMCs/well, add PHA to a final concentration of 10 &#181;g/ml.</li>
	<li>On the 3rd and 5th day of the co-culture experiment, assess proliferation of CFSE-labeled leukocytes (placed in round-bottom tubes) by flow cytometric analysis17 with 488 nm excitation and emission filters appropriate for fluorescein. 
	Note: Intracellular cytokine staining for flow cytometric analysis can also be performed to these CFSE-labeled leukocytes at this point to assess for changes in leukocyte cytokine expression profile as modulated by MSCs. Since CFSE is evaluated with a filter appropriate for fluorescein, the antibodies selected to assess various cytokines need to be conjugated to fluorochromes other than fluorescein or similar spectrum (i.e., phycoerythrin, peridinin chlorophyll protein complex (PerCP)).</li>
</ol>
6. Variation: Effector Suppression Assay Magnetic Bead-selected, MSC-induced Immunomodulatory Leukocytes on Activated CFSE-labeled Effector CD4 + T Cells
<ol>
	<li>Seed MSCs at 250,000 cells in 3&#160;ml of MSC complete medium in 6-well plates (MSC density: 25,000 cells/cm2) for attachment O/N in a 37 &#176;C incubator, allowing for the stem cells to reach 80% confluence. &#160;At least 3 6-well plates are necessary to ensure enough MSC-cocultured PBMCs for subpopulation selection.&#160;</li>
	<li>Aspirate medium, and add PBMCs separated as per Step 1&#160;but in 6-well plates at 2.5 x 106 cells/well (cell density: 250,000 leukocytes/cm2) with 3 ml of leukocyte complete medium. Co-culture for 48-72 hr in a 37 &#176;C incubator.</li>
	<li>Magnetic bead-select specific population of MSC-induced immunomodulatory leukocytes (i.e., CD14+ cells) as per Sections 2-3. 
	Notes: Intracellular cytokine staining for flow cytometric analysis can be performed at this point to assess for changes in leukocyte cytokine expression profile, i.e.,&#160;expression of interleukin-10-as modulated by MSCs.</li>
	<li>Add CFSE-labeled allogeneic CD4+ T cells generated as per Sections 2-4 in 24-well plates in 1&#160;ml of leukocyte complete medium (T cell density: 250,000 cells/cm2) to bead-selected MSC-induced leukocytes at various ratios, i.e., 1:10, 1:5, 1:2, and 1:1 (cell to cell) ratios.</li>
	<li>To stimulate CD4+ T cells, add &#945;-CD3/28 conjugated microbeads to obtain a bead-to-cell ratio of 1:1.</li>
	<li>For a negative control, plate 500,000 CD4+ T cells/well in a 24-well plate (T cell density: 250,000 cells/cm2) with 1 ml leukocyte complete medium only; for positive control, in addition to plating the same number of CD4+ T cells/well, add &#945;-CD3/28 conjugated microbeads to obtain a bead-to-cell ratio of 1:1.</li>
	<li>On the 3rd day of co-culture, assess proliferation of CFSE-labeled CD4+ T cells (placed in round-bottom tubes) by flow cytometric analysis16 with 488 nm excitation and emission filters appropriate for fluorescein.</li>
</ol>

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We have nothing to disclose.

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

Human mesenchymal stem cells (MSCs) are somatic progenitors that can differentiate into the paraxial mesodermal lineages of bone, cartilage, and adipose tissue1-4, as well as to a few extramesodermal lineages5. First isolated from the adult bone marrow, these multilineage progenitors have now been found in numerous tissues6-8 and, unexpectedly, shown to have strong immunomodulatory properties that appear highly amenable to clinical application9-12. Detailed mechanisms involved in the immunomodulatory effects are actively being investigated for effective application on specific disease entities. One of the most straightforward ways to evaluate immunomodulation is by assessing for the suppression of effector leukocyte proliferation13. Most effector leukocytes such as T lymphocytes and monocytes proliferate prodigiously when stimulated or activated. Immunomodulatory function can be assessed when suppression of proliferation is evidenced.
Traditionally, effector leukocyte proliferation has been evaluated by detection of [3H] thymidine incorporation into DNA. However, this method has significant drawbacks due to the concerns of radiation and post-use disposal, as well as the complex equipment needed. While there are non-radioactive assays to assess cell proliferation, the carboxyfluorescein succinimidyl ester (CFSE) assay has other advantages such as allowing for identification of specific cellular populations, which is especially useful in co-culture experiments involving multiple cell types. CFSE is a fluorescent cellular dye which can be assessed by flow cytometric analysis. As cells divide, the intensity of this cellular label is decreased proportionally; this not only enables determination of overall cell proliferation but also allows for the assessment on the number of cell divisions up to 8 divisions before the fluorescence becomes difficult to detect against background signal. Moreover, the stability of the fluorescent CFSE allows for in vivo tracking of labeled cells such that cells can be visualized up to many months14.
This assay can also be varied to evaluate specific types of effector leukocytes or the immunomodulatory function of specific populations of MSC-induced immunomodulatory leukocytes-such as interleukin-10 (IL-10) producing CD14+ monocytes15 by performing magnetic bead surface marker selection of cell populations of interest prior to or after co-culture as appropriate. Our protocol describes the basic assay of assessing the immunomodulatory effects of MSCs on effector leukocytes (flow chart shown in Figure 1) and a variation on this basic assay for evaluation of MSC-induced leukocyte immunomodulation on allogeneic CD4+ effector T lymphocytes (flow chart shown in Figure 4).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Ficoll-Paque PLUS</td>
			<td style="width:259px;">GE Healthcare</td>
			<td style="width:121px;">71-7167-00 AG</td>
			<td style="width:549px;">Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">V12883</td>
			<td>Cellular label for detection of cell division</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Phytoagglutinin (PHA)</td>
			<td style="width:259px;">Sigma</td>
			<td>L8902</td>
			<td>Activation of human PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Dynabeads Human T-Activator CD3/28</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">111.32D</td>
			<td style="width:549px;">Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">autoMACS&#8482; Separator</td>
			<td>Magnetic based cell separator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">autoMACS&#174; Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td>separation columns</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:276px;">CD14 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td style="width:549px;">For positive selection of CD14+ human monocytes and macrophages from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">130-045-101</td>
			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">RPMI 1640 Medium</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11875</td>
			<td>Human PBMC/leukocyte culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">DMEM, Low glucose, pyruvate</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11885</td>
			<td style="width:549px;">Human mesenchymal stem cell (MSC) culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">L-glutamine</td>
			<td style="width:259px;">Life Technologies</td>
			<td>25030-081</td>
			<td>Supplementation for MSC complete medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/Streptomycin</td>
			<td style="width:259px;">Life Technologies</td>
			<td>15070-063</td>
			<td>Supplementation for PBMC/leukocyte and MSC complete medium</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:276px;">Fetal bovine serum (FBS)</td>
			<td style="width:259px;">1) Hyclone, for MSC culture&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td>
			<td style="width:121px;">1) SH30070.03M 2) 10091-148</td>
			<td>Pre-test lots for support of MSC in vitro culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">24-well cell culture plate</td>
			<td style="width:259px;">Corning</td>
			<td>COR3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">50 mL centrifuge tube</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430291</td>
			<td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">15 mL centrifuge tube&#160;</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430766</td>
			<td>Collection of the labeled and unlabeled cell fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round-bottom tubes</td>
			<td style="width:259px;">BD Falcon&#160;</td>
			<td style="width:121px;">352008</td>
			<td>Collection of cells for flow cytometric analysis</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 denotes the overall schema of the experiment, and Figure 2 demonstrates the appearance of the various cell culture conditions as visualized by phase-contrast inverted microscopy. MSCs are adherent cells with a fibroblastic, spindle-shaped morphology, whereas PBMCs and leukocytes are small round non-adherent cells. These two morphologically different cell types can be clearly seen in the co-culture. At the end of the assay, when the PBMCs (or leuckotyes) are aspirated for flow cytometric analyses, ev...

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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.

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 ...

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Research

JoVE Journal

Immunology and Infection

12.1K Views.  National Health Research Institutes (NHRI). 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.

Video: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

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, ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...