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

必须使用人体细胞获得批准的机构审查委员会患者的知情同意书。 人外周血单个核细胞1.密度梯度分离（外周血单个核细胞） <ol><li>加25 ml的肝素化全血到50ml管由25毫升吸管。 <ol><li>稀释细胞用25ml磷酸盐缓冲盐水（PBS）的。 </li></ol></li><li>新增15 ml的菲可 - 帕克密度梯度到一个新的50ml管中，并在倾斜的管，非常缓慢并小心地在25毫升的稀释的细胞悬浮液在密度梯度的添加使得存在没有混合的全血与密度梯度， 即 ，无干扰的血液密度梯度的接口。 </li><li>离心从步骤1.2以400×g的30至40分钟，在温度控制的吊桶式转子无制动器在20℃的悬浮液中。 注意：在离心后三个不同的层应当是显而易见：第Ë上层存在等离子体;底部的透明层作为菲可 - 帕克密度梯度;和薄的，中间层的细胞作为PBMC中</li><li>吸液和丢弃通过抽吸上层用巴氏吸管，小心不要打扰单核细胞（ 即，淋巴细胞，单核细胞和血小板）的相间层。 </li><li>仔细收集这种单核细胞层用10毫升吸管到一个新的50ml离心管中。 </li><li>填充用20毫升的PBS管，混的不好，离心300×g的10分钟，在20°C。离心后完全除去上清液。 </li><li>重悬细胞沉淀在20ml的PBS中并在离心机200×g下在20℃下10-15分钟。离心后完全除去上清液。 注：此步骤除去血小板，这是不必要的，内部的外周血单个核细胞;这个步骤可以重复，以确保完全除去血小板。 </li><li>如果没有选择特定的人群是需要的话，在白细胞完全培养基重悬细胞沉淀（10％胎牛血清（FBS），1％L-谷氨酰胺和1％青霉素/链霉素的RPMI-1640培养基）用于培养进行细胞计数16后，暂停1毫升每10 7 PBMC的介质在进行下一步骤之前。 </li></ol>白细胞种群2.磁标记（跳至步骤4如果没有选择特定人群是必需的） <ol><li>离心机的PBMC悬浮液在300×g的10分钟，吸出上清液完全。 </li><li>悬浮细胞沉淀在80微升PBS每10 7个细胞。 </li><li>加入20微升指定磁珠（即，CD14磁珠隔离单核细胞，CD4磁珠的T淋巴细胞隔离）每10 7个细胞。 </li><li>拌匀，孵育15分钟，在2〜8℃的冰箱里。 </li><li>加1-2毫升的PBS每10 7个细胞进行冲洗，并且离心分离机在300×g的10分钟。 </li><li>吸上清完全和悬浮高达10 8个细胞在500微升PBS。 </li></ol>白细胞亚群的3磁选注意：各种磁珠分离器和列可用于白细胞的特定人口的隔离通过细胞表面标记从许多制造商。隔离可以通过基于一个或几个正标记或由意外的种群耗尽后负选择阳性选择。进行正选择使用一个标记（ 即，CD4的T辅助淋巴细胞或CD14的单核细胞）的一般方法如下所述。 <ol><li>制备和素磁性分离器包括根据制造商的说明分离柱。 </li><li>应用含有标记的PBMC的样品管中。提供两个15毫升离心管中收集的积极标记和unlabel的编细胞级分，按照生产商的说明。 </li><li>计数细胞数15，重悬在白细胞完全培养基的阳性选择的白细胞（例如，CD4 + T淋巴细胞或CD14 +细胞），使用1毫升培养基每10 7个细胞。 </li></ol>白细胞4. CFSE染色增殖的评估注意：CFSE已被广泛用于在免疫学调查，对于在体内和体外研究 。我们的协议已经优化，MSC / PBMC（或其他白细胞）相互作用的体外研究。而参与实施CFSE在体外标记的一般步骤是相似的，有可能是在协议14的特定剂量和定时差异。这可能是由于多种因素，例如，所用的特定的细胞类型，细胞数目用后可容易影响CFSE荧光信号的强度。 <ol&gt; <li>稀释CFSE库存溶液（10mM）的PBS，以10μM的（CFSE-工作溶液）所需的工作浓度。制备10 7个细胞（即，外周血细胞或T细胞），用于与所述的CFSE工作溶液标记。 </li><li>离心机在300×g的10分钟以获得细胞沉淀并吸出上清液。 </li><li>悬浮细胞轻轻在1ml预热（37℃）的CFSE工作溶液，并在37℃下孵育细胞10分钟。 </li><li>洗去过量的CFSE，（体积）的预冷（4℃）的RPMI培养基含有10％FBS稀释的细胞悬浮液具有10。通过离心沉淀细胞，在300×g离心5分钟，丢弃上清液。洗以这种方式将细胞沉淀两次。 </li><li>通过离心重新沉淀细胞并计数细胞数目15。悬浮10 7个细胞（无论的PBMC或T细胞）在1ml新鲜预热的白细胞的完全培养基。 </li></ol>的M 5，共培养种姓与白细胞和白细胞的活化<ol><li>预暖的MSC完全培养基（10％FBS（预测试以获得最佳的MSC生长），1％L-谷氨酰胺和1％青霉素/链霉素的DMEM低葡萄糖培养基）至37℃不超过30分钟。 </li><li>种子的MSC以50,000细胞在1ml的MSC完全培养基中24孔板（MSC密度：25,000个细胞/ cm 2）的用于附着O / N在37℃培养箱，允许干细胞达到80％汇合。 </li><li>吸出培养基，并根据种子的MSC号，添加CFSE标记的外周血单个核细胞（先前标记-请参阅协议上述步骤4）到培养的MSC（接种前一天在24孔平板）的1ml白细胞完全培养基中的一1时10（气泡率）MSCs向的PBMC共培养比率。 </li><li>添加分裂素植物血凝素（PHA），非特异性白细胞活化剂，以10微克/ ml的终浓度在1毫升白细胞完全培养基的总体积每孔。 <ol><li>可选择性伊利，刺激对T淋巴细胞的激活具体有：使用α-CD3 / 28微珠，并加入到二细胞共培养，以获得1珠对T淋巴细胞：1的比例。 </li></ol></li><li>为阴性对照，板500000的PBMC /孔（或特异性效应白细胞种群;细胞密度：250000白细胞/厘米2）在一个24孔板用1ml白细胞完全只介质;对于阳性对照，在除了电镀的PBMC /相同数量均匀，加入的PHA到10微克/毫升的最终浓度。 </li><li>在第三和共培养实验的第5天，评估CFSE标记的白细胞的增殖（置于圆底管）通过流式细胞仪分析17具有适用于荧光素488纳米激发和发射滤光片。 注：细胞内细胞因子染色的流式细胞术分析，也可进行对这些CFSE标记的白细胞在这一点上，以评估作为莫变化白细胞的细胞因子表达谱通过干细胞dulated。因为CFSE评价与过滤器适合于荧光素，选择的抗体，以评估各种细胞因子需要被缀合到荧光比荧光素或类似的频谱其他（即，藻红蛋白 ，多甲藻素叶绿素蛋白复合物（PerCP））。 </li></ol> 6.变异：效应抑制测定磁珠选择的，MSC诱导的免疫调节白细胞对活性CFSE标记的效应CD4 + T细胞<ol><li>种子的MSCs在250,000个细胞在3ml的MSC完全培养基中6孔板（MSC密度：25,000个细胞/ cm 2）的用于附着O / N在37℃培养箱，允许干细胞达到80％汇合。至少3 6孔板是必要的，以确保足够的MSC-共培养的PBMC为亚群的选择。 </li><li>吸出培养基，并添加的PBMC分离按步骤1，但在6孔板以2.5×10 6个细胞/孔（细胞密度：250,000勒ukocytes /厘米2）用3ml白细胞完全培养基。共培养48-72小时在37℃培养箱。 </li><li>磁珠选择MSC诱导免疫调节白细胞（ 即 CD14 +细胞）根据第2-3特定人群。 注：细胞内细胞因子染色的流式细胞术分析可以在此时被执行以评估更改中白细胞的细胞因子表达谱， 即，表达白介素-10-如由干细胞调制。 </li><li>在添加CFSE标记在24孔板产生为每第2-4节异源CD4 + T细胞 1 ml的白细胞完全培养基（T细胞密度：250000个细胞/厘米2），以珠选择的MSC诱导的白细胞在各种比例即，1：10,1：5，1：2和1：1（细胞与细胞）的比例。 </li><li>刺激CD4 + T细胞，加入α-CD3 / 28偶联的微珠，以获得1珠-细胞：1的比例。 </li><li>对于阴性对照，板500000 CD4 + T细胞/孔在24孔板（T细胞密度：250000个细胞/ cm 2）的用1毫升白细胞完全只介质;对于阳性对照，在除了电镀CD4 + T细胞的数量相同/孔，加入α-CD3 / 28偶联的微珠，以获得1珠-细胞：1的比例。 </li><li>上共培养的第 3天，通过流式细胞仪分析16具有适用于荧光素488纳米激发和发射滤光片评估CFSE标记的CD4 + T细胞（置于圆底管）的增殖。 </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...

人间充质干细胞（MSCs）是体祖细胞可以分化成骨，软骨和脂肪组织1-4的近轴中胚层谱系，以及几extramesodermal谱系5。首先从成人骨髓中分离，这些多谱系祖细胞现已发现在多种组织和6-8，出乎意料的是，显示出具有出现高度适合于临床应用9-12强免疫调节性能。参与免疫调节作用的详细机制正在积极研究对特定病种有效的应用。其中一个最简单的方式来评估免疫调节是通过评估的效应白细胞增殖13的抑制。大多数效应白细胞，如T淋巴细胞和单核细胞增生不歇刺激或激活时。免疫调节功能，可以评估时抑制增殖证明。 传统上，效应白细胞增殖进行了评价通过检测的 [3 H]胸苷掺入DNA中。然而，这种方法具有缺点显著由于辐射和后期使用处置的关注，以及所需要的复杂的设备。虽然有非放射性测定法来评估细胞增殖，所述羧基琥珀酰亚胺酯（CFSE）测定具有其它优点，如允许识别特定细胞群，这是在涉及多种细胞类型的共培养实验中特别有用的。 CFSE是荧光染料的细胞可以通过流式细胞术分析来评估。作为细胞分裂时，这种细胞标记的强度成比例地降低;这不仅使得能够确定的总的细胞增殖，但也允许对细胞分裂的高达8分割的数目评估前的荧光变得难以DET等针对背景信号。此外，该荧光CFSE的稳定性允许在体内跟踪，使得细胞可以可视化长达很多个月14标记的细胞。 该测定也可以改变，以评估特定类型的效应子白细胞或特定人群的免疫调节功能的MSC诱导的免疫调节白细胞，如白介素-10（IL-10）产生的CD14 +单核细胞 15通过执行的磁珠表面标记选择之前或共培养适当的利息后的细胞群。我们的协议描述评估对效应白细胞MSCs的免疫调节作用（ 图 1中所示的流程图）的基本测定，并在此基础测定法对异源CD4 +效应 T淋巴细胞的MSC诱导的白细胞免疫调节评价的变型（图中所示的流程图在图4中）。

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			<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>
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		<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>
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			<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>
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		<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>
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			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
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			<td>Magnetic based cell separator</td>
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			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
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			<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>
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			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
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			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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		<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>
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			<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>
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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.

图1表示了实验的总体架构，而图2展示了可视化作为用相差倒置显微镜的各种细胞培养条件的外观。的MSC是贴壁细胞与成纤维细胞，纺锤形的形态，而PBMC和白细胞小圆非贴壁细胞。这两个形态不同的细胞类型，可以清楚地看出，在共培养。在测定结束时，当PBMC中（或leuckotyes）吸出流式细胞仪分析，即使粘附的MSCs无意中包含（即，由于附接不良或移位通过剧烈抽吸），应?...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

人类骨髓间充质干细胞（MSC）的免疫调节属性显示越来越重要的临床应用。用MSC和外周血白细胞的共同培养系统的预染色的荧光染料羧基琥珀酰亚胺酯（CFSE），我们描述对效应白细胞增殖和特定亚群的体外评估的MSC免疫调节的。

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

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

assessment-immunomodulatory-properties-human-mesenchymal-stem-cells

Research

JoVE Journal

Immunology and Infection

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

12.1K 次观看.  National Health Research Institutes (NHRI). 人类骨髓间充质干细胞（MSC）的免疫调节属性显示越来越重要的临床应用。用MSC和外周血白细胞的共同培养系统的预染色的荧光染料羧基琥珀酰亚胺酯（CFSE），我们描述对效应白细胞增殖和特定亚群的体外评估的MSC免疫调节的。 

视频: 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.

人外周血NK细胞的扩展，纯化和功能评估

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

科研

免疫与感染

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.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估：一个细胞的分离纯化方法的新颖使用

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.

PRP作为一种新的方法，以防止感染的制备及<em在体外</em&gt;抗菌性能的PRP

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.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

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.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

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.

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

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.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

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.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

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.

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

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.

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

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 ...