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

Kurumsal inceleme kurulu tarafından onaylanan Hasta onam insan hücrelerinin kullanımı için alınması gerekir. İnsan Periferal Kan Tek Çekirdekli Hücreleri 1. yoğunluk gradyan izolasyonu (PBMC) <ol><li> 25 ml&#39;lik bir pipetle 50 ml&#39;lik bir tüp içine tam kan heparinize 25 ml ilave edilir. <ol><li> Fosfat tamponlu tuz (PBS), 25 ml hücreleri seyreltilir. </li></ol></li><li> Yeni 50 ml&#39;lik bir tüp içine Ficoll Paque yoğunluk gradyanı 15 ml ilave edilir, ve tüpü eğerken, tam kan herhangi bir karıştırma ile olduğu şekilde çok yavaş ve dikkatli bir şekilde yoğunluk gradyanı içinde seyreltilmiş hücre süspansiyonu, 25 ml eklemek yoğunluk gradyan, yani., kan-yoğunluk gradyan arayüzünün hiçbir rahatsızlık. </li><li> Santrifüj 20 ° C &#39;de fren olmayan 30 sıcaklık kontrollü bir sallanma-kepçeli rotor içinde 40 dakika için 400 x g&#39;de aşama 1,2 süspansiyon. Not: üç farklı tabakalar, santrifüj işleminden sonra belirgin olmalıdır: the üst katman varlık plazma; alt berrak tabaka Ficoll Paque yoğunluk gradyan olmak; ve ince, orta hücresel tabaka PBMC&#39;leri olmak </li><li> Aspire edin ve mononükleer hücrelerin (örneğin, lemfositler, monositler ve trombositler) interfaz tabakası bozmamaya dikkat sahip bir Pasteur pipeti ile emme ile üst tabaka atılır. </li><li> Dikkatli bir şekilde yeni bir 50 ml&#39;lik santrifüj tüpüne, 10 ml bir pipet ile bu tek çekirdekli hücre faz toplanır. </li><li> , 20 ml PBS ile tüp doldurun, iyice karıştırılmakta ve 20 ° C&#39;de 10 dakika boyunca 300 x g&#39;de santrifüje tabi tutun. Santrifüj sonra tamamen süpernatantı. </li><li> 20 ° C&#39;de 10-15 dakika boyunca 200 x g&#39;da hücre PBS, 20 ml pelet ve santrifüj yeniden süspanse edin. Santrifüj sonra tamamen süpernatantı. Not: Bu adım trombositler-istenmeyen-içinde PBMC&#39;ler vardır kaldırır; Bu adım, trombositlerin tam olarak çıkarılmasının sağlanması için tekrar edilebilir. </li><li> Belirli nüfusun herhangi bir seçim isebir hücre sayımı 16 gerçekleştirdikten sonra kültür için arzu edilen lökosit tam bir ortam içinde tekrar süspansiyon hücre topağı (% 10 fetal bovin serumu (FBS),% 1 L-glutamin ve% 1 penisilin / RPMI-1640 ortamı içinde streptomisin) içinde 1 ml askıya Bir sonraki adıma geçmeden önce 10 7 PBMC&#39;lerin başına ortam. </li></ol> Lökosit Populasyonlarının 2. Manyetik Etiketleme (Belirli popülasyonlarının hiçbir Seçim Gerekli ise 4. adıma atlayın) <ol><li> Tamamen 10 dakika ve aspirat süpernatant için 300 × g Santrifüj PBMC süspansiyon. </li><li> 10 7 toplam hücre başına PBS 80 ul süspanse hücre pelet. </li><li> 10 7, toplam hücre başına (T lenfositlerinin izolasyonu için monositler, CD4 manyetik boncuk izolasyonu için, yani, CD14 manyetik boncuklar) belirtilen manyetik boncuk 20 ul ekle. </li><li> İyice karıştırın ve buzdolabında 8 ° C&#39;de 2 ila 15 dakika inkübe edilir. </li><li> Yıkamak için 10 7 hücre başına PBS 1-2 ml ekleyin ve santrifüj10 dakika boyunca 300 x g&#39;de. </li><li> Süpernatant aspire tam ve 500 ul PBS içinde 10 8 hücrelere kadar yeniden süspanse edin. </li></ol> Lökosit alt popülasyonlarının 3. Manyetik Seçimi Not: manyetik boncuk ayırıcılar ve sütun çeşitli üreticileri bir dizi hücre yüzeyi işaretleyicisi tarafından lökositlerin özel nüfus izolasyonu için kullanılabilir. İzolasyon, bir veya bir kaç olumlu belirteçleri üzerine veya istenmeyen popülasyonlarının tükenmesi sonrasında negatif seleksiyon tarafından temelli pozitif seçimi ile olabilir. (Yani, monositler için CD4 T yardımcı lenfositler için veya CD14), bir işaretleyici kullanılarak pozitif seçim gerçekleştirmek genel bir yaklaşım, aşağıda özetlenmiştir. <ol><li> Hazırlama ve birinci manyetik ayırıcı üreticinin talimatlarına uygun ayırma kolonu da dahil olmak üzere. </li><li> Etiketli PBMC&#39;lerin örneğini içeren tüp uygulayın. Olumlu etiketli ve unlabel toplanması için iki adet 15 ml santrifüj tüpleri sağlayınimalatçının talimatlarına uygun olarak ed hücre fraksiyonları,. </li><li> 10 7 hücre başına ortam 1 ​​ml kullanılarak hücre sayısı 15 sayın ve lökosit tam bir ortam içinde pozitif seçilen lökosit (örneğin, CD4 + T lenfositleri veya CD14 + hücreler) yeniden süspanse edin. </li></ol> Proliferasyonunun Değerlendirme lökosit 4. KAKE Boyama Not: KAKE yaygın in vivo ve in vitro çalışmalar hem immünolojik araştırmalarda kullanılmaktadır. Bizim protokol MSC / PBMC (ya da diğer lökosit) etkileşimlerinin in vitro çalışma için optimize edilmiştir. KAKE in vitro etiketleme yürüten genel adımlar benzer olmakla birlikte, protokolün 14 özel doz ve zamanlama farklılıklar olabilir. Bu, bir dizi faktöre, örneğin kullanılan özel hücre tipi, hücre sayısı olabilir kullanılmış olan büyük olasılıkla CFSE floresan sinyalin şiddetini etkileyebilir. <ol&gt; <li> 10 uM (CFSE-çalışma çözeltisi), istenen bir çalışma yoğunluğuna PBS içinde CFSE stok solüsyonu (10 mM) ile seyreltilir. CFSE-çözelti ile etiketlenmesi için 10 7 hücreleri (örneğin, PBMC&#39;ler veya T hücreleri) hazırlayın. </li><li> 10 dakika için 300 × g Santrifüj hücre pelet elde etmek ve süpernatant aspire. </li><li> Yavaşça önceden ısıtılmış (37 ° C) CFSE-çalışma çözeltisi 1 ml hücreleri yeniden süspanse edin ve 37 ° C&#39;de 10 dakika boyunca hücreler inkübe edin. </li><li> Fazla CFSE yıkayın için, önceden soğutulmuş (4 ° C) RPMI ortamı,% 10 FBS ihtiva eden (hacim ile) 10x hücre süspansiyonu seyreltin. 5 dakika boyunca 300 xg&#39;de santrifüj hücreleri tortu ve supernatant atın. Iki kere daha bu şekilde hücre pelletini yıkayın. </li><li> Santrifüj hücreleri yeniden pelet ve hücre sayısını 15 saymak. 1 ml yeni, önceden ısıtılmış lökosit tam bir ortamda 10 7 hücreleri (PBMC&#39;ler veya T hücreleri olarak) yeniden süspanse edin. </li></ol> M 5. Co-kültürLökosit ve lökosit Aktivasyonu ile SC&#39;ler <ol><li> En fazla 30 dakika süreyle 37 ° C&#39;ye kadar ön-sıcak MSC komple ortam (optimum MSC büyümesi için önceden test% 10 FBS (),% 1 L-glutamin ve% 1 penisilin / DMEM-düşük glikoz ortamında streptomisin). </li><li> 24 oyuklu plakalar içinde MSC tam orta 1 ml 50,000 hücre (MSC yoğunluğu: 25.000 hücre / cm2) de tohum erişkin 37 ° C kuluçka makinesi içinde ek olarak O / N, kök hücre için izin% 80 konfluansa ulaşmalarına. </li><li> Tohumlanmış MSC numaraları göre orta aspire ve, CFSE etiketli PBMC ekleme (daha önce işaretlenmiş lütfen yukarıda protokolü Adım 4&#39;e bakınız), bir lökosit tam orta 1 ml (24 oyuklu plakalar içinde bir önceki güne tohumlanmış) kültürlenmiş MSC&#39;Iere 01:10 (hücre oranı) PBMC&#39;lerden için MKH ko-kültür oranı. </li><li> 1 ml lökosit tam orta toplam hacim içinde 10 ug / ml&#39;lik bir nihai konsantrasyona kadar, mitojen fitohemaglutinin (PHA), spesifik olmayan bir lökosit etkinleştirici Oyuk başına. <ol><li> Alternatively, özellikle T lenfositlerin aktivasyonu için uyarmak için: α-CD3 / 28, mikro-boncuklar kullanımı ve 1 bir kordon T&#39;ye lenfosit oranını elde etmek için, iki hücre ortak-kültürünün eklemek: 1 arasındadır. </li></ol></li><li> 500.000 PBMC / Negatif kontrol, levha (hücre yoğunluğu ya da belirli bir efektör nüfusunun lökosit: 250.000 lökosit / cm2) için, sadece 1 ml lökosit ortamla, 24 oyuklu bir plaka içerisinde, Pozitif kontrol için, ek olarak, iyi PBMC&#39;lerin / aynı sayıda kaplama için 10 ug / ml &#39;lik bir nihai konsantrasyona kadar PHA ekleyin. </li><li> 3 ve ko-kültür deney 5 gün, fluoresein için uygun 488 nm eksitasyon ve emisyon filtreleriyle akış sitometrik analizi 17 CFSE etiketli lökosit çoğalmasının (yuvarlak dipli tüplerine yerleştirilmiştir) değerlendirir. Not: Akış sitometri analizi için hücre içi sitokin boyaması da mo olarak lökosit sitokin ekspresyon profili değişiklikleri değerlendirmek için, bu noktada bu KAKE etiketli lökositlere gerçekleştirilebilirMKH tarafından dulated. KAKE fluoresein için bir filtre uygun değerlendirilmiştir olduğundan, antikorlar, çeşitli sitokinler floresein veya benzer yelpazenin dışındaki fluorochromes konjuge gereken değerlendirmek için seçilen (yani, fikoeritrin, klorofil protein kompleksinin (PerCP) peridinin). </li></ol> 6. Varyasyon: Efektör baskılama tahlili Manyetik Aktif CFSE etiketli Efektör CD4 + T hücreleri üzerinde bağışıklık ayarlayıcı Lökositler MSC ile indüklenen, Boncuk seçilen <ol><li> 6-yuvalı plakalar içinde MSC tam orta 3 ml 250,000 hücreler (MSC yoğunluğu: 25.000 hücre / cm2) de tohum erişkin 37 ° C kuluçka makinesi içinde ek olarak O / N, kök hücre için izin% 80 konfluansa ulaşmalarına. En az 3 ila 6 oyuklu plakalar alt popülasyonu seçimi için yeterli MSC-kültive PBMC&#39;ler sağlamak için gereklidir. </li><li> Kültür ortamı basınçla, ve Adım 1 &#39;deki gibi fakat / oyuk (hücre yoğunluğu 2.5 x 10 6 hücre 6 oyuklu plakalar içinde ayrılmış PBMC ekleyin: 250,000 leLökosit tam orta 3 ml ukocytes / cm2). 37 ° C kuluçka makinesi içinde 48-72 saat boyunca Co-kültür. </li><li> Manyetik Bölüm 2-3 uyarınca MSC kaynaklı immünomodülatör lökositlerin (yani, CD14 + hücreler) spesifik nüfusu boncuk seçin. Notlar: akış sitometri analizi için hücre içi sitokin boyaması lökosit sitokin ekspresyon profili, yani ekspresyonunda değişiklikler için değerlendirmek üzere bu noktada gerçekleştirilebilir interlökin-10-as MSC tarafından modüle. </li><li> 24 oyuklu plakalar içinde Bölüm 2-4 uyarınca üretilen allojenik CD4 + T hücrelerinin CFSE etiketli ekle 1 lökosit komple ortam (T hücre yoğunluğu: 250.000 hücre / cm2) arasında mi çeşitli oranlarda Boncuk seçilen MSC kaynaklı lökosit örneğin, 1:10, 1: 5, 1: 2 ve 1: 1 oranları (hücre hücre). </li><li> CD4 + T hücreleri teşvik etmek, 1 bir kordon-hücre oranı elde etmek α-CD3 / 28 konjuge mikroboncukları ekleyin: 1. </li><li> Negatif kontrol için, 5 plaka00.000 CD4 + T hücre / oyuk, 24 gözenekli bir plaka (T hücre yoğunluğu: 250.000 hücre / cm2) &#39;de, sadece 1 ml lökosit tam orta; Pozitif kontrol için, ek olarak 1, bir kordon-hücre oranını elde etmek için α-CD3 / 28 konjuge mikroboncukları eklemek / oyuk, CD4 + T hücreleri aynı sayıda kaplama: 1 arasındadır. </li><li> Eş-kültür 3. gününde, fluoresein için uygun 488 nm eksitasyon ve emisyon filtreleriyle akış sitometrik analizi ile 16 (yuvarlak tabanlı tüp içine yerleştirilmişlerdir) CFSE etiketli CD4 + T hücrelerinin proliferasyonunu değerlendirmek. </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...

İnsan mezenkimal kök hücreler (MKH) bir kaç extramesodermal soy 5 yanı sıra, kemik, kıkırdak ve yağ dokusunda 1-4 paraxial mezodermal soy içine ayırt edebilirsiniz somatik atalarıdır. İlk erişkin kemik iliğinden izole, bu mültilineaj atalarıdır artık klinik uygulama 9-12 için son derece müsait görünen güçlü bağışıklık düzenleyici özelliklere sahip oldukları gösterilmiştir, beklenmedik, çok sayıda dokularda 6-8 bulunabilir ve oylandı. Immünomodülatör etkileri katılan Detaylı mekanizmalar aktif spesifik hastalık varlıklar üzerinde etkili uygulama için araştırılmaktadır. Immünmodülasyonu değerlendirmek için en basit yollarından biri efektör lökosit çoğalması 13 bastırılması için değerlendirilmesi gereğidir. Uyarılmış ya da aktive olduğunda örneğin T lenfositler ve monositler gibi çoğu efektör lökositler müthiş eğlenir çoğalırlar. İmmünomodülatuar fonksiyonu değerlendirilebilir zaman bastırmaçoğalması kanıtlanmıştır. Geleneksel olarak, efektör lökosit proliferasyonu DNA içine [3H] timidin eklenmesi saptanması ile değerlendirilmiştir. Bununla birlikte, bu yöntem, radyasyon ve kullanım sonrası imha endişelerine önemli dezavantajları, hem de gereken karmaşık bir ekipmana sahiptir. Hücre çoğalmasını değerlendirmek için radyoaktif olmayan tahliller olmakla birlikte, carboxyfluorescein süksinimidil ester (CFSE) deneyi gibi birçok hücre tipini içeren ko-kültür deneyleri özellikle yararlı olan, özel hücresel popülasyonlar, tanımlanması için izin verilmesi gibi başka avantajlara da sahiptir. CFSE, akış sitometrik analizi ile tespit edilebilir bir flüoresan hücre boyadır. Hücre bölünmeleri gibi, bu hücresel etiketin yoğunluğu orantılı azalır; Bu sadece genel hücre çoğalmasının tespitine imkan değil, aynı zamanda floresan det zorlaşır önce 8 tümen kadar hücre bölünmeleri sayısına değerlendirilmesi için izin verirEKT arka plan sinyali karşı. Ayrıca, flüoresan CFSE stabilitesi hücreleri aylarca 14 kadar görselleştirilebilir şekilde etiketlenmiş hücrelerin in vivo izlenmesi için izin verir. Bu deney de efektör lökosit veya spesifik popülasyonlarının bağışıklık fonksiyonunun belirli türde değerlendirmek için değişik olabilir ve manyetik boncuk yüzey işaretleyici seçimi gerçekleştirerek bağışıklık lökosit-interlökin-10 (IL-10) üretilmesi, CD14 + monositleri 15 olarak MSC-kaynaklı önce veya uygun bir şekilde eş-kültür sonrası ilgi hücre popülasyonları. Bizim protokol (akış şeması gösterilmiştir (akış şeması Şekil 1 &#39;de gösterildiği gibi) efektör lökositler üzerinde MSC immünomodülatör etkileri belirlemek temel deneyi ve allojenik CD4 + efektör T lemfositlerinin MSC-uyarılan lökosit immünomodülasyon değerlendirilmesi için bu temel tahlilinde bir değişik biçimi tarif edilmiştir Şekil 4&#39;te).

<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>
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		<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.

Şekil 1, deney genel şema gösterir, ve Şekil 2, faz-kontrast ters mikroskobu ile gösterildiği gibi çeşitli hücre kültürü koşullarının görünümünü göstermektedir. PBMC&#39;ler ve lökositlerin küçük yuvarlak yapışmayan hücreler iken erişkin bir fibroblastik, iğ şeklinde morfolojiye sahip yapışmayan hücreler bulunmaktadır. Bu iki morfolojik olarak farklı hücre tipleri açık bir şekilde ko-kültür görülebilir. Testin sonunda, PBMC&#39;ler (veya leuckotyes) Akış sitomet...

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

İnsan mezenkimal kök hücrelerin (MSC) immünomodülatör özellikleri, klinik uygulama için giderek daha önemli görünmektedir. Floresan boya carboxyfluorescein süksinimidil ester (CFSE) ile ön-lekeli MSC ve periferal kan lökositlerinin bir ko-kültür sistemi kullanılarak, efektör lökosit çoğalmasının ve spesifik alt popülasyonları MSC immünomodülasyon in vitro değerlendirme tarif eder.

İnsan Mezenkimal Kök Hücre İmmünomodüler Properties (MKH) değerlendirilmesi

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 Görüntüleme.  National Health Research Institutes (NHRI).  İnsan mezenkimal kök hücrelerin (MSC) immünomodülatör özellikleri, klinik uygulama için giderek daha önemli görünmektedir. Floresan boya carboxyfluorescein süksinimidil ester (CFSE) ile ön-lekeli MSC ve periferal kan lökositlerinin bir ko-kültür sistemi kullanılarak, efektör lökosit çoğalmasının ve spesifik alt popülasyonları MSC immünomodülasyon in vitro değerlendirme tarif eder. 

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.

Genişleme, Saflaştırılması, ve İnsan Periferik Kan NK Hücrelerinin Fonksiyonel Değerlendirme

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

İmmünoloji ve Enfeksiyon

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.

Akış Sitometri Yaralı Omurilik Doku Bağışıklık Hücreleri Kantitatif Değerlendirilmesi: Hücre Arıtma Yöntemi Roman kullanın

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.

Hazırlama ve: Enfeksiyon Önleme Yeni Bir Yaklaşım olarak PRP<em&gt; In vitro</emPRP&gt; Antimikrobiyal Özellikleri

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.

Otoimmünite karşı Kök Hücre türetilmiş antijen-özgü düzenleyici T hücrelerinin gelişimini

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.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

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.

Tespiti için Manyetik Karıştırıcı Yöntemi<em&gt; Trichinella</emKas Samples&gt; Larva

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.

Akış Sitometresi tabanlı sitotoksisite tahlil insan NK hücre etkinliği değerlendirilmesi için

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.

Sitotoksik değerlendirilmesi ve insan hassas kesim akciğer dilimleri maddelerin etkileri Immunomodulatory

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.

Periferik kan ve lenf dokusu birincil insan T hücre sinaptik arabirimi değerlendirilmesi

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.

Insan kök hücresinde M hücre benzeri hücrelerin kaynaklı farklılaşma-türev ıleal 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 ...