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

기관 심사위원회가 승인 한 환자 동의서는 인간 세포의 사용을 취득해야합니다. 사람 말초 혈액 단핵 세포의 1. 밀도 구배 분리 (PBMC를) <ol><li> 25 mL를 피펫으로 50 ㎖ 튜브에 전체 혈액을 헤파린 25 ML을 추가합니다. <ol><li> 인산 완충 식염수 (PBS) 25 ㎖로 희석하여 세포. </li></ol></li><li> 새로운 50 ㎖ 튜브에 피콜 - 페이크 밀도 구배의 15 mL를 넣고 튜브 틸팅 동안 전혈의 믹싱으로 존재하지 않도록 매우 조심스럽게 천천히는 밀도 구배 위에 희석 세포 현탁액 25 ㎖에 추가 밀도 구배, 즉., 혈액 - 밀도 구배 인터페이스의 방해없이. </li><li> 원심 분리기 20 ° C에서 브레이크없이 30 온도 조절 스윙 버킷 로터에서 분 40 400 × g에서 1.2 단계에서 정지. 참고 : 세 가지 층은 원심 분리 후 명백하다 : 일즉 상층 존재 플라즈마; 바닥 클리어 층은 피콜 - 페이크 밀도 구배되며; 및 얇은 층은 중간 셀룰러 PBMC를 되 </li><li> 기음과는 단핵 세포 (즉, 림프구, 단핵구, 혈소판)의 계면 층을 방해하지 않도록주의로, 파스퇴르 피펫으로 흡입하여 상층을 버린다. </li><li> 조심스럽게 새로운 50 ㎖ 원심 분리 튜브에 10 ㎖ 피펫이 단핵 세포층을 수집한다. </li><li> , PBS의 20 ㎖와 튜브를 채우 잘 혼합하고, 20 ℃에서 10 분 동안 300 × g에서 원심 분리기. 원심 분리 후 완전히 뜨는을 제거합니다. </li><li> 20 ℃에서 10 ~ 15 분 동안 200 × g에서 세포 PBS 20ml에 펠렛을 재현 탁하고 원심 분리기. 원심 분리 후 완전히 뜨는을 제거합니다. 참고 :이 단계는 혈소판 - 원치 않는-내에서 PBMC를하다을 제거; 이 단계는 혈소판의 완전한 제거를 보장하기 위해 반복 될 수있다. </li><li> 특정 집단의 선택 범위가 없으면세포 수 (16)를 수행 한 후, 배양을 위해 바람직한, 백혈구 완전 배지에 재현 탁 된 세포 펠릿 (10 % 소 태아 혈청 (FBS), 1 % L- 글루타민, 1 % 페니실린 / RPMI-1640 배지에서 스트렙토 마이신)을 1 ml의 중단을 다음 단계로 진행하기 전에 10 7 PBMC를 당 매체. </li></ol> 백혈구 인구 2. 자기 라벨링 (특정 집단의 어떤 선택이 필요없는 경우 4 단계로 건너 뜁니다) <ol><li> 완전히 10 분, 흡인 상층 액 300 × g에서 원심 분리기 PBMC 서스펜션. </li><li> 10 7 총 세포 당 PBS의 80 μL에 재현 탁 세포 펠렛. </li><li> 10 7 총 세포 당 (T 림프구의 분리를위한 단핵 세포, CD4 자석 구슬의 격리를 위해 즉, CD14 자석 구슬) 지정된 자석 구슬의 20 μl를 추가합니다. </li><li> 잘 섞어 냉장고에 8 ° C에 2에서 15 분 동안 품어. </li><li> 씻어 10 7 세포 당 PBS의 1-2 ML을 추가하고, 원심 분리기10 분 동안 300 × g에서. </li><li> 대기음 상층 액을 완전히 PBS 500 μL에 10 8 셀을 재현 탁. </li></ol> 백혈구의 부분 집단 3. 자기 선택 참고 : 자기 비드 구분과 열의 다양한 제조 업체의 수와 세포 표면 마커에 의한 백혈구의 특정 인구의 분리에 사용할 수 있습니다. 격리 하나 또는 몇 가지 긍정적 인 마커 또는 원치 않는 인구의 고갈 후 음의 선택에 기반한 선택에 의해 할 수있다. (즉, 단핵구에 대한 CD4 T 헬퍼 림프구에 대한, 또는 CD14) 한 마커를 사용하여 긍정적 인 선택을 수행하는 일반적인 방법은 아래에 설명되어 있습니다. <ol><li> 준비 및 주요 마그네틱 분리기는 제조업체의 지침에 따라 분리 컬럼을 포함. </li><li> 표시 PBMC를의 샘플이 포함 튜브를 적용합니다. 긍정적으로 표지 및 unlabel의 수집이 15 ml의 원심 분리기 튜브를 제공합니다제조업체의 지침에 따라 에드 셀 분수,. </li><li> 107 세포 당 1 ml의 배지를 이용하여 세포 수를 카운트하고 15 백혈구 완전 배지에서 긍정적으로 선택된 백혈구 (예, CD4 + T 림프구 또는 CD14 + 세포)를 재현 탁. </li></ol> 대량 살상 무기 확산의 평가를위한 백혈구 4. CFSE 얼룩 주 : CFSE 널리 생체 내 및 시험 관내 연구에서 모두, 면역 학적 조사에 사용되었다. 우리의 프로토콜은 MSC / PBMC (또는 다른 백혈구)의 상호 작용의 체외 연구를 위해 최적화되었습니다. CFSE 체외 라벨을 수행에 관련된 일반적인 단계는 유사하지만, 프로토콜 (14)의 특정 용량과 타이밍의 차이가있을 수 있습니다. 이 때문에 다수의 인자, 즉, 사용 된 특정 세포 유형, 세포 수로 사용할 수 있고-있는 가능성 CFSE 형광 신호의 강도에 영향을 미칠 수있다. <ol&gt; <li> 10 μm의 (CFSE - 작업 솔루션)의 원하는 작업 농도로 PBS에서 CFSE 주식 솔루션 (10 mm)를 희석. CFSE-작동 용액으로 라벨링 10 7 세포 (즉, PBMC를 또는 T 세포)를 준비한다. </li><li> 10 분 동안 300 × g에서 원심 분리기는 세포 펠렛을 얻고 뜨는을 대기음합니다. </li><li> 부드럽게 예열 (37 ° C) CFSE-작동 용액 1 ㎖로 세포를 재현 탁하고, 37 ℃에서 10 분 동안 세포를 배양한다. </li><li> 과량 CFSE을 씻어, 예비 냉각 (4 ℃) RPMI 배지를 10 % FBS를 함유하는 (부피 기준)으로 10 배의 세포 현탁액을 희석. 5 분 300 XG에서 원심 분리하여 세포 침전물과 상층 액을 버린다. 두 번 더 이런 방식으로 세포 펠렛을 씻으십시오. </li><li> 원심 분리하여 세포를 다시 펠렛 세포 수 (15)를 계산. 1 ㎖의 백혈구 데워진 신선한 완전 배지에서 10 7 세포 (PBMC를 또는 T 세포를) 재현 탁. </li></ol> M 5. 공동 문화백혈구 및 백혈구의 활성화와 희주 <ol><li> 이하에서 30 분 동안 37 ° C까지 미리 따뜻한 MSC 완전 배지 (최적 MSC 성장을위한 테스트 된 10 % FBS (), 1 % L- 글루타민, 1 % 페니실린 / DMEM 낮은 포도당 배지에서 스트렙토 마이신). </li><li> 24 웰 플레이트에서 MSC 완전 배지 1 ㎖에 50,000 세포 (MSC 밀도 : 25,000 세포 / ㎠)에서 시드 엽 줄기 37 ° C의 배양기에 부착 O / N에 대한, 줄기 세포 수는 80 % 합류에 도달한다. </li><li> 시드 MSC 번호를 기반으로 대기음 매체와는 CFSE 표지 PBMC를 추가 (이전에 표시된 제발 위의 프로토콜 4 단계 참조)에 백혈구 완전 배지 1 ㎖에 (24 웰 플레이트에 전날 시드) 배양 된 중간 엽 줄기 세포에 1시 10분 (셀 비율)로 PBMC를 엽 줄기 세포의 공동 배양 비율. </li><li> 1 ㎖의 백혈구 완전 배지의 총 부피에서 10 μg의 / ml의 최종 농도로, 미토 겐의 피토 헤 마글 루티 닌 (PHA), 비특이적 백혈구 활성화 제를 추가 웰당. <ol><li> Alternativ엘리는, 구체적으로 T 림프구의 활성화를 자극하기 위해 : α-CD3 / 28 마이크로 비드를 사용하여 (1)의 비드 간 T 림프구의 비율을 얻기 위해 두 세포 공동 배양에 추가 1. </li></ol></li><li> 50 PBMC를 / 웰 음성 대조군, 플레이트 (; 세포 밀도 또는 특정 이펙터 백혈구 인구 : 25 만 백혈구 / ㎠)의 경우 만 1 ㎖의 백혈구 완전 배지와 24 웰 플레이트에; 양성 대조군을 위해, 추가로, PBMC를 웰 / 동일한 수의 도금을 10 μg / ML의 최종 농도로 PHA를 추가한다. </li><li> 3와 공동 배양 실험 5 일에, 형광에 적합한 488 nm의 여기 및 방출 필터와 유세포 분석 (17)에 의해 CFSE 표지 백혈구의 증식 (둥근 바닥 튜브에 배치)을 평가한다. 주 : 유세포 분석을 위해 세포 내 사이토 카인 염색은 또한 MO 같은 백혈구 사이토 카인 발현 양상의 변화를 평가하기 위해이 시점에서 이러한 CFSE로 표지 백혈구를 위해 수행 될 수있다중간 엽 줄기 세포에 의해 dulated. CFSE이 형광 용 필터에 적절한 평가되기 때문에, 항체는 각종 사이토 카인이 형광 또는 유사한 스펙트럼 이외의 형광 색소에 접합 될 필요가 평가하도록 선택 (즉, 피코 에리 트린, 엽록소 단백질 복합체 (를 PerCP) peridinin). </li></ol> 6. 변화 : 이펙터 억제 분석 마그네틱 활성화 CFSE 표지 이펙터 CD4 + T 세포에 면역 백혈구를 MSC 유도, 비드 선택 <ol><li> 6 웰 플레이트에 MSC 전체 매체의 3 ㎖에 25 만 세포 (MSC 밀도 : 25,000 세포 / ㎠)에서 종자 중간 엽 줄기 세포 37 ° C 배양기에 부착 O / N를 들어, 줄기 세포 수는 80 %의 합류에 도달합니다. 적어도 3 개의 6- 웰 플레이트 모집단 선택 충분한 MSC-PBMC를 공 배양을 보장하기 위해 필요하다. </li><li> 대기음 매체는, 및 1 단계에 따라하지만 / 웰 (세포 밀도 2.5 × 10 6 세포에서 6 웰 플레이트에서 분리 PBMC를 추가 : 250,000 르를백혈구 전체 매체의 3 ㎖와 ukocytes / ㎠). 37 ° C 배양기에서 48 ~ 72 시간 동안 공동 문화. </li><li> 자기 섹션 2-3에 따라 MSC에 의한 면역 백혈구 (즉, CD14 + 세포)의 특정 인구 비드-선택합니다. 주 : 유세포 분석을 위해 세포 내 사이토 카인 염색은 백혈구 사이토 카인의 발현 프로필, 즉, 발현의 변화를 평가하기 위해이 시점에서 수행 될 수있다 인터루킨 10와 같은 MSC들에 의해 변조. </li><li> 24 웰 플레이트 섹션 2-4에 따라 생성 된 동종 CD4 + T 세포를 CFSE 표지 된 추가 한 백혈구 완전 배지 (T 세포 밀도 250,000 세포 / ㎠) ml의 다양한 비율로 비드 선택된 MSC 유도 백혈구에 즉, 1:10, 1 : 5, 1 : 2, 1 : 1 비율 (셀로 셀). </li><li> CD4 + T 세포를 자극하기 위해, (1)의 비드 대 세포 비율을 구하는 α-CD3은 / 28 공액 마이크로 비드를 추가 1. </li><li> 음성 대조군은 5 판00,000 CD4 + T 세포 / 웰로 24- 웰 플레이트 (T 세포 밀도 250,000 세포 / ㎠)에 단 1 ㎖의 백혈구 완전 배지와; 양성 대조군을 위해, 추가로 (1)의 비드 대 세포 비율을 구하는 α-CD3은 / 공액 마이크로 비드 (28)를 추가 / 웰의 CD4 + T 세포의 수를 동일한 도금 : 1이다. </li><li> 공동 배양 3 일째에, 플루오 레세 적합한 488 nm의 여기 및 방사 필터를 사용하여 유세포 분석 (16)에 의해 (둥근 바닥 튜브에 배치) CFSE 표지 된 CD4 + T 세포의 증식을 평가. </li></ol>

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C., Lawrence, D. A., Reed, D. J., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNIT+18.8+Immune+cell+phenotyping+using+flow+cytometry.">UNIT 18.8 Immune cell phenotyping using flow cytometry.</a> Curr Protoc Toxicol. , 18.8.1-18.8.24 (2005).</li><li>Phelan, M. C., Lawler, G., Robinson, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APPENDIX+3A+Cell+counting.">APPENDIX 3A Cell counting.</a> Curr Protoc Cytom. , A.3A.1-A.3A.4 (2001).</li><li>Gebler, A., Zabel, O., Seliger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunomodulatory+capacity+of+mesenchymal+stem+cells.">The immunomodulatory capacity of mesenchymal stem cells.</a> Trends Mol Med. 18, 128-134 (2012).</li><li>Le Blanc, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+treatment+of+steroid-resistant,+severe,+acute+graft-versus-host+disease:+a+phase+II+study.">Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.</a> Lancet. 371, 1579-1586 (2008).</li><li>Tan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+therapy+with+autologous+mesenchymal+stem+cells+in+living-related+kidney+transplants:+a+randomized+controlled+trial.">Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial.</a> JAMA. 307, 1169-1177 (2012).</li><li>Le Blanc, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+inhibit+and+stimulate+mixed+lymphocyte+cultures+and+mitogenic+responses+independently+of+the+major+histocompatibility+complex.">Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.</a> Scand J Immunol. 57, 11-20 (2003).</li><li>Li, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+in+vitro+impairs+the+immunosuppressive+activity+of+mesenchymal+stem+cells+on+T+cells.">Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells.</a> Mol Med Rep. 6, 1183-1189 (2012).</li><li>Moll, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+cryopreserved+mesenchymal+stromal+cells+display+impaired+immunomodulatory+and+therapeutic+properties?.">Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?.</a> Stem Cells. 32, 2430-2442 (2012).</li><li>Ho, P. J., Yen, M. L., Tang, B. C., Chen, C. T., Yen, B. 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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...

인간 중간 엽 줄기 세포 (중간 엽 줄기 세포는) 몇 extramesodermal 계통 5뿐만 아니라, 뼈, 연골 및 지방 조직 1-4의 근축 중배엽 계통으로 분화 할 수 체세포 조상이다. 먼저 성인의 골수에서 분리,이 multilineage 조상은 지금 임상 응용 프로그램 9-12에 매우 의무 표시 강한 면역 특성을 보여, 예기치 않게, 많은 조직 6-8에서 발견되었다. 면역 효과에 관련된 자세한 메커니즘은 적극적으로 특정 질병의 실체에 효과적인 적용을 위해 연구되고있다. 면역을 평가하는 가장 간단한 방법 중 하나는 이펙터 백혈구 증식 (13)의 억제에 대해 평가하는 것이다. 자극 또는 활성화되면 이러한 T 림프구 및 단핵구와 같은 대부분의 이펙터 백혈구는 비범 증식. 면역 기능이 평가 될 수있는 경우의 진압확산은 입증된다. 전통적으로, 이펙터 백혈구 증식 DNA에 [3 H] 티미 딘의 결합 검출에 의해 평가되었다. 그러나,이 방법은 복사 현상 및 후 처리의 이용에 상당한 관심 단점뿐만 아니라, 필요한 복잡한 장비를 갖는다. 세포 증식을 평가하기위한 비 방사능 분석이 있지만, 카르복시 숙신 이미 딜 에스터 (CFSE) 분석은 여러 유형의 세포를 포함하는 공동 배양 실험에 특히 유용하다 특정 세포 개체수의 식별을 허용하는 다른 이점을 갖는다. CFSE은 유세포 분석에 의해 평가 될 수 셀룰러 형광 염료이다. 세포 분열,이 휴대 라벨의 강도는 비례 적으로 감소된다; 이뿐만 아니라 전반적인 세​​포 증식의 판정을 가능하게 할뿐만 아니라 형광 DET 곤란해진다 전에 8 분할 최대 세포 분열 횟수에 평가를 허용요법 배경 신호에 대하여. 또한, 형광 CFSE의 안정성은 많은 세포가 14 개월까지 가시화 될 수있는 표지 된 세포를 생체 내 추적을 허용한다. 이 분석은 또한 이펙터 백혈구 또는 특정 집단의 면역 기능의 특정 유형을 평가하기 위해 변화 될 수의 자성 비드 표면 마커의 선택을 행함으로써 면역 백혈구 - 인터루킨 10 (IL-10)의 제조 CD14 + 단핵구 15를 MSC를 유발 이전에 또는 적절한 공동 배양 한 후 관심의 세포 집단. 우리 프로토콜 (흐름도가 도시는 (플로우 차트는도 1에 도시) 이펙터 백혈구에서 중간 엽 줄기 세포의 면역 조절 효과를 평가의 기본적인 분석 및 동종 CD4 + 이펙터 T 림프구에 MSC 유도 백혈구 면역 평가를위한 기본적인 분석의 변형을 설명 그림 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.

도 1은 실험의 전반적인 스키마를 나타내고,도 2는 위상차 도립 현미경으로 가시화로서 다양한 세포 배양 조건의 모양을 보여준다. PBMC를하고 백혈구가 작은 원형 비 부착 세포 반면 중간 엽 줄기 세포는 섬유 모세포, 스핀들 모양의 형태와 부착 세포이다. 이 두 형태 학적으로 상이한 세포 유형은 명확 공존 배양에서 볼 수있다. 시험의 끝에, PBMC를 (또는 leuckotyes)를 유세포위한 흡인 될 ?...

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

인간 중간 엽 줄기 세포 (MSC)의 면역 특성은 임상 응용 프로그램에 대해 점점 더 관련이 나타납니다. 형광 염료 카르복시 숙신 이미 딜 에스터 (CFSE)로 미리 염색 엽 줄기 세포 및 말초 혈 백혈구의 공존 배양 시스템을 사용하여, 우리는 이펙터 백혈구 증식 및 특정 부분 집단에 MSC의 면역의 시험 관내 평가를 설명한다.

인간 중간 엽 줄기 세포의 면역 속성 (MSCS)의 평가

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

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JoVE Journal

Immunology and Infection

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

12.1K 조회수.  National Health Research Institutes (NHRI).  인간 중간 엽 줄기 세포 (MSC)의 면역 특성은 임상 응용 프로그램에 대해 점점 더 관련이 나타납니다. 형광 염료 카르복시 숙신 이미 딜 에스터 (CFSE)로 미리 염색 엽 줄기 세포 및 말초 혈 백혈구의 공존 배양 시스템을 사용하여, 우리는 이펙터 백혈구 증식 및 특정 부분 집단에 MSC의 면역의 시험 관내 평가를 설명한다. 

비디오: 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.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

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

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면역학 및 감염병학

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.

유동세포계측법에 의해 피해를 입은 척수 조직에서 면역 세포의 양적 평가 : 세포 정제 방법에 대한 소설을 사용

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.

준비를 한 다음 감염을 방지하기 위해 새로운 접근 방식으로 PRP<em&gt; 체외에서</emPRP의&gt; 항균 속성

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.

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

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 Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

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.

의 검출을위한 자석 교반기 방법<em&gt; 선모충</em근육 샘플에서&gt; 애벌레

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.

인간 NK 세포 활동의 평가 대 한 흐름 Cytometry 기반 세포 독성 분석 실험

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.

세포 독성의 평가 인간의 정밀 컷 폐 분할 영역에서 물질의 Immunomodulatory 효과

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.

말 초 혈액 및 림프 조직에서 기본 인간 T 세포의 시 냅 스 인터페이스의 평가

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.

인간 줄기 세포 유래 Ileal 장용 단층에서 M 세포 유사 세포의 분화 유도

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 ...