작품은 보건 국립 연구소 (R01GM079688, R21RR024439 및 R21GM075838), 국립 과학 재단 (CBET 0,941,055) 및 MSU 재단 일부 지원되었다.

1. 쥐 MSCS의 분리 Mesenchymal 줄기 세포는 이전에 다음 ID로 한 설명된대로 6-8주 오래된 스프 라그 - 돌리 좋아하지, 2 고립되었다. 절연 MSCS 플라스틱 표면을 준수하고 쉽게 체외 문화 중에 확장할 수 있습니다. <ol><li> 동물은 마취 챔버에 넣고 약 5 분 동안 anaesthetized했다. 마취 동안, 점멸, 호흡 및 운동 활동의 속도를 관찰합니다. 그것이 모터의 활동을 중지하고 깜빡 속도가 드문가 된 후 즉시 챔버에서 동물을 제거합니다. </li><li> 작업 역에있는 동물을 버리고, 경추 전위에 의해 동물을 죽이지. 다시 사지에서 대퇴골과 tibias 잘라, 피부와 근육을 제거하십시오. </li><li> 몇 초 동안 이소프로판올 70%에서 해부하는 대퇴골과 tibias를 넣고 1X D - PBS로 전송할 수 있습니다. </li><li> biosafety 캐비닛에서 대퇴골과 tibias는 DMEM을 포함 10cm 요리로 전송되었습니다. 각 뼈가 다음 핀셋 개최되었고 두 끝을는 가위로 잘라 오픈되었습니다. 3ml 주사기에 22G 바늘을 첨부하여 DMEM로 작성 후 뼈 중 하나 열 끝에 바늘을 삽입하여 50ml 튜브에 골수를 플러시. ~ 3 번 각각의 뼈에 반복 2. 모든 marrows가 취득되었을 때, 세포를 resuspend과 뼈가 파편과 혈액 집계를 제거하는 70μm 셀 스트레이너를 통해 세포 현탁액을 전달합니다. </li><li> 200g에 세포를 5 분 동안 4 ° C를 스핀 다운과 열망에 의해 표면에 뜨는를 제거합니다. 25ml MSC 매체에서 Resuspend 세포 (DMEM 10 % FBS, 1 % 펜 - Strep을 포함). 10ml의 세포 현탁액은 두 요리의 총 각 10cm 문화 요리에 씨앗을했다. 1 ~ 2 주 동안 37 ° C와 5% CO 2 배양기에서 문화 요리 보관하십시오. 매체는 2 ~ 3 일마다 변경되었습니다. </li></ol> 2. 쥐 MSCS의 농축 표면 단백질의 숫자는 CD54, CD90, CD73, CD105 CD271과 3-5를 포함하여 MSCS를 풍요롭게하는 데 사용되었습니다. 우리의 연구에서는, 우리는 자기 세포 정렬하여 MSCS를 풍부하게하는 표지로서 CD54과 CD90를 사용합니다. <ol><li> 세포 confluency 80 % 정도에 도달하면 매체를 대기음 각 접시에 4 ~ 5ml 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)를 추가합니다. 인큐베이터에 다시 요리를 넣고 세포 분리를 허용 5 분 동안 주변에 알을 품다. 일단 세포가 분리 트립신을 inactivate하는 문화 매체의 동일한 금액을 추가했다. 5 분 4 ° C, 200g에 15ml 튜브와 스핀 다운 세포에 세포 현탁액를 수집합니다. </li><li> 다음 단계는 BD IMagnet를 사용하여 세포 분리를위한 매뉴얼에 따라 두 표면 마커 CD54과 CD90에 의해 MSCS를 풍요롭게하는 방법에 대해 설명합니다. 세포 펠렛은 20,000,000 셀 / ML에서 세포 얼룩 버퍼 (1X D - PBS에서 3% 열 inactivated FBS)에 resuspended되었습니다. Biotinylated CD54 항체 (0.25μg 만명 당 세포)와 biotinylated CD90 항체가 (만 셀 당 0.15μg) 추가 및 세포 현탁액로 부드럽게 혼합했다. 15 분 동안 얼음에 부화 후, 표시된 전지 1X BD IMag 버퍼의 초과 볼륨 세탁했다. 분류 세포는 5 분, 4 ° C 200g에 아래 소용되었다. </li><li> 소용돌이 BD IMag streptavidin 입자 철저하게 모든 10000000 전지 40μl 입자를 추가합니다. 철저하게 표시된 세포와 입자를 섞어하고 ~ 12 ° C 30 분 6 세포를 품어. 이것은 streptavidin 입자에 바인딩할 수 있도록 각각 표면 단백질 CD54과 CD90에 바인딩되어있는, biotinylated 안티 CD54 및 안티 CD90. </li><li> 배양 시간 동안 긍정적인 분수를 수집하는 왕복 바닥 시험관에 라벨을 달고. 부화 후, 1X BD IMag 버퍼와 20000000 세포 / ML에 상표 볼륨을 가지고 있고 긍정 - 분수 수집 튜브에 표시된 세포를 전송합니다. BD IMagnet에 긍정적인 - 분수 튜브를 삽입 6 분 후, BD의 IMagnet 여전히 긍정적 - 분수 튜브와 유리 파스퇴르 피펫으로 뜨는을 제거 그것이가요. </li><li> BD의 IMagnet에서 긍정 - 분수 튜브를 제거하고 얼음에 놓으십시오. 1ml 얼음 차가운 1X BD IMag 버퍼와 부드러운 혼합하여 resuspend 세포를 추가합니다. BD IMagnet에 다시 튜브를 삽입하고 2 ~ 4 분 동안 머물게. 새로운 유리 파스퇴르 피펫으로 뜨는을 제거합니다. 2.5 한 번만 더에서 설명한대로 세탁을 반복합니다. </li><li> 문화 매체의 BD IMagnet과 resuspend 세포, 씨앗 하나 셀 및 유동세포계측법에 10cm의 접시를 유지하기위한 75cm 두 플라스크에서 튜브를 제거합니다. </li></ol> 3. 유동세포계측법에 의해 표면 마커의 표현의 확인 유동세포계측법 분석은 우리가 표현 CD54과 CD90를 얻은 세포를 확인하기 위해 수행되었다. HSC 마커 CD45는 MSCS가 HSCs의 소진 것을 확인하기 위해 사용되었다. <ol><li> 세포 ~ 80 %의 합류가 될 때, 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)과 세포를 trypsinize하고 15ml 튜브에 세포를 수집합니다. ° C 5 분 동안 세포를 수집 200g, 4 원심 분리기. </li><li> 뜨는을 대기음 있었고한번 1X PBS로 D - H 세포. 5 최종 농도 ~ 10000000 세포 / ML하고 얼음에 세포를 유지 세포 얼룩 버퍼에 Resuspend 세포 (1X D - PBS가 2퍼센트 FBS와 0.05 %의 나트륨 azide를 포함). (., 2 isotype 제어 IgG2a,.. 3 Isotype IgG1 제어, 4 CD45, 5. CD54,.. 6 CD90 세포 하나에만 해당) 여섯 표시 튜브로 나누어지는 100μl 세포 현탁액. </li><li> (:; : 0.5μg 만명 당 세포, CD54 : 0.25μg 만명 당 세포, CD90 : CD45 20μl 만명 당 세포 IgG2a 및 IgG1 0.15μg 만명 당 세포) 적절한 농도에 isotype 컨트롤 및 기본 항체를 추가하고 4 ° C에서 알을 품다 30 분 정도. </li><li> 100μl 세포 얼룩 버퍼에 두번 후 resuspend 세포 1X D - PBS로 세포를 씻으십시오. SA - PE (streptavidin - phycoerythrin, 만 셀 당 0.15μg를 사용)을 추가하고 4 품어 ° C를 어둠 속에서 30 분. </li><li> 두번 1X D - PBS로 표시된 세포를 씻으십시오. 400μl 세포 얼룩 버퍼에있는 세포를 Resuspend하고 유동세포계측법 분석을위한 팔콘 튜브로 전송할 수 있습니다. </li></ol> 4. MSCS를 유래 단일 식민지의 분리 MSCS 다른 세포 형태, 성장 속도뿐만 아니라 차별화 능력 6 가지 subpopulations 구성된 이기종 인구입니다. 그러나, 모든 subpopulations는 알려진 MSC 마커를 표현하기 때문에 그것이 subpopulations을 구분하는 마커를 사용 가능하지 않습니다. 따라서, 우리는 하나의 세포에 의해 형성된 콜로니의 다른 subpopulations를 따로 떼어 복제 실린더를 적용. <ol><li> 10cm 접시 당 약 50 ~ 100 세포에서 플레이트 세포. 1 ~ 2 주 동안 37 ° C와 5% CO 2 배양기에서 알을 품다. 이 기간 동안 거꾸로 현미경으로 잘 격리 식민지를 확인합니다. 식민지가 충분히 큰 크기 (더 나은 각 식민지에 100 개 이상의 세포)에 도달되면, 요리의 하단에 샤피로 식민지를 표시합니다. 표시하기 위해 식민지를 선택하면, 선택한 한 근처 둘러싼 식민지가없는 있는지 확인하십시오. </li><li> 매체 기음과 1X PBS로 D - 번 접시를 씻으십시오. 멸균 포셉와 살균 복제 실린더를 들고 부드럽게 표시 식민지 주위를 놓으십시오. 모두 표시 식민지 이상 배치 복제 실린더를 때까지이 과정을 반복합니다. 선택한 식민지 모든 복제 실린더가 하나의 식민지를 포함 각 등에서 멀리 떨어져 있어야합니다. </li><li> 각 복제 실린더에 100μl 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)을 추가하고 ~ 5 분 동안 인큐베이터에 다시 접시를 넣어. 5 분 후에, 그들이 돌고 있습니다 여부를 확인하기 위해 현미경으로 세포를 확인하십시오. 세포가 해제되면 트립신을 inactivate하는 문화 매체의 동일한 금액을 추가합니다. 200μl micropipettor으로 세포 현탁액을 혼합하고 3ml prewarmed 문화 매체를 포함하는 60mm 접시에 세포 현탁액을 전송할 수 있습니다. 제대로 요리를 라벨과 보육로 다시 넣어. 앞으로 몇 일 동안 형태학의 변화를 추적. </li></ol> 5. 대표 결과 쥐 MSCS 절연에 대한 부분에서 설명하는 프로토콜에 따라, 플라스틱 자기편 MSCS는 도금 후 다음날 표시해야합니다. 세포가 세포 분열 따위에 의해 번식을 계속로서, 합류 전지는 그림 1A에 표시된 셀에 같이한다. 세포에 도달하면 ~ confluency 80 % (그림 1B), subculturing이 수행하실 수 있습니다. subculture 동안, 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)은 세포를 분리하기 위해 사용되며 그림 1C 같이 해제 세포가 작고 둥근되었습니다. <img src="/files/ftp_upload/1852/1852fig1.jpg" alt="그림 1" /> 그림 1. 쥐 MSCS의 위상 콘트라스트 이미지를 표시합니다. (A) 콘플루앙 MSCS. 세포의 대다수는 스핀들과 같은, 별 같은 수 있습니다. (B) MSCS 80 % 정도 confluency. (C) trypsinization 후 찾았 는데요 세포가 작고 둥근 있습니다. 일단 MSCS가 자기 세포 분류에 의해 강화되고 유동세포계측법 분석은 표면 표시자 표현식을 확인하기 위해 수행됩니다. 농축 좋은 경우, 세포는 HSC 마커 CD45 (그림 2)에 대한 긍정적인 MSC 표시 CD54과 CD90에 대한 얼룩하지만 부정을 표시한다. Isotype 컨트롤 IgG2a과 IgG1은 부정적인 컨트롤로 사용됩니다. <img src="/files/ftp_upload/1852/1852fig2.jpg" alt="그림 2" /> 그림 2. 표면 마커에 대한 MSCS의 유동세포계측법 분석. MSCS는 IgG2a (isotype 제어 1), IgG1 (isotype 제어 2), CD45, CD54과 CD90에 대한 항체로 분류되었습니다. MSCS는 CD54과 CD90를 표현하지만 CD45. 세포가 적절한 clonal 밀도에 씨앗을 때, 식민지는 단일 세포에서 증가한다. 그림 3A는 하나의 세포에 의해 형성된 식민지를 나타냅니다. 복제 실린더는 다음 식민지에서 파생된 식민지와 세포가 별도로 양식 수있는 별도의하는 데 사용할 수 있습니다. 그림 3B와 3C는 두 개별 식민지에서 파생된 세포를 나타냅니다. 식민지 2에서 파생된 세포 (그림 3C) 라운드 반면 콜로니 1에서 파생된 세포는 같은 스핀들 (그림 3B)입니다. <p클래스 = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1852/1852fig3.jpg" alt="그림 3" /> 그림 3. MSCS와 단일 콜로니 파생 세포 콜로니 형성. MSCS는 clonal 밀도 양식 개별 식민지에서 교양. 이들 식민지는 별도 양식 수있는 복제 실린더와 다른 식민지에서 세포로 구분하실 수 있습니다. clonal 밀도에 도금하면 (A) 대표 식민지가 MSCS에 의해 형성. (B) 1 식민지에서 파생된 세포를 스핀들과 같은. (C) 라운드 전지는 다른 식민지에서 파생.

<ol>
	<li>de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+glial+glutamate+transporters+in+adult+mesenchymal+stem+cells.">Induction of glial glutamate transporters in adult mesenchymal stem cells.</a> J Neurochem. 91, 155-166 (2004).</li><li>Porter, R. M., Huckle, W. R., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dexamethasone+withdrawal+on+osteoblastic+differentiation+of+bone+marrow+stromal+cells.">Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells.</a> J Cell Biochem. 90, 13-22 (2003).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25, 2739-2749 (2007).</li><li>Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells:+from+basic+biology+to+clinical+applications.">Human mesenchymal stem cells: from basic biology to clinical applications.</a> Gene Ther. 15, 109-116 (2008).</li><li>Erica, L., Herzog, L. C., Diane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krause+Plasticity+of+marrow-derived+stem+cells.">Krause Plasticity of marrow-derived stem cells.</a> Blood. 102, 3483-3493 (2003).</li><li>Colter, D. C., Sekiya, I., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+subpopulation+of+rapidly+self-renewing+and+multipotential+adult+stem+cells+in+colonies+of+human+marrow+stromal+cells.">Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.</a> Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).</li></ol>

이 프로토콜은 격리와 MSCS를 풍요롭게하는 방법에 대해 설명합니다. 단일 식민지 파생 세포를 분리하는 방법도 포함될 수 있습니다. 성공적인 고립, 농축 및 식민지 분리에 중요한 몇 가지 단계가 있습니다. 쥐의 세포에서 분리하고 있지만, 그것은 셀 스트레이너 또는 혈전과 뼈 파편 제거와 비슷한 크기의 살균 나일론 메쉬를 통해 필터 권장합니다. 하룻밤 세포를 도금 후, 많은 죽은 세포는 매체...

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<td>1X D-PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14040-133</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>BD Imagnet</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552311</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG2a</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555572</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG1</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555747</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD54</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL054B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD90</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL005B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD45</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL001B</td>
<td>&nbsp;</td>
<tr>
<td>BD Imag buffer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552362</td>
<td>&nbsp;</td>
<tr>
<td>Round-bottom tube</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin particles</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>557812</td>
<td>&nbsp;</td>
<tr>
<td>SA-PE</td>
<td>&nbsp;</td>
<td>R&amp;D systems</td>
<td>F0040</td>
<td>&nbsp;</td>
<tr>
<td>Cloning cylinder</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2059</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs

MSCS는 치료 어플 리케이션을위한 유망한 원천이다 성인 줄기 세포의 인구입니다. 이러한 세포는 골수에서 격리 수 있으며, 쉽게 플라스틱 준수로 인해 조혈 줄기 세포 (HSCs)에서 분리될 수있다. 이 프로토콜은 쥐 대퇴골과 tibias에서 MSCS를 분리하는 방법에 대해 설명합니다. 분리된 세포는 더욱 자기 셀 정렬하여 두 MSCS 표면 마커 CD54과 CD90에 대한 풍부한되었습니다. 표면 마커 CD54과 CD90의 표현은 다음 유동세포계측법 분석에 의해 확인되었다. HSC 마커 CD45도 정렬 MSCS가 HSCs의 소진되었습니다 있는지 확인하기 위해 포함되었습니다. MSCS 자연 매우 이질 있습니다. 다른 형태, 증식 및 분화 능력을 가지고 세포의 subpopulations가 있습니다. 이러한 subpopulations는 모든 알려진 MSCS의 마커를 표현하고 더 독특한 마커는 아직 다른 subpopulations에 대한 확인되지 않았습니다. 따라서, 다른 subpopulations을 분리하는 대체 방법은 단일 식민지 파생 세포를 분리하는 복제 실린더를 사용하고 있습니다. 단일 식민지에서 파생된 세포는 다음 교양하고 별도로 평가하실 수 있습니다.

Watch this Scientific Journal Video about Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs at JoVE.com

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

쥐 MSCS는 대퇴골과 tibias으로부터 격리하고 자기 세포 분류에 의해 풍부하게되었다. 정렬 세포 유동세포계측법에 의해 표면 마커의 표현에 대해 확인했다. 이러한 세포는 하나의 식민지를 형성 clonal 밀도에 교양 있었다 그리고 이들 식민지는 복제 실린더로 구분했다.

분리 및 랫 Mesenchymal 줄기 세포의 농축 (MSCS)와 MSCS를 유래 단일 식민지의 분리

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

isolation-enrichment-rat-mesenchymal-stem-cells-mscs-separation

Research

JoVE Journal

Biology

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

28.3K 조회수.  City of Hope Cancer Center. 쥐 MSCS는 대퇴골과 tibias으로부터 격리하고 자기 세포 분류에 의해 풍부하게되었다. 정렬 세포 유동세포계측법에 의해 표면 마커의 표현에 대해 확인했다. 이러한 세포는 하나의 식민지를 형성 clonal 밀도에 교양 있었다 그리고 이들 식민지는 복제 실린더로 구분했다.

비디오: Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

분리 및 조혈 줄기 세포의 이식 (HSCs)

연구

생물학

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Hoechst의 분리 및 특성 낮은 CD45 부정적인 마우스 폐 Mesenchymal 줄기 세포

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

인간 Lipoaspirates에서 지방 유래 줄기 세포를 수동으로 분리

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146의 분리<sup&gt; +</sup쥐 폐에서&gt; 주민 폐 중간 엽 기질 세포

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

붉은털원숭이 지방유래 줄기세포의 분리, 증식 및 분화

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

배양된 인간 중간엽 줄기세포에서 세포외 소포의 분리, 특성화 및 치료적 적용

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

지방 조직 유래 줄기세포 분리 및 특성 분석

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

저자 스포트라이트: Sprague Dawley Rats의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Sprague Dawley 쥐의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...