作品は、国立衛生研究所（R01GM079688、R21RR024439、およびR21GM075838）、国立科学財団（CBET 0941055）、およびMSU財団によって部分的にサポートされていました。

1。ラットMSCの単離間葉系幹細胞は、以前は1で説明した6〜8週齢のSprague - Dawley雌ラット、2から分離された。隔離されたMSCは、プラスチック表面に付着し、容易にin vitro培養中に展開することができます。 <ol><li>動物は、麻酔室に入れ、約5分間麻酔した。麻酔中は、点滅して、呼吸と運動の速度を観察。それは運動を停止すると点滅速度が頻繁になった直後にチャンバーから動物を取り外します。 </li><li>操作ステーションで動物を下に置くと、頚椎脱臼により動物を殺す。バック手足から大腿骨と脛骨を切って、皮膚と筋肉を取り除く。 </li><li>数秒と1 × D - PBSに転送するためにイソプロパノール70％で解剖大​​腿骨と脛骨を置く。 </li><li>バイオセーフティキャビネットでは、大腿骨と脛骨をDMEMを含む10 cmディッシュに移した。それぞれの骨は、その後、ピンセットで開催され、両端はハサミで切開した。 3ミリリットルシリンジに22G針を添付し、DMEMでそれを埋める、そして骨の一つ開放端に針を挿入して50ミリリットルチューブに骨髄をフラッシュします。 〜3回、各骨を繰り返します2。すべての髄が得られたときに、細胞を再懸濁し、骨の破片と血液の凝集物を除去するために70μmセルストレーナーを通して細胞懸濁液を渡します。 </li><li> 200グラムで細胞を、5分間、4℃の回転を停止し、吸引により上清を取り除く。 25ミリリットルのMSC培地で再懸濁し、細胞（DMEM、10％FBS、1％ペン連鎖球菌を含む）。 10mlの細胞懸濁液は、2つの皿の合計のために各10cmの培養皿に播種した。 1〜2週間で37℃、5％CO 2インキュベーターで培養皿をおいてください。培地は2〜3日ごとに変更されました。 </li></ol> 2。ラットMSCの濃縮表面タンパク質の数は、CD54、CD90、CD73、CD105やCD271 3月5日を含めて、MSCを豊かにするために使用されている。私たちの研究では、磁気細胞選別によって、MSCを豊かにするためのマーカーとしてCD54とCD90を使用していました。 <ol><li>細胞がコンフルエントに約80％に達したとき、培地を吸引し、各シャーレに4〜5ミリリットルトリプシン- EDTAを加える。インキュベーターに戻し皿を置き、細胞の剥離を可能にするために約5分間インキュベートする。細胞が切り離された後、トリプシンを不活化するために培養液の同量を追加します。 5分間、4℃、200グラムで15ミリリットルチューブとスピンの細胞下に細胞懸濁液を収集する。 </li><li>次の手順では、BD IMagnetを用いた細胞分離のためのマニュアルに従って、2つの表面マーカーCD54とCD90でMSCを豊かにする方法について説明します。細胞ペレットを20万細胞/ mlで細胞を染色バッファー（1X D - PBSで3％の熱不活化FBS）中に再懸濁した。ビオチン化CD54抗体（0.25μg百万分の一の細胞）とビオチン化CD90抗体（万個の細胞あたり0.15μg）を添加し、細胞懸濁液を穏やかに混合した。 15分間氷上でインキュベートした後、標識された細胞を1X BD IMAGバッファの過剰量で洗浄した。標識された細胞を5分間、4℃200グラムでスピンダウンされました。 </li><li>渦BD IMAGストレプトアビジン粒子は完全に、すべての10万個の細胞に対して40μlの粒子を追加してください。徹底的に標識された細胞と粒子を混合し、6〜12で細胞をインキュベート℃で30分間。これにより、ストレプトアビジン粒子はそれぞれ表面タンパク質CD54とCD90にバインドされているビオチン化抗CD54および抗CD90に結合することができます。 </li><li>インキュベーション時間の間に、正の分数を収集するための丸底試験管にラベルを付けます。インキュベーション後、1 × BD IMAGバッファを20万細胞/ mlにラベリングのボリュームをもたらすと正のフラクションコレクションチューブに標識された細胞を移す。 BDのIMagnetにポジティブフラクションチューブを配置し、6分間滞在させ、その後も、BDのIMagnet上で正のフラクションチューブにガラスパスツールピペットで上清を取り除く。 </li><li> BDのIMagnetからポジティブフラクションチューブを外し、氷の上に置きます。 1ミリリットルの氷冷1 × BD IMAGバッファと穏やかに混合して再懸濁し、細胞を追加。 BD IMagnetに戻ってチューブを置き、それが2〜4分の滞在ができます。新しいガラスパスツールピペットで上清を取り除きます。 2.5つのより多くの時間で説明するように洗浄を繰り返します。 </li><li> 、培養培地中にBD IMagnetを再懸濁します細胞からチューブを外しシード細胞とフローサイトメトリー用の10cmディッシュを維持用の75センチメートル2フラスコ。 </li></ol> 3。フローサイトメトリーによる表面マーカーの発現の検証フローサイトメトリー分析は、我々が明示CD54とCD90を得られる細胞を確認するために実施した。 HSCマーカーCD45はMSCが造血幹細胞が枯渇していたことを確認するために使用されていました。 <ol><li>細胞がコンフルエント〜80％になると、トリプシン- EDTAで細胞をトリプシン処理し、15ミリリットルチューブに細胞を集める。 ° Cで5分間、細胞を収集するために200グラム、4で遠心分離します。 </li><li>上清を吸引していたかつて1X D - PBSでH細胞。 5の最終濃度が約10万細胞/ mlにし、氷上で細胞を維持する細胞の染色バッファーに再懸濁し細胞（1 × D - PBS、2％FBS、0.05％アジ化ナトリウムを含む）。 （。、2アイソタイプコントロール的なIgG2a、。。3アイソタイプコントロールIgG1を、4 CD45、5 CD54、。。6 CD90 1細胞のみ）6つのラベルの付いた試験管に分注し100μlの細胞懸濁液。 </li><li> （：;：0.5μg百万分の一の細胞、CD54：0.25μg百万分の一の細胞、CD90：CD4520μlの百万分の一の細胞IgG2aおよびIgG1の0.15μg百万分の一の細胞）を適切な濃度でアイソタイプコントロールおよび一次抗体を追加して4℃でインキュベートする30分。 </li><li>二回して、100μlの細胞の染色バッファーで細胞を再懸1X D - PBSで細胞を洗浄する。 SA - PE（ストレプトアビジン - フィコエリスリン、百万個の細胞あたり0.15μgを使用）を追加し、4℃でインキュベートCの暗所で30分間。 </li><li>倍1X D - PBSで標識した細胞を洗う。 400μlの細胞の染色バッファーで細胞を再懸濁し、フローサイトメトリー分析のためにファルコンチューブに移す。 </li></ol> 4。 MSCを派生シングルコロニーの分離 MSCは、異なるセルの形状、成長率だけでなく、分化能6の異なる亜集団から構成される不均一な集団である。しかし、すべての亜集団は、既知のMSCマーカーを発現し、従ってそれはこれらの亜集団を分離するためのマーカーを使用することは不可能です。したがって、我々は単一細胞によって形成されたコロニーな異なる亜集団を、分離するクローニングシリンダーを適用する。 <ol><li> 10 cmディッシュあたり約50〜100個の細胞で細胞を​​プレート。 1〜2週間で37℃、5％CO 2インキュベーター中でインキュベートする。この期間中、倒立顕微鏡でよく分離したコロニーを調べる。コロニーが十分に大きいサイズを（より各コロニーにおける100以上の細胞）に到達したら、皿の底にあるシャーピーとコロニーをマーク。マークされるコロニーを選択するときに、厳選されたGoogle Earthでも周囲のコロニーがないことを確認してください。 </li><li>培地を吸引し、1XのD - PBSで一度皿を洗う。滅菌ピンセットで滅菌クローニングシリンダーをピックアップし、優しく著しいコロニーの周りに置きます。すべてのマークされたコロニーは、上に配置、クローニングシリンダーになるまでこれを繰り返します。選んだコロニーは遠く離れてお互いからすべてのクロー​​ニングシリンダーが1つしかコロニーが含まれているようなものであるべきである。 </li><li>各クローニングシリンダーに100μlのトリプシン- EDTAを追加し、約5分間インキュベーターに皿を戻す。 5分後、彼らは切り上げかどうかを確認するために顕微鏡下で細胞を確認してください。細胞が持ち上げているときは、トリプシンを不活化するために培養液の同量を追加します。 200μlのマイクロピペッタで細胞懸濁液を混和し、3ミリリットル温めておいた培地を含む60ミリメートルディッシュに細胞懸濁液を移す。適切に皿にラベルを付け、インキュベーターにそれらを戻す。これから数日間にわたって形態学的変化を追跡。 </li></ol> 5。代表的な結果ラットMSCの分離のための部分で説明されているプロトコルによると、プラスチックの接着MSCは、めっき後の翌日に見えるはずです。細胞が増殖し続けているため、コンフルエントな細胞は、図1Aに示すように細胞のようになります。細胞がコンフルエント〜80％（図1B）に到達すると、継代培養を行うことができる。サブカルチャーの間に、トリプシン- EDTAは、細胞と持ち上げ細胞を分離するために使用され、図1Cに示すように小さくし、丸いです。 <img src="/files/ftp_upload/1852/1852fig1.jpg" alt="図1" /> 図1。ラットMSCの位相コントラスト像 。 （A）コンフルエントのMSC。細胞の大多数は、紡錘状または星状です。 （B）のMSC約80％コンフルエント。 （C）トリプシン処理後の細胞は小さく、丸い浮き。 一度のMSCは、フローサイトメトリー分析は表面マーカーの式を検証するために行われる、磁気細胞ソーティングによって濃縮される。濃縮が良ければ、細胞はHSCマーカーCD45（図2）に対してMSCマーカーCD54とCD90が陰性に対して陽性染色を示す必要があります。アイソタイプコントロールIgG2aおよびIgG1を陰性コントロールとして使用されています。 <img src="/files/ftp_upload/1852/1852fig2.jpg" alt="図2" /> 図2。表面マーカーのためのMSCのフローサイトメトリー分析。MSCはIgG2aの（アイソタイプコントロール1）、IgG1の（アイソタイプコントロール2）、CD45、CD54およびCD90に対する抗体で標識した。 MSCはCD54とCD90ではなくCD45を表明した。 細胞は適切なクローン密度で播種されると、コロニーは単一の細胞から立ち上がる必要があります。図3Aは、単一のセルによって形成されたコロニーを表します。クローニングシリンダーは、別々に培養することができるコロニー由来するコロニーと細胞を分離するために使用することができます。図3Bおよび3Cは、2つの個々のコロニーに由来する細胞を表しています。コロニー1から由来する細胞は、コロニー2由来の細胞のに対し（図3B）のようなスピンドルですラウンド（図3C）です。 <pクラス=&quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1852/1852fig3.jpg" alt="図3" /> 図3。 MSCおよび単一コロニー由来の細胞によるコロニー形成。MSCは、クローン密度のフォームの個々のコロニーで培養。これらのコロニーは別々に培養することができるクローニングシリンダーと異なるコロニー由来の細胞で区切ることができます。クローン密度で播種したとき（A）代表的なコロニーは、MSCによって形成。 （B）1つのコロニーに由来する細胞を紡錘状。 （C）円形細胞が別のコロニーから派生。

<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>

このプロトコルは、分離してMSCを豊かにする方法について説明します。シングルコロニー由来の細胞を分離する方法も組み込まれています。成功した分離、濃縮とコロニー分離する際に重要となるいくつかのステップがあります。ラットからの細胞単離を行っている間、それは、セルストレーナーまたは血液凝固と骨の破片を取り除くために同じようなサイズの滅菌ナイロンメッシュを介し?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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

MSCは、治療用途のための有望な源である成体幹細胞の集団である。これらの細胞は、骨髄から単離することができると簡単に、プラスチック接着による造血幹細胞（HSC）から分離することができる。このプロトコルは、ラットの大腿骨と脛骨からMSCを分離する方法について説明します。分離した細胞をさらに磁気細胞選別して、2つのMSCの表面マーカーCD54とCD90に対する濃縮した。表面マーカーCD54とCD90の発現は、フローサイトメトリー分析によって確認した。 HSCマーカーCD45はまた、ソートされたMSCのが造血幹細胞が枯渇していたかどうかを確認するために含まれていた。 MSCは、自然にかなり異質です。さまざまな形状、増殖および分化能力を有する細胞の亜集団がある。これらの亜集団は、すべての既知のMSCのマーカーを発現していないユニークなマーカーはまだ異なる部分母集団に同定されていない。したがって、別の亜集団を分離するための代替アプローチは、単一コロニー由来の細胞を分離するためにクローニングシリンダーを使用しています。シングルコロニーに由来する細胞を培養すると別々に評価することができます。

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

ラットMSCを、大腿骨と脛骨から分離し、磁気細胞ソーティングで濃縮した。ソートされた細胞はフローサイトメトリーによる表面マーカーの発現について確認した。これらの細胞はまた、単一のコロニーを形成するためにクローン密度で培養した後、これらのコロニーをクローニングシリンダーにより分離した。

ラット間葉系幹細胞（MSC）とMSCを派生シングルコロニーの分離の分離と濃縮

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

28.4K ビュー.  City of Hope Cancer Center. ラットMSCを、大腿骨と脛骨から分離し、磁気細胞ソーティングで濃縮した。ソートされた細胞はフローサイトメトリーによる表面マーカーの発現について確認した。これらの細胞はまた、単一のコロニーを形成するためにクローン密度で培養した後、これらのコロニーをクローニングシリンダーにより分離した。

ビデオ: 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.

造血幹細胞の分離と移植（造血幹細胞）

生物学

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.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 & 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.

ヘキストの分離および特性評価低 CD45負マウス肺間葉系幹細胞

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

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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.

間葉転換を誘導し、上皮の分析

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.

人間の脂肪吸引物からの脂肪由来幹細胞を手動で隔離

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

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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.

成体マウス切歯から歯科上皮幹細胞の単離および培養

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.

CD146の単離<sup&gt; +</supラット肺から&gt;常駐肺間葉系幹細胞

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

ラット間葉系幹細胞（MSC）とMSCを派生シングルコロニーの分離の分離と濃縮

要約

さらに動画を探す

この動画の章