我々は、このプロトコルの最初のバージョンをご提供するためスティーブンAustadに感謝。この作品は、NIHとエリソン医学財団からVGへの補助金によってと同様にサポートされて

1。始める前に<ol><li> 70％エタノールではさみや鉗子を滅菌する。 </li><li> 30mLのビーカー、箔の2層にカバーし、オートクレーブ内部の小さなマグネチックスターラーを置きます。 </li><li>滅菌水で28ワンチユニット/リベラーゼBlendzyme 3 mLの原液を準備します。 -20℃で0.5 mLを分注して凍結を行うあらゆる使用の前に新しいアリコートを解凍。解決策は融解後、混濁している場合があります。渦解が明確になるまで。 </li><li>細胞培養培地をウォームアップ。 </li></ol> 2。動物サンプルの調製 注意 ：野生動物が狂犬病ウイルスなどの病原体が含まれることがあります。常にオープン鋭利に注意してください。 <ol><li>動物を安楽死させると4で死体を置く℃に動物はフィールドで収集されているかのようにこのような実用的でない場合、それは、しかし、すぐに死体を使うのが最善である、死体は24時間以内に使用することができます。 24時間後の細胞収率が低下する。 </li><li> （ではない組織培養フードの）手術室で、または化学フードで動物を解剖。 </li><li>脇の下エリアでは、脇の下の皮膚が薄くなるように、脂肪分が含まれている皮膚のサンプルを収集するのに便利なサイトです、そして毛皮は密度が低いです。 70％エタノールで切開部位を清掃してください。毛皮は、エタノールに浸したされていることを確認します。 </li><li>鋭いメスで切開部位の周りに毛皮を剃る。希望の切開部位よりも大きい領域にひげをそる。皮膚へのカットを最小限に抑えるようにしてください。 70％エタノールで面積をスプレーし、乾燥させます。 </li><li>皮膚サンプルの消費税の約1 cm 2の断片を収集する。組織鉗子で皮膚をつまんで、はさみで切る。脂肪は、コラゲナーゼ消化を妨げるとして、皮膚と脂肪層を切らないようにしてみてください。肺のサンプルを収集するには、70％エタノールで胸の部分を洗浄し、T字型の切開を行うと皮膚のフラップを離れて引っ張って、ハサミで胸の皮膚を開きます。 70％エタノールで開かれた筋肉のエリアを洗い、乾燥させる。滅菌ピンセットやハサミを使用して胸郭をカット（大型動物のために骨のカッターを使用）。内臓の汚染を避けるために、無菌技術を使用してください。動物の毛皮を触る楽器で内臓を触れないでください。滅菌ピンセットやハサミを使って約1 cm 2の肺の断片をカット。 </li><li>乾燥を避けるために滅菌PBSで50 mLチューブに場所の組織片。 </li><li>エタノールによる組織片を50 mLチューブの外側を洗浄し、組織培養フードにそれらを取る。 </li><li>適切に動物の死体を処分。 </li></ol> 3。抽出セル<ol><li>滅菌メスを用いて10 cmの組織培養皿に組織片を転送する。サンプルであまりにも多くのPBSを転送しないでください。 </li><li> twoメスを用いて〜1mmの部分にティッシュを切る。シザー作用中心から開始して離れて引っ張っを使用して2つのブレードを使用してください。組織は、ピース単位でカットしていない、まで丸めたしてください。皮膚のパテに似てカットが十分であるときに、それは部分に分離することはありませんが薄いストレッチされます。肺組織では、カットする方が簡単です、そしてそれは、小さな断片に分割します。 </li><li>メスを使用して、滅菌攪拌棒で滅菌30mLのビーカーにカット組織を移す。 / 0.14ワンチ単位mLのリベラーゼBlendzyme 3、および1X抗生物質/抗真菌剤でDMEM/F12メディアの10mLで組織の切断に使用されるプレートを洗浄し、組織片を30 mLのビーカーにソリューションを追加。 </li><li>無菌箔でビーカーをカバーし、30〜90分間、ゆっくり攪拌し、37℃でインキュベートする。インキュベーションの長さは、種や組織型によって異なります。組織を過剰に消化しないように注意してください。組織片は、消化の終わりにまだ存在しているときに最高の収率が得られる。皮膚は肺よりも消化に時間がかかります。大型動物からの皮膚は、消化に時間がかかります。 30分後に消化し、10分ごとに確認してください。皮膚の消化が完了すると、メディアは曇りと互いに別々の皮膚の断片になり、作品の端は&quot;ファジー&quot;になる。肺の断片が赤から白への色を変更し、粘着性繊維を形成する起動時に肺消化を停止する必要があります。 </li><li>ピペットで塊を破壊するために上下組織断片を含む溶液を。滅菌50 mlチューブにソリューションを転送する。フラグメントは、10 mLのピペットの先端と消化によって容易に移動する場合はよく行われていた。 15％FBS、抗生物質/抗真菌剤1Xと暖かいDMEM/F12メディアの10mLでビーカーを3回リンスし、組織片を50 mLの試験管にメディアを追加します。 50 mLのチューブと反転数回転倒混和を閉じます。メディアのFBSは、リベラーゼ消化を停止します。 </li><li> 5分間、スイングバケット組織培養の遠心機で524グラムで回転する。上清を取り除く。 15％FBS、1X抗生物質/抗真菌剤で暖かいDMEM/F12メディア10mLにペレットを再懸濁する。ピペットでサスペンション付き組織片を破壊する力の最大値を設定します。 </li><li> 15％FBS、1X 5分間スイングバケット組織培養の遠心機で524グラムで、抗生物質/抗真菌剤、混合および遠心機でDMEM/F12メディアの別の30 mLを加え。リベラーゼの痕跡を削除する1つより多くの時間を繰り返します。 </li><li> 37 1X抗生物質/抗真菌剤および組織培養インキュベーターで10 cmの組織培養皿と場所への転送、15％FBSとDMEM/F12メディア10mLにペレットを再懸濁します° C、5％CO 2、3％O 2 。 </li><li>板の線維芽細胞とメディアの色のための毎日をチェックしてください。分離が成功した場合、線維芽細胞は組織片からはい出るとプレート（図1）に取り付けます。線維芽細胞は2-5日以内に組織片を終了し始めます。 </li><li>黄色へのメディアの色が変化した場合、これは潜在的な汚染またはセルの過密を示します。高倍率での顕微鏡でプレートを調べます。細菌、真菌、またはワームは、現在のプレート（これはほとんどの実験動物との問題がない、しかし、サンプルが野生から収集中に発生する可能性がある）破棄されている場合。ない汚染が存在しなくても、メディアが色を変更した場合、これはあまりにも多くの細胞や同じ皿に置かれた非常に多くの組織片のどちらかが原因です。プレートの60％以上が添付された線維芽細胞によって覆われている場合は、プレート上でメディアを変更し、新しいメディアと新しいプレートに組織片を転送する。プレートに接続されていない多くの線維芽細胞の場合、メディアを変更すると2-4プレートに組織片を分割。 </li><li>メディアが先に変更されていた場合、7日後、、メディアを変更し、新しいメディアと新しいプレートに組織片を転送する。 </li><li>追加の7日間の細胞および組織断片をインキュベートする。 14日目では、すべての実行可能な線維芽細胞は、組織断片を終了した。 </li><li>細胞分離の初めから14日後、古いメディアと組織断片を破棄、5 × 10 5細胞/プレートで、新しいプレートに15％FBS、1Xペニシリン/ストレプトマイシンをEMEMを細胞やプレート、それらを収穫する、非必須アミノ酸、およびナトリウムピルビン酸。 EMEMのメディアは、他の種類の細胞が死ぬか、増殖を停止される線維芽細胞の成長をサポートします。 </li><li>細胞が80〜90％コンフルエントに達した後、将来の使用のための細胞のアリコートを凍結する。 </li><li>細胞が80〜90％コンフルエントに達したときに5 × 10 5細胞/プレートで分割しで培養細胞を続ける。 </li></ol> 4。代表的な結果正常な線維芽細胞は顕著な突起（葉状仮足）（図2）を持つ大規模な細胞である。線維芽細胞は単層で増殖。健全な成長の文化は、Mステージの1-10％の細胞が含まれていますように板の表面上に上昇して細胞を切り上げ、ただしプレート（図3）からデタッチされていない認識。通常は、5 × 10 5細胞を接種した10cmディッシュは3-4日でコンフルエントになる。倍加時間は、種間で大きく異なり、そして長寿命のげっ歯類1のいくつかのために大きくなることがあります。細胞は彼らがG1の舞台での増殖を阻止プレートを入力するとき。線維芽細胞の典型的なコンフルエントプレートは、細胞（図4）の密集層が含まれています。細胞が90％コンフルエントに達すると、彼らは、分割するための準備が整いました。必要に応じて、細胞は通常のメディアの変更（週1〜2回）で長時間のための逮捕コンフルエントプレート上で維持することができます。 <img src="/files/ftp_upload/2033/2033fig1.jpg" alt="図1" /> 図1。マウスの皮膚（）と肺（B）から線維芽細胞の分離。メディアは7日目で写真を撮る前にアタッチされていない細胞と破片を除去するために変更されました。 <img src="/files/ftp_upload/2033/2033fig2.jpg" alt="図2" /> 図2。マウスの肺線維芽細胞。 <img src="/files/ftp_upload/2033/2033fig3.jpg" alt="図3" /> 図3。 10倍の倍率で撮影したM - STAGE内の細胞を含むマウスの線維芽細胞のフィールド、。 <img src="/files/ftp_upload/2033/2033fig4.jpg" alt="図4" /> 図4。 10倍の倍率で撮影したマウス線維芽細胞のコンフルエントプレート、。

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> Cell. 7, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5, 741-747 (2003).</li></ol>

利害の衝突は宣言されません。

通常の主な線維芽細胞は、生物学の研究で​​確立された癌細胞株に対する優れた代替手段を提供しています。線維芽細胞の重要な利点は、彼らが癌遺伝子および腫瘍抑制遺伝子の変異を運ぶし、無傷の細胞周期チェックポイントを維持していないということです。これは正常な線維芽細胞周期調節、DNA修復、およびアポトーシスの研究に適したシステムです。ここで説明するプロトコルは?...

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<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>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TM Research Grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05401127001</td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

establishing primary adult fibroblast cultures from rodents

生物学的研究のために、初代培養細胞ではなく癌細胞株を使用することの重要性は広く認識になりつつあります。がん細胞は、これらのプロセスに関与する遺伝子の変異を運ぶように主細胞は、細胞周期の制御、アポトーシス、およびDNA修復の研究で好まれている。初代細胞は無限に複製老化または異数性発現の発現のために培養することができません。したがって、新しい文化は、定期的に確立する必要があります。齧歯類の胚性線維芽細胞の単離のための手順は十分に確立されているが、成人線維芽細胞培養を単離することはしばしば課題となっています。ヒトの患者から線維芽細胞にそれらを比較するときに、ヒト疾患のモデルマウスから分離された成体げっ歯類の繊維芽細胞は、優先制御があります。妊娠中の女性を容易に得ることができない野生のげっ歯類を操作するときまた、成人線維芽細胞でのみ利用可能な材料です。ここでは、げっ歯類の皮膚や肺から成人線維芽細胞の単離および培養のためのプロトコルを提供しています。我々は、実験用マウスおよびラットからそのようなビーバー、ヤマアラシ、やリスなどの野生げっ歯類20種のげっ歯類上から線維芽細胞を分離するためにこのプロシージャを正常に使用する。

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents

この記事では野生のげっ歯類の皮膚および肺組織からの初代線維芽細胞培養物の分離および保守のためのプロトコルについて説明します。

げっ歯類からプライマリ大人の線維芽細胞培養の確立

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

establishing-primary-adult-fibroblast-cultures-from-rodents

Research

JoVE Journal

Biology

49.2K ビュー.  University of Rochester. この記事では野生のげっ歯類の皮膚および肺組織からの初代線維芽細胞培養物の分離および保守のためのプロトコルについて説明します。

ビデオ: Establishing Primary Adult Fibroblast Cultures From Rodents

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

後期ステージショウジョウバエ蛹の脳からの初代神経培養

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

成体ラットの心筋細胞の初代培養

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

高密度ヒト胚性幹細胞の培養線維芽細胞からの迅速な除去

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

基礎脊椎動物の系統からの一次筋原前駆細胞/筋芽細胞培養液の調製

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

初代マウス骨芽細胞から骨細胞培養物を入手するレチノイン酸のアプリケーション

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...