この作品は、キー研究と開発中国プログラム (2017YFA0104604)、国家自然科学基金の中国 (NSFC、81772093)、一般的なプログラムによって支持された科学および技術開発プログラムの蘇 (ZXL2015128)江蘇省 (BK20161241) の自然科学財団と山東省泰山学者賞 (tshw201502065)。

このプロトコルで使用される人間の組織は、金融機関の人間研究倫理委員会のガイドラインによると処理されている (NO.2015120401、日: 2015 年 5 月 12 日)。
1. 準備
<ol><li>冷たいダルベッコの 10 mL と 50 mL のチューブの病院で整形手術から破棄収集新鮮な大人の腹部皮膚組織のイーグル培地 (DMEM)、変更されました。供試体は、細胞生存率を大きく影響せず 72 h まで 4 ° C で保存できます。</li>
	<li>下記のとおり、試薬、培地を準備します。<ol>
<li>(100 U/mL) ペニシリンとストレプトマイシン (100 mg/L) の 1 mL を 50 mL のリン酸緩衝液 (PBS) 洗濯ソリューションを準備するのに追加します。</li>
		<li>酵素消化液の 2 つのセットを準備: (1) 当期 (DMEM で 2.5 mg/mL) の 50 mL を準備し、50 mL のタイプ I はコラゲナーゼ (DMEM で 2.5 mg/mL) 別に従来法;(2) 当期の混合物の 50 mL を準備し、コラゲナーゼをタイプ I (ディゾルブ 125 mg 当期粉末、125 mg の I 型コラゲナーゼ粉 DMEM の 50 mL の) 新しいメソッドの。 注: すべての酵素のソリューション必要がありますフィルタ リング 0.22 μ m ストレーナー付けや at4 ° C を保持</li>
		<li>両方のメソッドに対して消化ソリューションを準備: 0.05% トリプシンと 0.25% トリプシンに商業的に購入しました。10 mg/ml DNase 私私粉末の 5 mL の PBS、DNase の 50 mg を溶解することによりソリューション 0.22 μ m ストレーナーとフィルター、-20 ° C で保存</li>
		<li>酵素の消化力を中和するために媒体の 500 mL を準備: 10% 牛胎児血清 (FBS) と 1% ペニシリン/ストレプトマイシン DMEM 培。</li>
		<li>500 ml 接種培地 (成長因子を含む中、G の中と呼ばれる) の: 40 ng/mL の線維芽細胞成長因子 2 (FGF2) 20 ng/mL 上皮成長因子 (EGF)、40 μ g/mL fungizone 1% ペニシリン/ストレプトマイシンを含む DMEM/F12 (3:1) 培地と2 %b27 を補足します。</li>
		<li>100 mm 細胞培養を前処理料理型を含むコーティング マトリックスの 2 mL の私部屋の温度で 30 分のコラーゲン。</li>
	</ol>
</li>
</ol>2. 従来の方法
<ol><li>
皮膚組織の前処理方法
	<ol>
<li>皮膚組織の 1.5 cm2を取るし、それを洗って 1 x 10 の mL の PBS。30 の 70% エタノール 10 mL 洗い流し、s それを孵化と 10 ml の洗浄ソリューション (PBS 2% ペニシリンとストレプトマイシンを含む)、5 分の 2 倍。 注: すべての洗浄は、層流フードの内部 100 mm 細胞培養皿で行われます。</li>
		<li>100 mm 細胞培養ディッシュで皮下脂肪層を削除し、(重量はこのプロトコルでは 1 g) 皮膚組織を推し量る滅菌はさみで組織をトリミングします。 注: 十分なトリミングは真皮から表皮の効率的な分離のため非常に重要です。黄色の脂肪層は、視覚的に簡単に区別できます。</li>
		<li>ダウン組織を別 100 mm 滅菌皿真皮側に転送、その後、皮膚組織をメス刃を使用して 3-4 mm 幅の短冊状にカットします。 注: 脂肪層と真皮側が視覚的に認識されます簡単に。</li>
		<li>100 mm ディッシュの皮膚組織に 2.5 mg/mL 当期溶液 10 mL を追加し、その後、4 ° C (20 h 以上) で一晩中皿をインキュベートします。 注: 当期溶液の量は、組織の各グラムの消化のため当期溶液 10 mL を使用して計算できます。</li>
	</ol>
</li>
	<li>
皮膚組織からの表皮の分離
	<ol>
<li>2 日目、良いピンセットを使用して真皮から表皮をはがします。 注: 表皮を剥がしにくい場合は、当期の消化が足りなかった、ティッシュの不十分なトリミングのため通常であります。この場合、組織より長い培養時間を必要があります。</li>
		<li>メス刃; を使用して組織液に細胞培養液 500 μ L で皮をむいた表皮をミンチします。10 mL を 50 mL チューブに 0.25% トリプシンとそれを再懸濁し、それの揺れで、37 ° c の水浴中 20 分を孵化させなさい。</li>
	</ol>
</li>
	<li>
コレクション、初代培養細胞の培養
	<ol>
<li>トリプシン活性を中和する中和液 (このプロトコルでは 10 mL) の等しい量を追加することによって。</li>
		<li>20 x 上下ソリューションを 10 mL の血清ピペットでピペッティングによる表皮細胞を分離し、残存組織の残骸を削除する 100 μ m セル ストレーナー携帯ソリューションを渡します。</li>
		<li>ろ過の後 200 x gで 5 分で細胞液を遠心分離します。洗浄用中和培地で細胞ペレットを再懸濁します、その後、細胞ペレットを取得する 200 x gで再度遠心。</li>
		<li>低カルシウム、無血清ケラチノ サイト媒体 (SFM) の 10 mL の細胞ペレットを再懸濁します。このソリューション総細胞数をカウントし、コーティングのマトリックスで前処理した 100 mm 細胞培養皿に SFM の 10 mL で約 2 x 10 の6セルをプレートの 10 μ L を取る (を含むタイプ I コラーゲン)。 注: コーティングは、従来の方法に必要なステップです。</li>
		<li>培養培地ごとの 2 d を変更します。 注: は前に、と培養液を変更した後、顕微鏡の下で細胞粘着をチェックします。</li>
		<li>通路、細胞の約 80% に達すると合流。 注: すべての培養皿はコーティング行列で前処理しました。</li>
	</ol>
</li>
</ol>3 新しい方法
<ol><li>
皮膚組織の前処理方法
	<ol>
<li>組織に従って上記 (ステップ 2.1) 従来の方法を収集します。</li>
		<li>皮膚組織 1 を洗って 10 ml の PBS の x (重量はこのプロトコルでは 1 g 程度) 電子スケールで滅菌したプラスチック容器の重さと。 注: このプロトコルに必要な組織の最小重量は 0.1 g 使用組織標本が小さすぎる場合、十分な細胞数を得ることが困難だからです。オペレーターの処理能力に依存する最終的にこのプロトコルのための組織の最大重量はありません。</li>
		<li>鉗子を使用して、30 の 70% エタノール 10 mL で皮膚組織を洗浄する s。</li>
		<li>2 組織をインキュベート洗浄ソリューション (2.1.1 の手順で準備)、5 分の各時間の 10 ml x。 注: 上記すべての洗浄手順は、100 mm 細胞培養皿 (ティッシュ文化フード) の層流フードの内部で実行されます。</li>
		<li>滅菌 100 mm セル ディッシュを別組織に転送します。</li>
		<li>メスのブレードを使用すると、すると、組織は組織スラリーに徹底的にミンチします。</li>
		<li>濡れている組織を維持する 200 μ L の PBS 5 分ごとを追加します。 注: は、手順 3.1.5 - 3.1.7 約 15 分かかります。上記のすべての手順は、部屋の温度で行われます。</li>
	</ol>
</li>
	<li>
皮膚組織の消化
	<ol>
<li>50 mL チューブに均質化組織ソリューションを転送します。皮膚組織のすべての 1 g の酵素の混合物の 10 mL を追加します。均質化組織に酵素を徹底的にミックスします。</li>
		<li>1 h の 37 ° C の水浴の揺れとの混合物を孵化させなさい。</li>
		<li>37 ° C の水浴中で 30 分間別消化混合物に 1/5 量 0.25% トリプシン (このプロトコルで 2.5 mL) を追加します。</li>
		<li>DNase を追加私ソリューション 1: 100 (v/v) 比率 (このプロトコルで 250 μ L) で酵素の混合し、室温で 5 分間インキュベートします。</li>
	</ol>
</li>
	<li>
コレクション、初代培養細胞の培養
	<ol>
<li>1:1 の比率で中和中 12.5 mL を追加することによって消化力プロセスを停止します。約 20 x、10 mL の血清ピペットを使用してセルを分離するソリューションを上下にピペットします。</li>
		<li>ティッシュの残骸を削除する 100 μ m セル ストレーナー解離細胞をフィルターします。200 x gで 5 分間観察チューブの下部にペレットで上清を遠心します。</li>
		<li>上澄みを廃棄し、200 x g細胞を収集するために 5 分間で 10 mL、中和中、遠心力で細胞ペレットをもう一度洗浄します。</li>
		<li>10 mL 接種培地 (G ステップ 1.2.5 から媒体) の Y-27632 の 10 μ m で細胞ペレットを再懸濁します。</li>
		<li>総セル数をカウントし、100 mm 細胞培養ディッシュに G の中の 10 mL で約 2 x 10 の6セルをプレートに溶液 10 μ L を取る。 注: プリコートのステップは新しいメソッドに必要ななく、真皮層の除去は不要ですか。</li>
		<li>3 d の後、接種培地を低カルシウム SFM 媒体に置き換えます。培養培地ごとの 2 d を変更します。 注: は前に、と媒体を変更した後に細胞接着をチェックします。</li>
		<li>通路、細胞の約 80% に達すると合流。 注: 必要に応じて、2 分の 0.05% トリプシンで細胞を前処理し、表皮細胞をトリプシン前に皮膚細胞を汚染する PBS のセルを洗浄します。</li>
	</ol>
</li>
</ol>4. 細胞の継
<ol><li>メディアを取り出して、洗浄、pbs セルと、(各 100 mm 皿) あたり 0.05% トリプシンの 2 mL を加えます。</li>
	<li>5 分のためのインキュベーター (5% CO2と 37 ° C) のセルを孵化させなさい。</li>
	<li>すべての細胞が培養皿から切り離されるかどうかを確認する顕微鏡を使用しているセルを確認します。</li>
	<li>10 %dmem の 8 mL を加えて酵素反応を中和し、15 mL チューブに細胞を収集し、FBS。</li>
	<li>200 x g細胞ペレットを取得する 5 分間の遠心分離機します。</li>
	<li>ゆっくりと上澄みを除去、その後、SFM 培地 10 ml 細胞ペレットを再懸濁し、セルの数をカウントします。</li>
	<li>1 x 10 の6セルは、各セル 100 mm 皿に SFM の 10 mL でプレートします。</li>
	<li>媒体ごとの 2 d を変更します。</li>
</ol>

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著者が明らかに何もありません。

培養主 HKCs は、3 年以上の診療所で傷を治療するために広く利用されているし、その時間以来常にタイムリーで効率的に臨床応用のための細胞の十分な数を取得することが重要です。したがって、実習では、真皮から表皮の分離を必要とする従来の分離法が難しく細胞と成体細胞を通過する低能力の低収量のためのこれらの要求を満たすために。ここでは、効率的に研究室と臨床応用の両方?...

目標は、臨床応用の特に成体からプライマリ人間ケラチノ サイト (HKCs) を分離する簡単で効率的なプロトコルを開発することでした。皮膚表皮幹細胞、皮膚の基底層に局在を持って増殖し区別するため、皮膚1,2,3,の機能を維持するケラチノ サイトを提供する可能性の高い4. HKCs 組織は皮膚組織工学と再生のために特に痛んだ肌の修復と遺伝子治療臨床応用5,6広く皮膚から分離しました。HKC ベースのアプリケーションにとって重要な問題は、効率的に分離し、高い潜在的な体外7,8で HKCs の大きな数字を展開することです。これらのメソッドは時々 時間がかかり、実行および低細胞収率と皮膚標本の種類によって限定されているなど、その他の制限に関して複雑なさまざまな研究グループは、幹のような HKCs の文化を生産する方法を開発しているが、9を使用します。例えば、皮膚組織中の HKCs を分離する従来の方法は、6真皮から表皮の分離と 2 段階の酵素消化を含まれます。そのメソッドは通常新生児の組織のためによく働くが、成体組織から細胞を分離するために使用するとき非常に困難します。
Y-27632、Rho 関連キナーゼ (ロック) の阻害剤は、表皮幹細胞の分離と植民地成長10、11,12の効率を大きく向上する報告されています。以前の研究では、我々 は Y-27632 は表皮細胞のクローナルな増殖が容易になりますが、特異的接着分子13式を制御することによって皮膚細胞の収量が減少を発見しました。また、G の中、成長と一次表皮細胞の収量をサポートすると呼ばれる新しいエアコン接種媒体を確立しました。Y-27632 を G の中を組み合わせることによりこの手法自発的に表皮真皮分離13,14のステップを除去する酵素の消化力の後表皮及び真皮細胞を区切ることができます。大人の皮膚組織から HKCs を分離するこの新しい方法の詳細な手順を述べる今以前の報告に基づき、.

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			<td height="23" style="height:23px;width:263px;">Countess automated cell counter</td>
			<td style="width:291px;">Invitrogen Inc.&#160;</td>
			<td style="width:264px;">C10227</td>
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			<td>Thermo Scientific</td>
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			<td>Life Technologies</td>
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a simplified and efficient method to isolate primary human keratinocytes from adult skin tissue

新鮮な皮膚組織とその拡大培養から分離されたプライマリ ヒト角は実験室の研究および臨床応用、広く使用されています。ヒト角化従来の分離方法は、低細胞回収率、減らされた細胞による成体組織から細胞を生成する効率的に証明されている 2 つの連続酵素消化手順生存率。我々 は最近、媒体の Rho キナーゼ阻害剤 Y-27632 を活用した皮膚組織中の人間の主な表皮の前駆細胞を分離する高度な方法を報告しました。従来のプロトコルと比較して、この新しいメソッドは、単純な簡単に、そして少ない時間のかかる、上皮幹細胞収量が増加し、その幹細胞の特性が向上します。さらに、新しい方法論は真皮から表皮の分離を必要としないし、したがって、異なったタイプの大人のティッシュからの細胞を隔離するために適しています。この新規分離培養手法は従来の方法の主な欠点を克服、高能室と臨床応用の両方表皮細胞の大量生産に適しています。ここでは、詳細に新しい手法について述べる。

(図 1 b) の従来の方法と新しい方法 (図 1 a) の回路図は図 1に示した。従来は、2 日目の手順を必要とする 2 段階の消化です。対照的に、新しいメソッドは、実行に約 3 時間かかりますワンステップ消化です。重要なは、ワンステップの新しいメソッドは接種培地に Y-27632 の存在に依存する同時に 2 つの...

Watch this Scientific Journal Video about A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue at JoVE.com

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

大人の皮膚組織からプライマリ ヒト角を効率的に分離するためのプロトコルをご紹介します。このメソッドは、自発的に皮膚細胞から表皮細胞を分離する接種培地で岩阻害剤の Y-27632 を使用して従来の手順を簡素化します。

大人の皮膚組織から人間ケラチノ サイト主を特定する簡易で効率的な方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

a-simplified-efficient-method-to-isolate-primary-human-keratinocytes

Research

JoVE Journal

Developmental Biology

10.0K ビュー.  Shandong University. 大人の皮膚組織からプライマリ ヒト角を効率的に分離するためのプロトコルをご紹介します。このメソッドは、自発的に皮膚細胞から表皮細胞を分離する接種培地で岩阻害剤の Y-27632 を使用して従来の手順を簡素化します。

ビデオ: A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

発生生物学

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

トランスフェクション、選択、およびAAVS1セーフハーバーへのGFP遺伝子とTALENを標的とする人間の人工多能性幹細胞のコロニーピッキング

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

ヒト胚性幹細胞から生成された内胚葉細胞の精製のための迅速かつ効率的な方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

大人の皮膚線維芽細胞からのインスリン分泌細胞を得るために安全かつ簡便な方法として、エピジェネティック変換

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

背マウス皮膚から毛包幹細胞と表皮角化細胞の単離

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

脱分化脂肪細胞を得るために効率的な方法

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

単離およびヒト胎盤組織由来の間葉系幹/間質細胞の増殖

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

世代、大人の肌からプライマリ ヒト角化培養

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

ショウジョウバエメラノガスターの胚から成人への生存率を評価する直接的かつ簡単な方法

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

大人の皮膚組織から人間ケラチノ サイト主を特定する簡易で効率的な方法

大人の皮膚組織から人間ケラチノサイト主を特定する簡易で効率的な方法