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>

<ol><li>Ojeh, N., Pastar, I., Tomic-Canic, M., Stojadinovic, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+Cells+in+Skin+Regeneration%2C+Wound+Healing%2C+and+Their+Clinical+Applications%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Stem Cells in Skin Regeneration, Wound Healing, and Their Clinical Applications.</a> International Journal of Molecular Sciences. 16 (10), 25476-25501 (2015).</li>
<li>Sotiropoulou, P. A., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+homeostasis+of+the+skin+epidermis%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectives+in+BIology%22%5BJournal%5D)">Development and homeostasis of the skin epidermis.</a> Cold Spring Harbor Perspectives in BIology. 4 (7), a008383(2012).</li>
<li>Kamstrup, M., Faurschou, A., Gniadecki, R., Wulf, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+stem+cells+-+role+in+normal%2C+wounded+and+pathological+psoriatic+and+cancer+skin%5BTitle%5D)+AND+%22Current+Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin.</a> Current Stem Cell Research & Therapy. 3 (2), 146-150 (2008).</li>
<li>Blanpain, C., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+stem+cells+of+the+skin%5BTitle%5D)+AND+%22Annual+Review+of+Cell+and+Developmental+Biology%22%5BJournal%5D)">Epidermal stem cells of the skin.</a> Annual Review of Cell and Developmental Biology. 22 (22), 339-373 (2006).</li>
<li>Guo, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Building+a+microphysiological+skin+model+from+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Building a microphysiological skin model from induced pluripotent stem cells.</a> Stem Cell Research & Therapy. 4 (S1), S2(2013).</li>
<li>Aasen, T., Izpisua Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+cultivation+of+human+keratinocytes+from+skin+or+plucked+hair+for+the+generation+of+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells.</a> Nature Protocols. 5 (2), 371-382 (2010).</li>
<li>Bayati, V., Abbaspour, M. R., Neisi, N., Hashemitabar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skin-derived+precursors+possess+the+ability+of+differentiation+into+the+epidermal+progeny+and+accelerate+burn+wound+healing%5BTitle%5D)+AND+%22Cell+Biology+International%22%5BJournal%5D)">Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.</a> Cell Biology International. 41 (2), 187-196 (2017).</li>
<li>Hirsch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+the+entire+human+epidermis+using+transgenic+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Regeneration of the entire human epidermis using transgenic stem cells.</a> Nature. 551 (7680), 327-332 (2017).</li>
<li>Hentzer, B., Kobayasi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+human+epidermal+cells+from+fibroblasts+in+primary+skin+culture%5BTitle%5D)+AND+%22Archiv+f%C3%BCr+dermatologische+Forschung%22%5BJournal%5D)">Separation of human epidermal cells from fibroblasts in primary skin culture.</a> Archiv für dermatologische Forschung. 252 (1), 39-46 (1975).</li>
<li>Terunuma, A., Limgala, R. P., Park, C. J., Choudhary, I., Vogel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+procurement+of+epithelial+stem+cells+from+human+tissue+specimens+using+a+Rho-associated+protein+kinase+inhibitor+Y-27632%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.</a> Tissue Engineering Part A. 16 (4), 1363-1368 (2010).</li>
<li>Zhou, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ROCK+inhibitor+Y-27632+increases+the+cloning+efficiency+of+limbal+stem%2Fprogenitor+cells+by+improving+their+adherence+and+ROS-scavenging+capacity%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C%3A+Methods%22%5BJournal%5D)">ROCK inhibitor Y-27632 increases the cloning efficiency of limbal stem/progenitor cells by improving their adherence and ROS-scavenging capacity.</a> Tissue Engineering Part C: Methods. 19 (7), 531-537 (2013).</li>
<li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+Rho-associated+protein+kinase+%28ROCK%29+inhibitor+to+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Journal+of+Bioscience+and+Bioengineering%22%5BJournal%5D)">Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells.</a> Journal of Bioscience and Bioengineering. 114 (6), 577-581 (2012).</li>
<li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells%5BTitle%5D)+AND+%22Journal+of+Tissue+Engineering+and+Regenerative+Medicine%22%5BJournal%5D)">Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (2), e1251-e1255 (2018).</li>
<li>Zou, D., Pan, J., Zhang, P., Wu, X. <a target="_blank" href="https://www.researchgate.net/publication/306307941_A_new_method_to_isolate_human_epidermal_keratinocytes">A new method to isolate human epidermal keratinocytes.</a> Journal of Clinical Dermatology (in Chinese). 6, 424-429 (2016).</li>
<li>Cerqueira, M. T., Frias, A. M., Reis, R. L., Marques, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interfollicular+epidermal+stem+cells%3A+boosting+and+rescuing+from+adult+skin%29%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Interfollicular epidermal stem cells: boosting and rescuing from adult skin).</a> Methods in Molecular Biology. 989, 1-9 (2013).</li>
<li>Candi, E., Schmidt, R., Melino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cornified+envelope%3A+a+model+of+cell+death+in+the+skin%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">The cornified envelope: a model of cell death in the skin.</a> Nature Reviews Molecular Cell Biology. 6 (4), 328-340 (2005).</li>
<li>Breyer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+Rho+kinases+increases+directional+motility+of+microvascular+endothelial+cells%5BTitle%5D)+AND+%22Biochemical+Pharmacology%22%5BJournal%5D)">Inhibition of Rho kinases increases directional motility of microvascular endothelial cells.</a> Biochemical Pharmacology. 83 (5), 616-626 (2012).</li>
<li>Wozniak, M. A., Modzelewska, K., Kwong, L., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focal+adhesion+regulation+of+cell+behavior%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Focal adhesion regulation of cell behavior.</a> Biochimica et Biophysica Acta. 1692 (2-3), 103-119 (2004).</li>
</ol>

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、.

<table><tbody><tr><td>Countess automated cell counter</td><td>Invitrogen Inc.&amp;nbsp;</td><td>C10227</td><td>Automatic cell counting&amp;nbsp;</td></tr><tr><td>CO2 Incubator</td><td>Thermo Scientific</td><td>51026333</td><td>For cell incubating</td></tr><tr><td>Sorvall ST 16R Centrifuge</td><td>Thermo Scientific</td><td>75004380</td><td>Cell centrifuge</td></tr><tr><td>Constant Temperature Shaker</td><td>Shanghai Boxun</td><td>150036</td><td>For water bath</td></tr><tr><td>Electronic Scale</td><td>Harbin Zhonghui</td><td>1171193</td><td>For tissue weighing</td></tr><tr><td>Cell Culture Dish</td><td>Eppendorf</td><td>30702115</td><td>For cell culture</td></tr><tr><td>50ml Centrifuge Tube</td><td>KIRGEN</td><td>171003</td><td>For cell centrifuge</td></tr><tr><td>Cell Strainer</td><td>Corning incorporated</td><td>431792</td><td>Cell filtration</td></tr><tr><td>Phosphate buffered solution</td><td>Solarbio Life Science&amp;nbsp;</td><td>P1020-500</td><td>Washing solution</td></tr><tr><td>DMEM</td><td>Thermo Scientific</td><td>C11995500</td><td>Component of neutralization medium</td></tr><tr><td>Defined K-SFM</td><td>Life Technologies</td><td>10785-012</td><td>Epidermal cells culture medium</td></tr><tr><td>Penicillin Streptomycin</td><td>Thermo Scientific</td><td>15140-122</td><td>Antibiotics</td></tr><tr><td>Fetal Bovine Serum</td><td>Biological Industries</td><td>04-001-1AC5</td><td>Component of neutralization medium</td></tr><tr><td>0.05% Trypsin</td><td>Life Technologies</td><td>25300-062</td><td>For HKC dissociation</td></tr><tr><td>0.25% Trypsin&amp;nbsp;</td><td>Beijing Solarbio Science &amp;amp; Technology</td><td>T1350-100</td><td>For HKC dissociation</td></tr><tr><td>Coating Matrix Kit</td><td>Life Technologies</td><td>R-011-K</td><td>For coating matrix</td></tr><tr><td>Dispase</td><td>Gibco</td><td>17105-041</td><td>For HKC isolation</td></tr><tr><td>Collagenase Type I</td><td>Life Technologies</td><td>17100-017</td><td>For HKC isolation</td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma</td><td>9003-98-9</td><td>For HKC isolation</td></tr><tr><td>F12 Nutrient Mix, Hams</td><td>Life Technologies</td><td>31765035</td><td>Component of G-medium</td></tr><tr><td>B27 Supplement</td><td>Life Technologies</td><td>17504044</td><td>Growth factor in G-medium</td></tr><tr><td>FGF-2 Millipore</td><td>Merck Biosciences</td><td>341595</td><td>Growth factor in G-medium</td></tr><tr><td>Y-27632</td><td>Sigma-Aldrich</td><td>Y0503</td><td>ROCK inhibitor</td></tr><tr><td>Fungizone</td><td>Gibco</td><td>15290026</td><td>Preparation for G-medium</td></tr><tr><td>EGF Recombinant Human Protein</td><td>Gibco</td><td>PHG0311</td><td>Growth factor in G-medium</td></tr><tr><td>Cell Counting Kit-8</td><td>Thermo Scientific</td><td>NC9864731</td><td>cell proliferation and cytotoxicity assays</td></tr><tr><td>Mouse Anti-Human Cytokeratin5</td><td>Hewlett-Packard Development Company</td><td>MA-20142</td><td>For immunofluorescence staining to check differentiation marker of HKCs</td></tr><tr><td>Rabbit Anti-Human Loricrin</td><td>Covance</td><td>PRB-145p</td><td>For immunofluorescence staining to check differentiation marker of HKCs</td></tr><tr><td>Mouse anti-human Vimentin</td><td>Cell Signaling Technology</td><td>3390</td><td>For immunofluorescence staining of dermal fibroblasts</td></tr><tr><td>Integrin &amp;alpha;6(GOH3)</td><td>Santa Cruz&amp;nbsp;</td><td>SC-19622</td><td>flow cytometry analysis of HKCs</td></tr><tr><td>Rat IgG2a FITC</td><td>Santa Cruz&amp;nbsp;</td><td>SC-2831</td><td>negative control antibody of &amp;alpha;6-integrin&amp;nbsp; in flow cytometry analysis&amp;nbsp;</td></tr></tbody></table>

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

10.1K 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

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

黒色腫切片 3 D 回転楕円体の皮膚モデル

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

がん研究

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. ...

発生生物学

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

ケラチノサイト幹細胞の可塑性をモデル化する高スループット表皮球面培養システムの確立

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

生物学

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 ...

Generating Primary Cultures for Keratinocyte Live Cell Imaging

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro.&#160;Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.

This protocol outlines the culture of freshly obtained (passage 0) keratinocytes for use in live cell imaging.

ケラチノサイト生細胞イメージングのための初代培養物の生成

Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in ...

生物工学

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

神経科学

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 ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

マウス表皮ケラチノサイトの単離とインビトロクロノジェニック培養

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

成人マウス口蓋からの一次口腔角化細胞の分離と培養

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

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

要約

さらに動画を探す

黒色腫切片 3 D 回転楕円体の皮膚モデル

世代、大人の肌からプライマリヒト角化培養

ケラチノサイト幹細胞の可塑性をモデル化する高スループット表皮球面培養システムの確立

ヒト皮膚から大人上皮幹細胞の単離と培養

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

ケラチノサイト生細胞イメージングのための初代培養物の生成

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

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

マウス表皮ケラチノサイトの単離とインビトロクロノジェニック培養

成人マウス口蓋からの一次口腔角化細胞の分離と培養

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

要約

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黒色腫切片 3 D 回転楕円体の皮膚モデル

世代、大人の肌からプライマリ ヒト角化培養

ケラチノサイト幹細胞の可塑性をモデル化する高スループット表皮球面培養システムの確立

ヒト皮膚から大人上皮幹細胞の単離と培養

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

ケラチノサイト生細胞イメージングのための初代培養物の生成

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

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

マウス表皮ケラチノサイトの単離とインビトロクロノジェニック培養

成人マウス口蓋からの一次口腔角化細胞の分離と培養

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

世代、大人の肌からプライマリヒト角化培養