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プラスチック製の手術健康な成人ボランティアの皮膚のサンプルのコレクションには、ホスト機関の倫理委員会の承認が必要です。このプロトコルは、地域倫理委員会の地域中央ユラン地域、デンマーク (M-20110027) によって承認されました。ここで説明したメソッドが Maciaqらによって同様の研究から派生します。10劉と Karasek11。
1. 人間の皮膚からケラチノ サイトの分離
<ol>
	<li>次の解決策をすることから始めます: DPBS 0.25% トリプシンと 0.1% ブドウ糖 50 mL ソリューション。ミックスとフィルターは殺菌ソリューション (0.2 μ m) です。RPMI 1640 + 2 %fbs ソリューション、DPBS と表皮細胞無血清培地 (KSFM) の 50 mL の 10 mL を準備します。KSFM 500 mL に KSFM サプリメントとゲンタマイシンの 250 μ L を追加します。あらかじめ使用前に 37 ° C への解決策を温めます。</li>
	<li>プラスチック製の手術健康な成人ボランティアから皮膚を収集します。皮膚の試料約 10 cm × 15 cm がサイズが異なります。冷却要素を含む発泡スチロール ボックスで皮膚検体の移送によって交通機関の下で、皮膚を冷却を保ちます。必要な場合、皮膚のサンプルと一晩 4 ° C で保存できます。</li>
	<li>滅菌ハサミ、メス、鉗子を使用して皮膚のセクションの下にから脂肪を除去します。</li>
	<li>針を使用して板の上に滅菌カバーの肌のセクション (約 10 cm × 15 cm) をバックルします。まず、乾燥滅菌ガーゼのパッドと肌をきれい。その後、70% エタノールで滅菌ガーゼのパッドを使用して肌をきれい。</li>
	<li>足の平面を使用して、皮膚のセクションの上部層 (表皮) のカットし、9 cm シャーレに入れてください。ペトリ皿に (手順 1.1 で準備) DPBS/トリプシン/グルコース溶液の 25 mL をすぐに追加します。37 ° C で 30 分間インキュベートします。</li>
	<li>ピペットを使用して DPBS/トリプシン/グルコース溶液をペトリ皿から削除し、RPMI 1640 + 2% トリプシンを不活化する政府短期証券の 10 mL を追加します。</li>
	<li>2 つの鉗子を使用して、表皮の細胞の優しくこすると、表皮と皮膚部分の皮膚のコンパートメントを攪拌によって今媒体に解放します。</li>
	<li>金属フィルター (1 mm 穴の大きさ) を介して表皮細胞懸濁液をフィルタ リングし、50 mL のチューブで収集します。表皮細胞懸濁液を含む 50 の mL の管に金属を介して懸濁液フィルター 10 s. フィルターの FBS と渦なし RPMI 1640 の 10 mL を含む 50 mL のチューブに残りの皮膚セクションを追加します。50 mL の容量に DPBS を追加します。</li>
	<li>室温で 450 x g で 10 分間の細胞懸濁液を遠心します。</li>
	<li>上澄みを除去し、37 ° C KSFM 細胞ペレットのサイズによって約 10 mL で表皮細胞ペレットを再懸濁します。トリパン ブルー染色顕微鏡法を使用してセルをカウントします。10 cm x 15 cm 肌セクションから得られた細胞の数は約 50-100 x 10 の6セルです。各 75 cm2培養フラスコに一緒に 12 mL 37 ° C KSFM の 8 x 10 の6セルを転送します。細胞の均一な分布を確実に培養フラスコを軽く振る。</li>
	<li>100% 湿度と 5% CO2と 37 ° C の定温器でケラチノ サイトを孵化させなさい。2 日間、週 3 回後に媒体を変更します。通路細胞文化が 70-80% 合流するケラチノ サイトの増殖率によって約 2 週間かかります。</li>
</ol>
2. ケラチノ サイトの継
<ol>
	<li>インキュベーターの 37 ° C に 0.05% トリプシン EDTA 溶液の適切な量の熱。75 cm2培養用フラスコの 4.5 mL の 0.05% トリプシン-EDTA 溶液を使用します。</li>
	<li>細胞からメディアを削除し、4.5 mL/75 cm2培養用フラスコ、細胞に 0.05% トリプシン EDTA 溶液で加温を追加します。インキュベーター (37 ° C) で培養用フラスコを置き、約 5 分後に顕微鏡下で細胞が緩み始めている場合を確認します。</li>
	<li>ときに細胞の約 50% は消え、残りのセルを緩める手に対する培養用フラスコは優しくヒットします。トリプシンを不活化できる、75 cm2フラスコに RPMI 1640 + 2 %fbs (37 ° C) の 6 mL を追加し、50 mL のチューブに細胞懸濁液を転送します。</li>
	<li>RPMI 1640 + 2% の 3 ml 培養フラスコを洗い FBS 50 mL チューブに追加。室温で 450 x g で 10 分の細胞を遠心します。
	<ol>
		<li>KSFM (37 ° C) の 10 mL の小球形にされた細胞を再懸濁します。セルをカウントし、3 x 10 の6セルを KSFM (37 ° C) の 20 mL と 150 cm2培養用フラスコに転送します。細胞の均一な分布を確実に培養用フラスコを軽く振る。合流を文化が 80-90% の場合、細胞を凍結、(セクション 3、凍結プロトコル下記参照) ケラチノ サイトの増殖率によって約 4-6 日かかります。適切な凍結する在庫を生成するケラチノ サイトを展開します。</li>
	</ol>
	</li>
</ol>
3. ケラチノ サイトの凍結
<ol>
	<li>インキュベーターの 37 ° C に 0.05% トリプシン EDTA 溶液の適切な量の熱。150 cm2培養用フラスコに 0.05% トリプシン-EDTA 溶液 9 mL を使用します。</li>
	<li>細胞からメディアを削除し、9 mL/150 cm2培養用フラスコ、細胞に 0.05% トリプシン EDTA 溶液で加温を追加します。インキュベーター (37 ° C) で培養用フラスコを置き、約 5 分後に顕微鏡下で細胞が緩み始めている場合を確認します。</li>
	<li>細胞の約 50% は消え、残りのセルを緩める手に対する培養用フラスコ ヒット優しく。トリプシンを不活化できる、150 cm2フラスコに RPMI 1640 + 2 %fbs (37 ° C) の 12 の mL を追加し、50 mL のチューブに細胞懸濁液を吸引します。</li>
	<li>RPMI 1640 + 2% の 6 mL の培養フラスコを洗い FBS 50 mL チューブに追加。4 ° C で 450 x g で 10 分間のチューブを遠心分離します。冷たいセルフリーズの媒体 (KSFM + 10 %dmso) を準備します。</li>
	<li>KSFM + 10 %dmso 細胞ペレットを再懸濁します、セルをカウントし、標準的な遅く凍結凍結保存方法12を使用して 6 × 106セル/mL の濃度で細胞を凍結します。</li>
	<li>使用する準備ができるまでには、液体窒素 (気相) のケラチノ サイトを格納します。</li>
</ol>
4. 解凍・冷凍ケラチノ サイトを培養
<ol>
	<li>37 ° C の水浴の適切な凍結バイアルを急速に解凍します。70% エタノールを使用、クリオバイアルの外側をきれいにします。培養用フラスコ細胞 (約 15,000 のセル/cm2) の適切な番号を転送し、すぐに培養用フラスコに予め温めておいた培 (KSFM) を追加します。 実験のセットアップによっては、培養フラスコの任意のサイズを使用できます。</li>
	<li>25 cm2培養用フラスコの培地 5 mL を使用します。</li></ol>セルの数は追加培養用フラスコが細胞の成長変化ドナーからドナーにするために異なる場合があります。100% 湿度と 5% CO2と 37 ° C の定温器で細胞を孵化させなさい。
	<li>変更後 2 日後と 3 回中毎週新鮮な予め温めておいた KSFM を使用します。 注: 我々 のデータは、平均でここで説明したプロトコルによって分離した細胞の 95% が陽性表皮細胞マーカー cytokeratine 1413を実証しています。</li>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocytes:+key+immunocytes+of+the+integument.">Keratinocytes: key immunocytes of the integument.</a> Am J Pathol. 143 (2), 325-331 (1993).</li><li>Fuchs, E., Raghavan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+under+the+skin+of+epidermal+morphogenesis.">Getting under the skin of epidermal morphogenesis.</a> Nat Rev Genet. 3 (3), 199-209 (2002).</li><li>Bos, J. D., Kapsenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+skin+immune+system+Its+cellular+constituents+and+their+interactions.">The skin immune system Its cellular constituents and their interactions.</a> Immunol Today. 7 (7-8), 235-240 (1986).</li><li>Nestle, F. 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.著者には、利害の対立はないです。

ここでは、大人の皮膚から人間ケラチノ サイト主を簡単に分離する方法、およびそれらを体外培養する方法について述べる。このモデルは、表皮の細胞生物学の調査のための広範なアプリケーションを持つことができます、皮膚病の研究に興味を持って研究に便利することができます。
ここで説明したプロトコルの利点のいくつかは新生児の男性の割礼を受けて?...

皮膚は人体の最大の臓器であり、外部環境に対する保護バリアとして機能します。皮膚は 2 つの主要な層で構成される: 真皮と表皮が皮膚の最外層を構成する表皮。表皮の最も豊富なセル型はケラチノ サイト細胞質量1,2の 95% 以上を構成します。ケラチノ サイトは、表皮の分化の各段階で維持され、分化3の特定の段階に対応する基底、棘、粒状、角質層に分類されます。ケラチノ サイトの主な機能は、それにより外の世界にそのままバリアを作り出す表皮の構造的な整合性を提供することです。
ケラチノ サイトも皮膚の病原体に対する防御の最初の行を表す、したがって自然免疫応答4,5で重要な役割を果たします。ケラチノ サイトの外部からの刺激への暴露は、サイトカイン、ケモカイン、および抗菌ペプチドを含む様々 な炎症性メディエーターの数の生産をその後、細胞内のシグナル伝達経路の活性化につながります。これら表皮細胞由来のタンパク質は募集し、特定の T 細胞6、7好中球、樹状細胞などの免疫細胞を活性化して炎症反応に参加します。したがって、ケラチノ サイトは、多数の生物学的プロセスに重要な役割を果たす、ので、ここで示した手法の背後にある理論的根拠は皮膚生物学を勉強するための in vitroモデルを生成します。新生児包皮から得られるプライマリ ケラチノ サイト培養皮膚生物学8,9の研究に使われます。ただし、ここで説明する手法に男女両方からケラチノ サイトは、細胞の高い生物学的多様性の結果取得されます。
ここでは、メンテナンスを含むとケラチノ サイトの凍結分離と大人の皮膚からプライマリ ヒト角化世代の詳細なプロトコルを提案します。このメソッドの全体的な目標は、 in vitro皮膚の生物学を研究するモデルとして使用することができますプライマリの人間ケラチノ サイトを生成することです。

<table><tbody><tr><td>KSFM</td><td>ThermoFisher Scientific</td><td>17005-034</td><td>Cell culture medium</td></tr><tr><td>KSFM supplements</td><td>ThermoFisher Scientific</td><td>37000-015</td><td>Supplements for KSFM</td></tr><tr><td>DPBS</td><td>ThermoFisher Scientific</td><td>14190-144</td><td>DPBS without Calcium and Magnesium</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>Dimethyl sulfoxid</td></tr><tr><td>Gentamycin</td><td>ThermoFisher Scientific</td><td>15710-049</td><td>Cell culture medium additive</td></tr><tr><td>Sterilization filter</td><td>Sartorius</td><td>16534</td><td>Syringe filter with a pore size of 0.2 &amp;micro;m</td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T7409</td><td>Used to trypsinize cells</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>-</td></tr><tr><td>RPMI-1640</td><td>ThermoFisher Scientific</td><td>61870-010</td><td>-</td></tr><tr><td>FBS</td><td>ThermoFisher Scientific</td><td>16000044</td><td>Used to inactivate trypsin</td></tr><tr><td>Forceps</td><td>-</td><td>-</td><td>Forceps from any company can be used</td></tr><tr><td>Scissors</td><td>-</td><td>-</td><td>Scissors from any company can be used</td></tr><tr><td>Scalpel</td><td>Swann Morton</td><td>0501</td><td>Scalpels from any company can be used</td></tr><tr><td>70% ethanol</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Gauze pads</td><td>NOBAMED</td><td>875420</td><td>Gauze pads from any provider can be used</td></tr><tr><td>Foot planer</td><td>Credo Solingen</td><td>1510</td><td>Foot planer from any provider can be used</td></tr><tr><td>Petri dishes</td><td>TPP</td><td>93100</td><td>Petri dishes from any provider can be used</td></tr><tr><td>Metal filter</td><td>-</td><td>-</td><td>In-house 1 mm hole size metal filter</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>NUNC</td><td>156499</td><td>-</td></tr><tr><td>150 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>TTP</td><td>90151</td><td>-</td></tr><tr><td>0.05% Trypsin-EDTA solution</td><td>ThermoFisher Scientific</td><td>25300-062</td><td>Used to trypsinize cells when passaging</td></tr></tbody></table>

generation and culturing of primary human keratinocytes from adult skin

ケラチノ サイトの主な機能は、それにより外の世界に機械的な障壁を維持する表皮の構造的な整合性を提供することです。加えて、ケラチノ サイトは、高速、非特異的に抗原刺激に応答する生得の免疫組織の部分であることによって開始、保守、および表皮の免疫応答の調節に重要な役割を再生します。ここで、成人の皮膚から人間ケラチノ サイト主の隔離のためのプロトコルを記述し、これらの細胞はカルシウム誘発性分化、分化マーカー involucrin の発現の増加によって測定されるように応答を示します。さらに、分離のケラチノ サイトが p38 MAPK 経路の活性化による細胞内シグナル伝達経路の IL 1 β による活性化に敏感であることを示します。一緒に取られて、分離、成人の皮膚からプライマリ ヒト角化培養するための方法について述べる。優勢な細胞型表皮ケラチノ サイトであるために、このメソッドは皮膚の生物学体外の分子メカニズムの研究に有用です。

カルシウム誘発性分化 ヒト角は、カルシウム14,,1516と分化を受けます。プライマリ ヒト角が分離・培養上のプロトコルで説明されているようです。約 50-60% 合流がカルシウム (1.2 mM) と細胞が刺激され、車両とセルの写真は、0、1、および 2 の日に撮影されました。カルシウム刺激時に観察されるケラチノ サ...

Watch this Scientific Journal Video about Generation and Culturing of Primary Human Keratinocytes from Adult Skin at JoVE.com

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

人間の皮膚は外部環境に対する防御の最初の行として機能します。大人の皮膚から人間ケラチノ サイト主を分離する手法を提案します。これらの孤立したケラチノ サイトは、多数の実験のセットアップに便利、皮膚の生物学の in vitroの分子機構を研究するための非常に適してモデル

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. ...

generation-culturing-primary-human-keratinocytes-from-adult

Research

JoVE Journal

Developmental Biology

16.3K ビュー.  Aarhus University Hospital. 人間の皮膚は外部環境に対する防御の最初の行として機能します。大人の皮膚から人間ケラチノ サイト主を分離する手法を提案します。これらの孤立したケラチノ サイトは、多数の実験のセットアップに便利、皮膚の生物学の in vitroの分子機構を研究するための非常に適してモデル

ビデオ: Generation and Culturing of Primary Human Keratinocytes from Adult Skin

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

発生生物学

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

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

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 ...

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

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

3次元の再構築されたヒト表皮を大規模に育成

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

生物工学

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 ...

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 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 ...

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

ヒト皮膚ケラチノサイト単離:皮膚サンプルから初代ケラチノサイトを培養する方法

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...

世代、大人の肌からプライマリヒト角化培養

要約

さらに動画を探す

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

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

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

3次元の再構築されたヒト表皮を大規模に育成

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

成人マウス口蓋からの一次口腔角化細胞の分離と培養

ヒト皮膚ケラチノサイト単離:皮膚サンプルから初代ケラチノサイトを培養する方法

世代、大人の肌からプライマリ ヒト角化培養

要約

さらに動画を探す

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

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

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

3次元の再構築されたヒト表皮を大規模に育成

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

成人マウス口蓋からの一次口腔角化細胞の分離と培養

ヒト皮膚ケラチノサイト単離:皮膚サンプルから初代ケラチノサイトを培養する方法

世代、大人の肌からプライマリヒト角化培養

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