Name: isolation and culture of primary human gingival epithelial cells using y-27632
Uploaded: 2021-11-06T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632 at JoVE.com

この研究は、山東省自然科学財団(ZR2019ZD36)の主要プログラムと山東省の主要研究開発プログラム(2019GSF108107)からX.W.に支援されました。山東省の主要研究開発プログラム(2018GSF118240)からJ.G.へ山東省医療・健康科学技術開発プロジェクト(2018WS163)からZ.Xへ、山東省医療・健康科学技術開発プロジェクト(2019WS045)からJ.S.

このプロトコルで使用されるヒト組織は、本学の人間研究倫理委員会のガイドラインに従って、顎顔面外科の影響を受けた歯の抽出から廃棄された新鮮な成人の歯肉組織である(プロトコルNo.GR201711、日付:02-27-2017)。
1. 準備
<ol><li>10 mLのリン酸緩衝生理食塩水(PBS)を含む15 mLチューブに新鮮な成人歯肉組織を採取し、ペニシリン/ストレプトマイシン(P/S)を3%補?...

<ol>
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P., Grenier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+anti-inflammatory+activity+of+the+antimicrobial+peptides+human+beta-defensin-3+(hBD-3)+and+cathelicidin+(LL-37)+in+a+three-dimensional+co-culture+model+of+gingival+epithelial+cells+and+fibroblasts.">Synergistic anti-inflammatory activity of the antimicrobial peptides human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts.</a> PloS one. 9, 106766 (2014).</li><li>Sciezynska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+primary+keratinocytes-a+methods+review.">Isolation and culture of human primary keratinocytes-a methods review.</a> Experimental Dermatology. 28, 107-112 (2019).</li><li>Bryja, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+different+methods+used+in+the+primary+culture+of+oral+mucosa+cells.">Overview of the different methods used in the primary culture of oral mucosa cells.</a> Journal of Biological Regulators and Homeostatic Agents. 33, 397-401 (2019).</li><li>Kedjarune, U., Pongprerachok, S., Arpornmaeklong, P., Ungkusonmongkhon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+primary+human+gingival+epithelial+cells:+comparison+of+two+isolation+techniques.">Culturing primary human gingival epithelial cells: comparison of two isolation techniques.</a> Journal of Cranio-Maxillo-Facial Surgery: Official Publication of the European Association for Cranio-Maxillo-Facial Surgery. 29, 224-231 (2001).</li><li>Klingbeil, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+cellular+harvesting+methods+for+primary+human+oral+culture+of+keratinocytes.">Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes.</a> Cell and Tissue Banking. 10, 197-204 (2009).</li><li>Qian, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+simple+isolation+method+to+obtain+both+epidermal+and+dermal+stem+cells+from+human+skin+specimen.">One-step simple isolation method to obtain both epidermal and dermal stem cells from human skin specimen.</a> Methods in Molecular Biology. 1879, 139-148 (2019).</li><li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells.">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, 1251-1255 (2018).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+and+efficient+method+to+isolate+primary+human+keratinocytes+from+adult+skin+tissue.">A simplified and efficient method to isolate primary human keratinocytes from adult skin tissue.</a> Journal of Visualized Experiments: JoVE. , (2018).</li><li>Lauer, G., Wiedmann-Al-Ahmad, M., Otten, J. 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H., Forest, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratin+patterns+of+human+oral+mucosae+in+histiotypic+culture.">Cytokeratin patterns of human oral mucosae in histiotypic culture.</a> Archives of Oral Biology. 36, 747-758 (1991).</li><li>Kasai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+human+oral+mucosal+epithelial+cells+as+a+quality+control+of+the+cell+source+for+fabrication+of+transplantable+epithelial+cell+sheets+for+regenerative+medicine.">biopsy of human oral mucosal epithelial cells as a quality control of the cell source for fabrication of transplantable epithelial cell sheets for regenerative medicine.</a> Regenerative Therapy. 4, 71-77 (2016).</li><li>Oda, D., Dale, B. A., Bourekis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oral+epithelial+cell+culture.+II.+Keratin+expression+in+fetal+and+adult+gingival+cells.">Human oral epithelial cell culture. II. Keratin expression in fetal and adult gingival cells.</a> In Vitro Cellular &amp; Developmental Biology: Journal of the Tissue Culture Association. 26, 596-603 (1990).</li><li>Guarino, M., Tosoni, A., Nebuloni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+contribution+of+epithelium+to+organ+fibrosis:+epithelial-mesenchymal+transition.">Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition.</a> Human Pathology. 40, 1365-1376 (2009).</li><li>Hatakeyama, S., Yaegashi, T., Takeda, Y., Kunimatsu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+bromodeoxyuridine-incorporating,+p63-+and+p75(NGFR)-+expressing+cells+in+the+human+gingival+epithelium.">Localization of bromodeoxyuridine-incorporating, p63- and p75(NGFR)- expressing cells in the human gingival epithelium.</a> Journal of Oral Science. 49, 287-291 (2007).</li><li>Bayar, G. 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歯肉組織は歯周の完全性と健康を維持する重要な構造です。歯肉上皮細胞は歯周組織の修復および再生に重要な役割を有し、経口生物学、薬理学、毒物学、および口腔粘膜欠乏症を含む科学的研究および臨床応用および関連分野で使用することができる18。したがって、経口上皮細胞19を収穫するために安定かつ効率的な方法を開発する必要がある。初代上...

ヒト歯肉は、歯周組織を保護する第1線防御構造であり、物理的および化学的障壁1であるだけでなく、免疫応答に関与し、免疫バリア2,3を構成する炎症性メディエーターの異なるクラスを分泌する。歯肉上皮は歯周組織の修復および再生に重要な役割を果たす。したがって、歯肉上皮の防御と免疫を研究することは、歯膜炎の発生、診断、および治療を理解する上で非常に重要である。ヒト歯肉組織からの歯肉上皮細胞の単離および培養は、歯肉上皮の研究に必要な第一歩である。このような手順は、組織工学のための種子細胞の生産、歯周関連疾患のインビトロモデル、および歯周欠損を修復するための材料などの基本的な操作を必要とします。
初代歯肉上皮細胞は、vitro4における低い分裂率によって特徴付けられるが、研究者たちは何十年もの間、最適な単離および培養法を探してきた。現在までに、2つの異なる技術、直接外植法および酵素的方法は、一般的に、インビトロ4、5で一次歯肉上皮細胞を得るために実験室で使用されている。この直接外植法は、より低い量の組織標本および単純な単離手順の要件などの利点を有するが、汚染5に対する培養時間および感受性が長いという欠点を有する。酵素法は必要な培養時間を短縮するが、効率は比較的低く、使用する酵素や培地によって異なる。Kedjaruneら 6は、サブカルチャー(2週間)前により多くの時間を要する直接外植法が、酵素法と比較して歯肉上皮細胞の培養に成功したように見えることを示した。しかし、これら2つの方法を比較すると、Klingbeilら7は、酵素法が経口上皮細胞の一次培養に最良の結果を有し、最短時間(11.9日対14.2日)以内に最適な細胞収量を得ることが可能であることを発見した。
したがって、口腔上皮細胞4の単離および培養に対して、より便利で効果的な方法を開発することが重要であった。我々は、以前、Y-27632、Rho関連タンパク質キナーゼ(ROCK)の阻害剤を添加すると、成人の皮膚組織8、9、10からのヒト初表皮細胞およびケラチノサイトの単離手順を簡素化することを報告した。我々は、表皮を皮膚細胞から自然に分離し、初発表皮細胞8,9,10の増殖と収率を支える新しい調整接種培地であるG培地を開発した。本研究では、G培地とY-27632を組み合わせることにより、歯肉上皮細胞の新しい無血清分離および培養技術を開発した。本質的に、我々の方法は従来の2段階の酵素法の単純化に基づいているので、我々は新しい方法を直接外植法と比較した。この修飾酵素法は、歯肉の上皮細胞を歯肉組織から分離するのに必要な時間を大幅に短縮し、歯肉上皮細胞の培養の効率を高める。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Names</td>
			<td></td>
			<td></td>
			<td>Abbreviations &#38; Comments</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Shanghai Ruiyu Bio-science&#38;Technology Co.Ltd.</td>
			<td>BBA0218AC</td>
			<td>Automatic cell counting</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>51026333</td>
			<td>For cell incubation</td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75004380</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Eppendorf</td>
			<td>30702115</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>50 ml Centrifuge Tube</td>
			<td>KIRGEN</td>
			<td>171003</td>
			<td>For cell centrifugation</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge Tubes</td>
			<td>KIRGEN</td>
			<td>190691J</td>
			<td>For cell digestion</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</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>Gingival epithelial&#160; 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 HGGEPCs dissociation</td>
		</tr>
		<tr>
			<td>Dilution Medium</td>
			<td>Life Technologies</td>
			<td>50-9701</td>
			<td>For coating matrix</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>For HGGEPCs isolation</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Life Technologies</td>
			<td>17100-017</td>
			<td>For HGGEPCs 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>Gene Operation</td>
			<td>IAD1011</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>Dojindo Molecular Technologies</td>
			<td>CK04</td>
			<td>For Cell proliferation assay</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human CK18</td>
			<td>Abcam</td>
			<td>ab82254</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human Cytokeratin10</td>
			<td>Abcam</td>
			<td>ab76318</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>Mouse anti-human Vimentin</td>
			<td>Cell Signaling Technology</td>
			<td>3390</td>
			<td>For immunofluorescence staining of Gingival fibroblasts</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Human pan-ck</td>
			<td>BD</td>
			<td>550951</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-Ki67</td>
			<td>Abcam</td>
			<td>15580</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-p63</td>
			<td>Biolegend</td>
			<td>619002</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
		<tr>
			<td>rabbit anti-p75NGFR</td>
			<td>Abcam</td>
			<td>ab52987</td>
			<td>For immunofluorescence staining to check differentiation marker of HGGEPCs</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary human gingival epithelial cells using y-27632

歯肉組織は歯周組織を保護し、多くの口腔機能において意味のある役割を果たす最初の構造である。歯肉上皮は歯肉組織の重要な構造であり、特に歯周組織の修復および再生において重要である。歯肉上皮細胞の機能を研究することは、口腔欠損の修復や生体物質の適合性の検出など、科学的価値が重要です。ヒト歯肉上皮細胞は高度に分化された角化細胞であるため、その寿命は短く、通過が困難です。これまでのところ、歯肉上皮細胞を単離して培養する方法は2つしかなく、直接外植法と酵素法です。しかし、直接外植法を用いて上皮細胞を得るのに要する時間は長く、酵素法の細胞生存率は低くなる。臨床的には、歯肉組織の獲得は限られているため、安定、効率的、簡便 なインビトロ 分離および培養システムが必要です。我々は、上皮細胞の増殖を選択的に促進することができるRho関連キナーゼ(ROCK)阻害剤であるY-27632を添加することで、従来の酵素的方法を改善した。当社の改変酵素法は、従来の酵素法のステップを簡素化し、上皮細胞の培養効率を高め、直接外植法および酵素法に対して大きな利点を有する。

図1 は、直接外植法及び改変酵素法の概略図を示す。直接の外植法は、全体のプロセス中に任意の消化酵素を必要としません。対照的に、伝統的な酵素法は通常、消化酵素の2組、ディスパスおよびコラゲアーゼを必要とし、下層の線維芽細胞層から上皮シートを分離し、次にトリプシンを使用して上皮細胞を懸濁液に放出する。我々の新しい方法は分離のステップを省?...

Watch this Scientific Journal Video about Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632 at JoVE.com

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

ここでは、従来の方法にロック阻害剤Y-27632を添加することにより、ヒト歯肉上皮細胞の単離および培養のための改変方法を提示する。この方法は、より簡単で、時間のかかるおらず、幹細胞の特性を高め、実験室と臨床用途の両方で、より多くの高電位上皮細胞を産生します。

Y-27632を用いた初原ヒト歯肉上皮細胞の単離と培養

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

したがって、この細胞ベースのプロトコルの一般的な目標は、直接ナス法の欠点を改善するためにヒトギンジバル上皮細胞を単離して遭遇させることです。新しい修飾された内皮的方法から、我々の材料と機器に基づいて、比較的短時間でより多くの一次歯肉上皮細胞を得ることができます。したがって、このプロトコルで使用されるヒト組織は、施設の人間研究倫理委員会のガイドラインに従って顎顔面外科の影響を受けた歯の抽出から廃棄された新鮮な成人の歯肉組織でした。そして、それぞれ5分間に2回洗浄液を用いた。歯肉組織の消化.2つの外科用ブレードで組織を小さく切る。2.5mg/ミリリットルのディスパーゼとコラゲナーゼ-1溶液を1.5ミリリットル遠心分離チューブに加えます。1.5ミリリットル遠心分離チューブに組織片を入れます。37分で15~20分間インキュベート

Research

JoVE Journal

Bioengineering

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

ヒト細胞培養のための回転細胞培養システム：モデルとしてのヒト栄養膜細胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

光音響流量測定法を用いて循環メラノーマ細胞の検出と分離

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

細胞の脂質滴の分離：酵母細胞とヒト胎盤から2精製技術

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

外科的組織からの初代ヒト結腸腫瘍細胞の単離およびトラクションサイトメトリーのためのソフト弾性基板上にそれらを直接培養

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

2Dと3Dの肌の改善<em&gt;インビトロ</em分子クラウディングの使用&gt;モデル

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

マイクロ流体チャンバーモデル止血や血小板輸血に再構成された血液を使用しました<em&gt;インビトロ</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

脱細胞肺足場を使用してESC由来のマウス気道上皮細胞の生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人間歯槽骨膜から骨膜由来細胞の骨形成促進活性に及ぼすビタミン D の間葉系幹細胞の分離

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Handling and Assessment of Human Primary Prostate Organoid Culture

処理と人間プライマリ前立腺培養の評価

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...