Name: primary culture of porcine retinal pigment epithelial cells
Uploaded: 2022-09-23T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

著者らは、この研究で細胞に貢献しているすべての動物に感謝と敬意を表したいと思います。この研究の一部は、中国の国家重点研究開発プログラム(2019YFA0111200、Yi Liao & Yuan Gao、およびGrant nos. 2018YFA0107301、Wei Li)からの助成金によって支援されました。著者らは、共焦点イメージングの技術サポートを提供してくれた厦門大学医学部中央研究所のJingru HuangとXiang Youに感謝しています。

実験動物の使用は、視覚眼科学研究協会(ARVO)の規則に準拠し、厦門大学の実験動物管理の倫理委員会によって承認されました。
1. 実験用手術器具、組織消化酵素、細胞培養バッファーの調製
<ol><li>実験用手術器具を準備し、眼球解剖の前日に2組の眼科手術用ハサミ及び鉗子を洗浄及びオートクレーブ処理し、その後、手術器具の入った箱を一般プロトコー...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanin+pigment+in+the+pigmented+epithelium+of+the+retina+of+the+embryo+chick+eye+studied+in+vivo+and+in+vino.">Melanin pigment in the pigmented epithelium of the retina of the embryo chick eye studied in vivo and in vino.</a> The Anatomical Record. 18, 260-261 (1920).</li><li>Schnichels, S., 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=Retina+in+a+dish:+Cell+cultures,+retinal+explants+and+animal+models+for+common+diseases+of+the+retina.">Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina.</a> Progress in Retinal and Eye Research. 81, 100880 (2021).</li><li>D'Antonio-Chronowska, A., D'Antonio, M., Frazer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+iPSC-derived+retinal+pigment+epithelium+cells+(iPSC-RPE).">In vitro differentiation of human iPSC-derived retinal pigment epithelium cells (iPSC-RPE).</a> Bio-Protocol. 9 (24), 3469 (2019).</li><li>Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+of+the+human+RPE+cell+line,+ARPE-19,+induced+by+nicotinamide.">Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.</a> Experimental Eye Research. 179, 18-24 (2019).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. 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ここでは、ブタ眼球からのRPE細胞の単離、培養、および特性評価のための詳細で最適化されたプロトコルが、RPE細胞のin vitro特性評価およびRPE関連障害研究のための優れたモデルを生成することが記載されている。ヒト、マウス、およびラットの眼からRPEを単離するための方法は、以前に記載されている23、24、25。?...

網膜色素上皮(RPE)は、網膜1の外層の光受容体と脈絡毛細血管の間に位置し、血液網膜関門の形成、栄養素と網膜代謝物の輸送と交換、正常な視覚サイクルを維持するためのビタミンAのリサイクル、脱落した光受容体外側セグメント(POS)の食作用とクリアランスなど、複数の機能を持っています2,3。.POSは視力を生成するために絶え間ない自己複製を必要とするため、RPE細胞は網膜の恒常性を維持するために剥離したPOSを継続的に飲み込む必要があります4。したがって、RPE機能障害は、加齢黄斑変性症(AMD)4、網膜色素変性症(RP)5、レーバー先天性無力症6、糖尿病性網膜症7など、多くの失明性眼疾患を引き起こします。今まで、これらの病気のほとんどの正確な病因はとらえどころのないままです。その結果、RPE細胞生物学、病理学的変化、およびその根底にあるメカニズムを研究するためにRPE細胞培養が確立されます。
細胞生物学を研究するための最も単純なモデルとして、RPE細胞の培養は早くも1920年代に開始されました8。ARPE-19はRPE細胞として広く用いられているが、色素沈着の喪失、石畳の形態、特にこの細胞株におけるバリア機能は、多くの懸念を引き起こす9。それに比べて、初代ヒトRPE細胞の培養は、生理学的および病理学的研究のためのより現実的なシナリオを提供します9。ただし、可用性が比較的限られているため、使用が制限され、倫理的な問題が常に存在します。さらに、いくつかのグループは、RPE細胞を培養するためにマウスモデルを使用しました。しかし、マウスの目のサイズは小さく、1回の培養には通常多くのマウスが必要であり、これは便利ではありません9。最近、科学者は、ヒト胚性幹細胞または人工多能性幹細胞を使用してRPE細胞を誘導する新しい方法を開発しました。この技術は、遺伝性RPE障害の治療に特に可能性を秘めているが、時間がかかり、通常、成熟RPE細胞を生成するのに数ヶ月を要する10。これらの問題を克服するために、ここでは、実験室で高純度RPE細胞を日常的に分離および培養するためのわかりやすいプロトコルを紹介します。適切な培養条件下では、これらの細胞は典型的なRPE機能を示し、典型的なRPE形態を示すことができます。したがって、この培養法は、RPE生理学を理解し、細胞毒性を研究し、関連する眼疾患の病理学的メカニズムを調査し、薬物スクリーニングを実施するための優れたモデルを提供することができます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARPE-19 cells</td>
			<td>CCTCC</td>
			<td>GDC0323</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Yeasen</td>
			<td>36101ES60</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM 880 with Airyscan</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch</td>
			<td>Bio-Rad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sangon</td>
			<td>F619301</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>NEST</td>
			<td>121621EH01</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>NEST</td>
			<td>29821075P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12 Medium</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td>For TER measurement</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ExCell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>0300-0108P</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Rabbit IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>5196-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>MCE</td>
			<td>HY-N0583/CS-2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution (20 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 Instrument</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Liothyronine</td>
			<td>MCE</td>
			<td>HY-A0070A/CS-4141</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM(1X)+GlutaMAX Medium</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA(100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N1</td>
			<td>Sigma-Aldrich</td>
			<td>SLCF4683</td>
			<td></td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>New Cell&#38;Molecular Biotech</td>
			<td>P10300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat Powdered Milk</td>
			<td>Solarbio</td>
			<td>D8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>SparkJade</td>
			<td>SJ-MV0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+-K+ ATPase antibody</td>
			<td>Abcam</td>
			<td>ab76020</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparation Kit(10%)</td>
			<td>Epizyme</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>primary Human RPE cells&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Generous gift from Shoubi Wang lab&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad by Dotmatics</td>
			<td>version 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktails</td>
			<td>APExBIO</td>
			<td>K1024</td>
			<td></td>
		</tr>
		<tr>
			<td>PRE65 antibody</td>
			<td>Proteintech</td>
			<td>17939-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PEDF antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-390172</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>100 x penicillin/streptomycin&#160;</td>
			<td>Biological Industries</td>
			<td>03-031-1BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>RARBIO</td>
			<td>RA-9005</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>Toyobo</td>
			<td>FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Damas-beta</td>
			<td>107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo-Fisher&#160;</td>
			<td>15596026</td>
			<td>RNA extraction solution</td>
		</tr>
		<tr>
			<td>TB Green Fast qPCR Mix</td>
			<td>Takara</td>
			<td>RR430A</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well transwell inserts</td>
			<td>Labselect</td>
			<td>14212</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF antibody</td>
			<td>Proteintech</td>
			<td>19003-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>VEGF ELISA kit</td>
			<td>Novusbio</td>
			<td>VAL106</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>ABclonal</td>
			<td>A0659</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
	</tbody>
</table>

primary culture of porcine retinal pigment epithelial cells

網膜色素上皮(RPE)は、網膜の脈絡膜と神経網膜の間に位置する偏光色素上皮細胞の単層です。食作用、栄養素/代謝物輸送、ビタミンA代謝などを含む複数の機能がRPEによって日常的に行われています。RPE細胞は、再生能力がほとんどまたはまったくない最終分化上皮細胞です。RPE細胞の喪失は、加齢黄斑変性症などの視覚障害につながる複数の眼疾患をもたらします。したがって、細胞株よりもin vivoのRPEによく似た初代RPE細胞のin vitro培養モデルの確立は、RPE細胞の特徴的かつ機構的な研究にとって重要です。ヒトの眼球の供給源が限られていることを考慮して、我々は初代ブタRPE細胞を培養するためのプロトコルを作成します。このプロトコルを使用することにより、RPE細胞は成体のブタ眼球から容易に解離することができる。その後、これらの解離した細胞は培養皿/インサートに付着し、増殖してコンフルエントな単層を形成し、in vivoで上皮組織の重要な特徴を2週間以内に迅速に再確立します。qRT-PCRにより、初代ブタRPE細胞は、天然RPE組織と同等のレベルで複数のシグネチャ遺伝子を発現する一方で、ヒトRPE様細胞ARPE-19では、これらの遺伝子のほとんどの発現が失われたり、大幅に減少したりすることが実証されています。さらに、免疫蛍光染色は、培養初代細胞におけるタイトジャンクション、組織極性、細胞骨格タンパク質の分布、およびビタミンA代謝に重要なイソメラーゼであるRPE65の存在を示しています。全体として、高純度でネイティブなRPE機能を備えた初代ブタRPE細胞を培養するためのわかりやすいアプローチを開発し、RPE生理学を理解し、細胞毒性を研究し、薬物スクリーニングを容易にするための優れたモデルとして役立つ可能性があります。

初代ブタRPE(pRPE)細胞を10%FBSを含むDMEM/ベーシック培地で培養し、播種後2日目(図2A)、6日目(図2B)、および10日目(図2C)に光学顕微鏡下で細胞形態を撮影しました。1週間後、石畳の形態を有する色素性pRPE細胞のコンフルエント単層が観察された。
初代pRPE細胞をよりよく特徴付けるために、継代3(P3)18の初代ヒ?...

Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Primary Culture of Porcine Retinal Pigment Epithelial Cells

ここでは、初代ブタ網膜色素上皮細胞を 試験管内で 培養するためのわかりやすい方法が提示されている。

ブタ網膜色素上皮細胞の初代培養

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

RPE関連障害は、依然として疾患の混合の主要な部分を占めていますが、RPE関連障害の生理学的および病理学的研究の優れたモデルとして役立つ初代RPE細胞の仮想培養における効果的な残骸はありません。このプロトコルでは、初代IPCを培養するためのわかりやすいプロトコルを提供し、後のRP特性を迅速に回復および維持することができ、この方法を使用することにより、人々はマシステート研究および保護薬物スクリーニングからRP細胞を迅速に生成でき、RPE障害の新しい治療法の開発を促進することができると考えています。段階的な価値のデモンストレーションにより、RP細胞を研究したいさまざまなラボでこの方法を再現するのがはるかに簡単になります。組織消化酵素マラクアドの解凍を開始し、1X PBSを滅菌します。0.22マイクロメートルのシリンジフィルターユニットを通して溶液をろ過することにより、2%ペニシリンと2%ストレプトマイシンを補給します。培養プレートとトランスウェルインサートのウェルを1X PBSで洗浄します。PBSをウェルから取り出し、ピペットでトランスウェルし、1ミリリットルの新鮮な1X PBSを下チャンバに加え、600マイクロリットルの10マイクログラム/ミリリットルのラミニン溶液を上部チャンバに加えます。プレートとトランスウェルインサートをこのヘリシカルチャーインキュベーターで摂氏37度、二酸化炭素5%で一晩インキュベートします。その後、ラミネート溶液を上部チャンバーから取り出し、1ミリリットルの冷たい1X PBSで2回洗浄してから、セルを装着します75%エタノールとUV光でラミネートフローフードを清掃します。約25ミリリットルの75%エタノールを含む50ミリリットルの滅菌遠心分離管3本、約25ミリリットルの1X PBSを含む50ミリリットルの滅菌遠心分離管3本、および約15ミリリットルの1X PBSを含む3つの10センチメートル滅菌細胞培養皿を準備します。10センチのペトリ皿に約15ミリリットルの75%エタノールに4頭のブタの眼球を浸し、はさみと鉗子を使用して、残っている結合組織と筋肉をすべて切り取ります。眼球を除染するには、75%エタノールで満たされた3つの50ミリリットルの滅菌遠心チューブに4つの眼球を浸して洗浄します。次に、1X PBSで満たされた3つの50ミリリットルの滅菌遠心チューブで眼球を順番に洗浄します。眼球を完全に洗うために毎分チューブを反転させます。4つの眼球を1X PBSを含む10センチメートルの滅菌細胞培養皿に移します。各眼球の外面を再度トリミングして、視神経と小さな破片を取り除きます。はさみを使用して、辺縁と強膜の交差点に小さな切り込みを入れます。次に、角膜、虹彩、水晶体、硝子体、神経網膜を取り除きます。RPE脈絡膜強膜複合体を新しい10センチメートルの細胞培養皿に移し、4つのカットをしてアイカップを平らにし、四つ葉のクローバーの形にします。4つのRPE脈絡膜強膜複合体を新しい10センチメートルの皿に入れ、20ミリリットルの新鮮なトリプシンEDTA溶液を注いでRPE脈絡膜強膜複合体をマージすることにより、EDTA溶液消化のトリップを実行します。皿を摂氏37度の細胞培養インキュベーターにほぼ30分間入れます。30分後、インキュベーターから皿を取り出し、10%FBSを含む20ミリリットルのプレウォーム細胞培養培地を皿に加えます。EDTA溶液中のトリップを中和するには、5ミリリットルのトランスファーピペットを使用して、数回穏やかにピペッティングしてRPE細胞を解離させます。細胞懸濁液を15ミリリットルの遠沈管に集め、さらに10ミリリットルの新鮮な培養培地をFBSで静かにピペットでピペットで留めて、皿上のRPE脈絡膜強膜複合体を洗浄します。チューブを300 Gで室温で5分間遠心分離することにより、RPE細胞を収集します。ピペットを使用してスーパーエージェントを吸引し、FBSを含むさらに5ミリリットルの培養培地を加えて細胞を再懸濁します。さらに300 Gで5分間遠心分離します。スーパーエージェントをデカントし、12ミリリットルの培地で細胞を再懸濁します。次に、ウェルあたり100, 000〜200, 000個の細胞を12ウェル培養プレートまたはトランスウェルインサートに播種します。2日ごとに培地を交換し、細胞がインサートごとにウェルの表面を完全に覆ったら、血清濃度を1%に下げます。細胞が回収されるまで、2日ごとに培地を交換してください。2日目、6日目、10日目の初代ブタRPE細胞の代表的な画像をここに示します。初代ブタRPE細胞、初代ヒトRPE細胞、ARPE 19細胞およびブタRPE脈絡膜組織におけるシグネチャ遺伝子のmRNAレベルのQRTPCR分析は、このグラフィック画像によって検出されます。ここでは、GAP DHをRTPCRのハウスキーピング遺伝子として使用しました。初代ブタRPE細胞には4つの生物学的複製が使用され、RPE 19細胞には3つの生物学的複製が初代ヒトRPE細胞およびブタRPE脈絡膜組織に使用された。ARPE 19細胞における遺伝子発現レベルを対照として設定した。ARPE 19およびブタRPE細胞ならびに初代ヒトRPE細胞およびブタRPE脈絡膜組織におけるRPE 65タンパク質の西洋血液分析をこの図に示す。ビンキュリンはここでローディングコントロールとして使用されました。この図は、1%FBSを含むDMEMベーシック培地中のブタRPE細胞の2週間の培養を示しています。ラミンの有無にかかわらず培養した細胞をRPE 65、ナトリウムカリウムAPTA、Z01、およびHexで染色しました。ラミニンの有無にかかわらず培養した細胞シートにおけるTR測定値がこの図に描かれている。初代ブタRPEおよびARPE19細胞における血管内皮増殖因子またはVEGFおよび色素上皮由来因子またはPEDFの西部血液分析をこの図に示す。培養初代ブタRPEおよびARPE 19細胞のセクター機能をこれらの画像に示します。このプロトコルの大部分は、RPEチデグリア複合体からRP細胞を溶解することです。毎回新鮮なトリップと溶液を使用し、消化時間を適切に制御して、8つのRP細胞を溶解することをお勧めします。

primary-culture-of-porcine-retinal-pigment-epithelial-cells

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

初代ヒト卵管上皮細胞のex vivoでの文化

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

生物学

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

ヒトiPS細胞のマイクロRNA発現プロファイル、IPから派生網膜色素上皮、および胎児の網膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

地図状委縮の目の大きいモデルにヒト胚性幹細胞由来網膜色素上皮細胞の網膜下移植

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

マウス網膜色素上皮からの一次電池文化

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

初代マウス網膜色素上皮細胞の単離

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

高分解能肺活量測定法を用いた初代ヒト網膜色素上皮細胞における生体エネルギーのリアルタイム解析

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

モルモットの目からの網膜色素上皮細胞の分離

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

人間のティッシュからの乳腺の上皮細胞を単離するためのプロトコル

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

著者スポットライト:肉芽腫性乳房炎の治療のための東洋医学と西洋医学の統合

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

初代ヒト乳腺上皮細胞の単離と培養

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...