Name: transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes
Uploaded: 2017-06-03T14:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

この研究は、国立健康研究所（05A1-CSPP16-014、HJL）および科学技術省（MOST 103-2320-B-400-015-MY3、HJL）からの助成金によって支えられた。

動物を含むすべての研究は、動物管理機関委員会によって承認されたプロトコールを遵守した。 1.細胞外小胞/エキソソームの単離および検証<ol><li>培養乳房上皮基底細胞、NAMEC 4、500mLのMCDB170、pH7.4 + 500mLの重炭酸ナトリウム（0.2438％）を含むDMEM / F12からなる新鮮な無血清培地; EGF（5ng / mL）。ヒドロコルチゾン（0.5μg/ mL）;インスリン（5μg/ mL）。ウシ下垂体抽出物（BPE;35μg/ mL）;およびGW627368X（1μg/ mL）を15cmディッシュに入れた。 </li><li>血球計数器で細胞を計数した後、0日目に15cmディッシュあたり12mLの培地に1.2x10 6個の細胞を4日間播種する4 。 </li><li>培養4日後、卓上遠心分離機を用いて300 xgで5分間遠心します。上清をコニカルチューブに移す（ 図1 ）。 </li><li>上清を遠心分離する。卓上遠心分離機で2,000 xg、20分間。上清を超遠心分離チューブに移し、死んだ細胞と細胞破片を残す（ 図1 ）。 </li><li>上清を10,000 xgで30分間4℃で遠心分離します。上清を新しい超遠心チューブに移す（ 図1 ）。 </li><li>上清を110,000 xgで60分間4℃で遠心分離する。上清を除去し、EV /エキソソームペレットをPBS（ 図1 ）に再懸濁する。 </li><li>上清を110,000 xgで60分間4℃で遠心分離する。上清を除去する。 EV /エキソソームペレットをPBSに再懸濁する（ 図1 ）。 NAMEC条件培地240〜480mLから単離したペレットを100μLで再懸濁する。 </li><li> BC懸濁液のタンパク質濃度をBCAタンパク質アッセイで測定する。濃度が約20〜40μg/ 100μLであることを確認してください。 -20℃での保存さらなる分析。 </li><li>先にGardiner らによって記載されているように、ナノ粒子追跡分析（NTA）によってEV /エキソソームの濃度およびサイズを測定する。 11 。 NTA分析のために、EV /エキソソーム（20μg/100μL）をPBSで10,000倍に希釈する。 注：NTA分析の結果は、分析された小胞の数およびサイズを反映する。 </li><li>以前にLin ら 4に記載されているように、透過電子顕微鏡（TEM）を用いてEV /エキソソームを画像化する。 </li></ol> 2.密度勾配を用いたエキソソーム精製<ol><li>ステップ1.7の110,000gペレットをPBS（2mL）中の40％（w / v）イオジキサノールに再懸濁する。 PBS（それぞれ2mL）中の30％、20％、10％、および5％（w / v）のイオジキサノールのアリコートを順番に混合し、超遠心チューブに密度勾配を形成する。 </li><li>混合物を200,000×gで4時間、8時間遠心分離する。 </li><li>各グラジエントフラクションを収集する（10フラクションons; 1mL /画分）をピペットでチューブの上部から取り出します。 </li><li> SDS-PAGE 12およびウェスタンブロットによって、各画分のエキソソームマーカー（ 例えば、 CD81、CD9、CD63およびTsg101）の存在を分析する。各画分の50μL懸濁液を0.1％（w / v）SDSを含む10％ゲルにロードし、タンパク質をゲル電気泳動で画分に分離する。 </li><li>タンパク質をゲルからPVDFメンブレンに移し、4℃で一晩エキソソームマーカー（ 例えば CD81、CD9、CD63、およびTsg101）およびハウスキーピングタンパク質GAPDH（ Table of Materials ）に対する抗体と膜をインキュベートする。 注：結果は、エキソソームを含有する画分を同定する。 </li></ol> 3.細胞外小胞/エキソソーム標識<ol><li>ステップ1.7で得られたEV /エキソソームを20μgのエキソソームタンパク質/100μLで10μMカルボキシフルオレセインスクシンイミジルジアセテートエステル（CFSE）に懸濁する。並列サンプルを準備する後のEV /エキソソーム取り込みアッセイのネガティブコントロールとして、同じ方法で処理されたCFSEおよびPBSのみを含む。混合物を37℃で30分間放置する。 </li><li> 50倍量のPBSにEV /エキソソームを懸濁し、4℃で60分間110,000×gで懸濁液を遠心分離する。上清を除去し、EV /エキソソームペレットをPBSで再懸濁する。ステップ3.2を1回繰り返します。 </li><li> EVs /エキソソームを20μgのエキソソームタンパク質/100μLの濃度でPBS中に懸濁させ、次にEVs /エキソソームを0.22μmの膜を通して濾過し、細胞にEV /エキソソームを添加する。 </li></ol> 4.細胞外小胞/エキソソーム取り込みアッセイ<ol><li>培養培地を作るために、500mLのMCDB170、pH7.4 + 500mLのDMEM / F12を重炭酸ナトリウム（0.2438％）と混合する。 EGF（5ng / mL）。ヒドロコルチゾン（0.5μg/ mL）;インスリン（5μg/ mL）。およびBPE（35μg/ mL） 4 。ヒトの乳房上皮HMLE細胞を6ウェルディッシュ（1×10 6細胞//細胞&gt; /ウェル）をEV /エキソソーム処理の1日前に投与する。ステップ3.3で得られた2μg/ mLのCFSE蛍光標識EV /エキソソームをHMLE細胞の培地に2〜6時間添加する。陰性対照群のHMLE細胞を、ステップ3.1で説明した平行調製物で処理する。 </li><li> 2〜6時間のインキュベーション後、室温で4mLのPBSで細胞を2回洗浄する。 </li><li> 0.25％トリプシンで10分間細胞を分離し、0.2％FBSを含むPBS中で細胞を再懸濁する。蛍光細胞分析装置4を使用して、細胞内の蛍光強度からのEV /エキソソーム取り込みを測定する。共焦点顕微鏡法を用いてEV /エキソソームまたはネガティブコントロールで処理した細胞を画像化する。 注：細胞の緑色の蛍光は、EV /エキソソームの取り込みによって引き起こされます。顕微鏡の設定については、 図6の凡例を参照してください。 </li></ol>原発性マウス乳腺上皮細胞の単離<ol><li&gt; 0.1cmのゼラチン溶液12mlを10cmの皿に加えてゼラチン被覆皿を調製する。プレートを37℃のインキュベーターに30分間置く。ゼラチン溶液を除去し、ゼラチンコーティングが乾燥するまで4時間層流フード中で蓋を皿から離しておく。 </li><li>はさみを使用して12週齢の処女雌C57BL / 6マウスから2番、3番、4番、5番の乳腺（ 図2 ）を解剖し、剃刀を用いて小塊（2mm 2 ）に切る。 </li><li> 0.2％コラゲナーゼIV型、0.2％トリプシン、5％FBS、5μg/ mLゲンタマイシンを含有する50mLのDMEM / F12を用いて、乳腺のスラリー（10匹のマウスから）を37℃で60分間、120rpmで攪拌しながらさらに解離させる。 、および1x pen-strep。 </li><li> 350×gで10分間遠心分離することにより、混合物から上皮オルガノイドをペレット状にペレット化する。 </li><li> 0.1mg / mL DNase Iを含む4mLのDMEM / F12に上皮オルガノイドを室温で5分間懸濁する。 6mLのDMEM / F12を最終10mLの容量。 </li><li>懸濁液を室温で10分間400xgで遠心分離し、上清を捨てる。 </li><li>ペレットを10mLのDMEM / F12に再懸濁する。サスペンションを遠心分離機に乗せるが、速度が400 x gに達するとブレーキが掛かる。上清を捨てる。 注：ブレーキを叩くことによって、上皮オルガノイドは速やかにペレット化されますが、線維芽細胞は単細胞として上清中に残ります。 </li><li>手順5.6と5.7を5〜7回繰り返します。血球計にサスペンションの一滴を加え、遠心分離の各ラウンド後に顕微鏡下でオルガノイド混合物から線維芽細胞のクリアランスをチェックする（ 図3 ）。 </li><li> 1x ITS、5％FBS、50μg/ mLゲンタマイシン、10ng / mL EGFおよび1xペン - ストレプを含有するDMEM / F12で、ステップ5.1で得られたゼラチンコートディッシュ中のオルガノイドを48時間培養する。 </li><li> 48時間後、FBSを含まない新鮮な培養培地で培地を置換することにより、培養中の浮遊細胞を除去する（1×ITS、50μg/ mLゲンタマイシン、10ng / mL EGF、および1×ペン - ストレプトマイシンを含むDMEM / F12）で洗浄した。 </li><li>乳房上皮細胞の単層が、付着した上皮オルガノイドから3日間で移動し、成長することを確認する。 </li></ol>原発性マウス基礎/管腔上乳腺上皮細胞の分離<ol><li> 3日間の培養後、20分間、タンパク質分解酵素およびコラーゲン分解酵素活性を有する天然酵素混合物でマウス初代乳腺細胞を分離する。 PBS中の5％FBSで酵素活性を中和する。 </li><li>懸濁液を室温で10分間450xgで遠心分離し、上清を捨てる。細胞ペレットを5mg / mLディスパーゼに20分間再懸濁する。 PBS中の5％FBSで酵素活性を中和する。 </li><li>懸濁液を室温で10分間450xgで遠心分離し、上清を捨てる。 </li><li>希釈して10 7細胞/ mLの濃度で0.2％FBSを含む100μLのPBSに細胞を再懸濁した抗CD49fおよび抗EpCAM抗体を氷上で暗所で1時間インキュベートした。 </li><li> PBSで細胞を洗浄し、暗所で30分間氷上でフルオロフォア結合二次抗体で細胞をインキュベートする。 </li><li> 0.2％FBSを含むPBS中で細胞を洗浄し、再懸濁する。 </li><li> EpCAM hi / CD49f lo管腔上乳腺上皮細胞を細胞選別機4で選別する。 </li></ol> 7.細胞外小胞/エキソソーム処理<ol><li>ゼラチンでコートした6ウェルディッシュ（2×10 5細胞/ウェル）上に、ステップ6.7からの選別したEpCAM hi / CD49f lo管腔上乳腺細胞を播種し、PBSまたは2μg/ mL NAMEC由来EV /エキソソームで処理するステップ1.7で得られた。 </li><li>長期培養処理におけるEV /エキソソームの生物学的有効性を確実にするために、PBSまたは2μg/ mLのNAMEC由来のEV /エキソソームを含む新鮮な培地で2日ごとに細胞培養培地を交換する。マウスプリマを分割しないでください10日間の処置の間に細胞内の細胞を除去する。 </li></ol>乳腺上皮細胞の脂肪パッド注入<ol><li>イソフルラン2〜3％吸入剤を含む3週齢の雌性C57BL / 6マウスを麻酔する。 </li><li>麻酔したマウスをその背中に置く。中腹部の毛皮をカミソリ/ヘアクリームで取り除き、70％アルコールとポビドンヨードの3回の交互サイクルで手術部位をきれいにする。 </li><li>はさみで腹側胸骨 - 鼠径部に沿って皮膚を通して1.5cmの垂直切開を行い、次に右と左の第4乳房脂肪パッドを交互に露出させる。 </li><li>はさみで腺実質を取り除くことによって各脂肪パッドをきれいにする。リンパ節を脂肪パッドに置き、リンパ節の下の腺実質全体を除去する。 注記：脂肪パッドの3分の2はそのまま残してください。 </li><li>工程7.2から得られた細胞を、天然の酵素混合物（ 表の表を参照 ）でタンパク質分解性aコラーゲン分解酵素活性を10分間測定した。 PBS中の5％FBSで酵素活性を中和する。 </li><li>懸濁液を室温で10分間450xgで遠心分離し、上清を捨てる。血球計数器で細胞を計数し、15μLに所望の細胞用量（10 4 -10 2細胞/パッド）が含まれるような濃度のPBS中で細胞を懸濁させる。 </li><li>乳腺上皮細胞浮遊液15μLを、27G針に取り付けられた100μLガラスシリンジを用いて脂肪パッドに注入する。 </li><li>もう片方の脂肪パッドについては、手順8.4-8.7を繰り返します。 </li><li>創傷クリップで皮膚を閉じます。 </li></ol> 9.乳腺完全マウント<ol><li>細胞注入後8週間でCO 2 +頚部脱臼でマウスを安楽死させる（ステップ8.7）。 </li><li>はさみを使用して、胸骨領域から鼠径領域への皮膚層を垂直に切開し、次に右と左の両方を露出させる。大麻の脂肪パッド。第4乳腺を除去する（ 図2 ）。 </li><li>ガラス顕微鏡スライド上の脂肪パッドを広げ、室温で一晩、Kahle固定液（4％ホルムアルデヒド、30％EtOH、および2％氷酢酸）で脂肪パッドを固定する。 注意：Kahleの固定剤は刺激剤です。ケミカルフードでこの手順を実行します。 </li><li>ファットパッドを250mLの70％EtOHで15分間、次いで250mLのdH 2 Oで5分間洗浄する。ファットパッドをカルミンミョウバン（1gのカルミンおよび2.5gの硫酸アルミニウムカリウムを500mLのdH 2 O中に）で室温で一晩染色する。 </li><li>脂肪パッドを250mLの70％EtOHで15分間、250mLの95％EtOHで15分間、および250mLの100％EtOHで15分間洗浄する。 </li><li>ファットパッドが透明になったら、キシレンの脂肪パッドを数日間清掃し、キシレンインキュベーションを停止します。 注意：キシレンは刺激剤です。ケミカルフードでこの手順を実行します。 </li><li>スライドをマウントするデジタルスライドスキャナを使用して脂肪パッドの画像（2,400dpi）を撮影する。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+nano-sized+vesicles+released+by+cells+and+subsequent+quantitative+and+qualitative+analysis+by+high-resolution+flow+cytometry.">Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.</a> Nat Protoc. 7 (7), 1311-1326 (2012).</li><li>Kowal, J., 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=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proc Natl Acad Sci U S A. 113 (8), E968-E977 (2016).</li><li>Li, D., 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=ADVANCED+IMAGING.+Extended-resolution+structured+illumination+imaging+of+endocytic+and+cytoskeletal+dynamics.">ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.</a> Science. 349 (6251), (2015).</li><li>Outzen, H. C., Custer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+normal+and+neoplastic+mammary+tissues+in+the+cleared+mammary+fat+pad+of+the+nude+mouse.">Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.</a> J Natl Cancer Inst. 55 (6), 1461-1466 (1975).</li><li>Sheffield, L. G., Welsch, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+breast+epithelia+to+mammary-gland-free+fat-pads+of+athymic+nude+mice:+influence+of+mammotrophic+hormones+on+growth+of+breast+epithelia.">Transplantation of human breast epithelia to mammary-gland-free fat-pads of athymic nude mice: influence of mammotrophic hormones on growth of breast epithelia.</a> Int J Cancer. 41 (5), 713-719 (1988).</li></ol>

著者は何も開示することはない。

エキソソームは、それらを放出した細胞の特徴を有することが多く、放出されたエキソソームの量は、刺激によって誘発され得る4 。細胞の培養培地を回収し、EV /エキソソーム収集のための示差的超遠心分離に供することができる（ 図1 ）。現在のところ、EV /エキソソームを単離する理想的な方法に関する一般的な合意はない。ここで使用される最適...

エキソソームは細胞間の膜とサイトゾルタンパク質、脂質、RNAを伝達することで細胞間の伝達を媒介することができます1 。エキソソーム媒介性の伝達は、多くの生理学的および病理学的プロセス（ すなわち、抗原提示、寛容2の発生および腫瘍進行3 ）に関与することが実証されている。エキソソームはしばしば、それらを放出するソース細胞のものと同様の内容を有する。したがって、エキソソームは、供給源細胞から特定の細胞特性を運び、それらを摂取する細胞にこれらの特性を伝達することができる4 。 エキソソームは、50〜150nmの二重層膜ベシクルであり、特異的マーカー（ 例えば、 CD9、CD81、CD63、HSP70、Alix、およびTSG101）を提示する。したがって、エキソソームは、異なる側面のための様々な方法によって特徴付けられなければならない。透過電子顕微鏡法を用いて膜小胞を可視化することができる例えば、エキソソーム4,5 。精製されたエキソソームのサイズおよび数を測定するために、ナノ粒子追跡分析（NTA）および動的光散乱分析（DLS）が使用される。エキソソームの脂質膜含量は、密度勾配によって確認することができる。 CD9、CD81、CD63、HSP70、Alix、およびTSG101,6,7などのエキソソームマーカーは、ウェスタンブロッティングによって測定することができる。 乳房基底細胞は、脂肪パッドに移植されたとき乳腺を生成する能力を有するが、内腔細胞は8,9,10 であり得ない。従って、乳房基底細胞は、乳房再増殖単位とも呼ばれる。乳房基底および管腔細胞のモデルを用いることにより、EVs /エキソソームが異なる細胞集団間で細胞特性を伝達する能力を調べることができる。この作品乳房基底上皮細胞由来のEV /エキソソームを用いて乳腺上皮細胞から乳房内腔上皮細胞への腺形成能を伝達する方法を実証する。ルーメン乳房上皮細胞は、基底細胞から分泌されたEV /エキソソームの摂取後に基底細胞特性を獲得し、次に乳腺4を形成することができる。

<table><tbody><tr><td>MCDB 170&amp;nbsp;</td><td>USBiological</td><td>M2162</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo</td><td>1250062</td><td></td></tr><tr><td>Optima L-100K ultracentrifuge</td><td>Beckman</td><td>393253</td><td></td></tr><tr><td>SW28 Ti Rotor</td><td>Beckman</td><td>342204</td><td></td></tr><tr><td>SW41 Rotor</td><td>Beckman</td><td>331306</td><td></td></tr><tr><td>NANOSIGHT LM10</td><td>Malvern</td><td>NANOSIGHT LM10</td><td>for nanoparticle tracking analysis (NTA)</td></tr><tr><td>Optiprep&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D1556</td><td>60% (w/v) solution of iodixanol in water (sterile).</td></tr><tr><td>CD81 antibody</td><td>GeneTex</td><td>GTX101766</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD9 antibody</td><td>GeneTex</td><td>GTX100912</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD63 antibody</td><td>Abcam</td><td>Ab59479</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>TSG101 antibody</td><td>GeneTex</td><td>GTX118736</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>GAPDH</td><td>GeneTex</td><td>GTX100118</td><td>1:6,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td><td>Thermo</td><td>V12883</td><td></td></tr><tr><td>FACSCalibur</td><td>BD Biosciences</td><td></td><td>fluorescence cell analyzer</td></tr><tr><td>collagenase Type IV&amp;nbsp;</td><td>Thermo</td><td>17104019</td><td></td></tr><tr><td>trypsin</td><td>Thermo</td><td>27250018</td><td></td></tr><tr><td>&amp;nbsp;ITS</td><td>Sigma-Aldrich</td><td>I3146</td><td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td></tr><tr><td>accutase</td><td>ebioscience</td><td>00-4555-56</td><td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td></tr><tr><td>dispase&amp;nbsp;</td><td>STEMCELL</td><td>7913</td><td>5 mg/mL = 5 U/mL</td></tr><tr><td>anti-CD49f antibody</td><td>Biolegend</td><td>313611</td><td>1:50</td></tr><tr><td>anti-EpCAM antibody</td><td>Biolegend</td><td>118213</td><td>1:200</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>cell sorter</td></tr><tr><td>carmine alum</td><td>Sigma-Aldrich</td><td>C1022</td><td></td></tr><tr><td>human mammary epithelial cells (HMLE cells, NAMECs)</td><td></td><td></td><td>gifts from Dr. Robert Weinberg</td></tr><tr><td>permount</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SP15-500</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Zymeset&amp;nbsp;</td><td>BSB101</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-015</td><td></td></tr><tr><td>Hydrocoritisone</td><td>Sigma-Aldrich</td><td>SI-H0888</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SI-I9278</td><td></td></tr><tr><td>BPE (bovine pituitary extract)</td><td>Hammod Cell Tech&amp;nbsp;</td><td>1078-NZ</td><td></td></tr><tr><td>GW627368X&amp;nbsp;</td><td>Cayman</td><td>10009162</td><td></td></tr><tr><td>15 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353025</td><td></td></tr><tr><td>table-top centrifuge</td><td>Eppendrof&amp;nbsp;</td><td>Centrifuge 3415R</td><td></td></tr><tr><td>ultracentrifuge tube</td><td>Beckman</td><td>344058</td><td></td></tr><tr><td>PBS (Phosphate-buffered saline)&amp;nbsp;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>23228</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>HT7700</td><td></td></tr><tr><td>gelatin&amp;nbsp;</td><td>STEMCELL</td><td>7903</td><td></td></tr><tr><td>10 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353003</td><td></td></tr><tr><td>6-well culture dish</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>female C57BL/6 mice</td><td>NLAC (National Laboratory Animal Center</td><td></td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>BioWest&amp;nbsp;</td><td>S01520</td><td></td></tr><tr><td>gentamycin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15710072</td><td></td></tr><tr><td>Pen/Strep</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>DNase I</td><td>5PRIMER</td><td>2500120</td><td></td></tr><tr><td>isofluorane&amp;nbsp;</td><td>Halocarbon</td><td>NPC12164-002-25</td><td></td></tr><tr><td>formaldehyde</td><td>MACRON</td><td>H121-08</td><td></td></tr><tr><td>EtOH (Ethanol)</td><td>J.T. Baker</td><td>800605</td><td></td></tr><tr><td>glacial acetic acid</td><td>Panreac</td><td>131008.1611</td><td></td></tr><tr><td>aluminum potassium sulfate</td><td>Sigma-Aldrich</td><td>12625</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Leica</td><td>3803665</td><td></td></tr><tr><td>0.22 &amp;mu;m membranes</td><td>Merck Millipore</td><td>Millex-GP</td><td></td></tr><tr><td>AUTOCLIP Wound Clips, 9 mm</td><td>BD Biosciences</td><td>427631</td><td></td></tr><tr><td>AUTOCLIP Wound Clip Applier</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>CellMask&amp;trade; Deep Red</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>C10046</td><td>plasma membrane stain</td></tr></tbody></table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

細胞は、エキソソームを介して、タンパク質、脂質、および核酸を含む約100nmの細胞外小胞（EV）と通信することができる。非接着/間葉系乳房上皮細胞（NAMEC）由来の細胞外小胞は、示差的超遠心分離によってNAMEC培地から単離することができる。それらの密度に基づいて、EVは110,000×gでの超遠心分離によって精製することができる。超遠心分離からのEV調製物は、可溶性タンパク質の混入を防ぐために連続密度勾配を用いてさらに分離することができる。次いで精製されたEVを、調製物中の小胞のサイズおよび数を測定するナノ粒子追跡分析を用いてさらに評価することができる。 50〜150nmの範囲の細胞外小胞はエキソソームである。 NAMEC由来のEV /エキソソームは、乳腺上皮細胞によって摂取され得、これは、フローサイトメトリーおよび共焦点顕微鏡によって測定され得る。いくつかの乳腺幹細胞の特性（ 例えば、乳腺形成能）は、NAMEC由来EVs /エキソソームを介して、幹様NAMECから乳房上皮細胞に移され得る。単離された一次EpCAM hi / CD49f lo管腔上乳腺細胞は、マウス脂肪パッドに移植された後、乳腺を形成することができず、EpCAM lo / CD49fは、移植後に乳腺を形成する。 EpCAM hi / CD49f lo管腔上乳腺上皮細胞によるNAMEC由来EVs /エキソソームの取り込みは、脂肪パッドに移植された後に乳腺を発生させる。幹様乳腺上皮細胞に由来するEV /エキソソームは、EpCAM hi / CD49f lo管腔上乳腺上皮細胞に乳腺形成能を伝達する。

PGE 2 / EP 4シグナル伝達を遮断することにより、乳房基底様幹細胞4からのEV /エキソソーム放出が誘発されることが示されているので、この研究は、誘発されたEV /エキソソームを乳房上皮基底細胞（NAMEC）培養から単離する方法を提示する。 NAMECは無血清培地で培養されるため、血清由来のEV /エキソソームは存在しない13...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

このプロトコルは、非接着性/間葉性乳房上皮細胞からの細胞外小胞（EVs）/エキソソームの精製、定量化および特徴づけ方法、ならびにこれらを用いて乳腺上皮細胞に乳腺形成能を転移させる方法を記載する。幹様乳腺上皮細胞に由来するEV /エキソソームは、この細胞特性をEV /エキソソームを摂取する細胞に移入することができる。

細胞外小胞/エキソソームを介した乳腺上皮細胞と乳房管腔細胞との間の乳腺形成能の伝達

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

transfer-mammary-gland-forming-ability-between-mammary-basal

Research

JoVE Journal

Developmental Biology

11.6K ビュー.  National Health Research Institutes. このプロトコルは、非接着性/間葉性乳房上皮細胞からの細胞外小胞（EVs）/エキソソームの精製、定量化および特徴づけ方法、ならびにこれらを用いて乳腺上皮細胞に乳腺形成能を転移させる方法を記載する。幹様乳腺上皮細胞に由来するEV /エキソソームは、この細胞特性をEV /エキソソームを摂取する細胞に移入することができる。 

ビデオ: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6Jマウスにおける間質細胞と癌細胞の乳腺移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

医学

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

間肩甲膜白脂肪パッドへのラット乳腺上皮細胞移植

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

がん研究

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

生物学

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植の手順

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

微小トリプシン法によるモザイク乳腺オルガノイドの生成

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

発生生物学

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

マウスモデルにおける乳腺の発達と機能の評価

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

マウス乳腺の凍結切片の間接免疫

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

細胞外小胞/エキソソームを介した乳腺上皮細胞と乳房管腔細胞との間の乳腺形成能の伝達

要約

さらに動画を探す

この動画の章

C57BL/6Jマウスにおける間質細胞と癌細胞の乳腺移植

間肩甲膜白脂肪パッドへのラット乳腺上皮細胞移植

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

乳がん研究のための潜在的機能インビトロモデルとしての乳腺上皮および内皮細胞スフェロイド

乳腺上皮移植の手順

微小トリプシン法によるモザイク乳腺オルガノイドの生成

マウスモデルにおける乳腺の発達と機能の評価

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

マウス乳腺の凍結切片の間接免疫

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

細胞外小胞/エキソソームを介した乳腺上皮細胞と乳房管腔細胞との間の乳腺形成能の伝達

要約

さらに動画を探す

この動画の章

C57BL/6Jマウスにおける間質細胞と癌細胞の乳腺移植

間肩甲膜白脂肪パッドへのラット乳腺上皮細胞移植

マウス乳腺上皮細胞は乳腺刺激分化の過程でMammospheresを形成し、

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

乳腺上皮移植の手順

微小トリプシン法によるモザイク乳腺オルガノイドの生成

マウスモデルにおける乳腺の発達と機能の評価

マンモスフィアの自然発生の事前侵襲乳房病変の非酵素的、無血清組織培養

マウス乳腺の凍結切片の間接免疫

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

乳がん研究のための潜在的機能インビトロモデルとしての乳腺上皮および内皮細胞スフェロイド