著者は、UCSFリソース割当プログラムにより提供される資金調達を承認したいと思います。

全ての動物の作品は、国立衛生研究所の実験動物の管理と使用に関する指針の推奨事項に厳密に従って下で行った。プロトコルは、カリフォルニア大学の施設内動物管理使用委員会（IACUC）、サンフランシスコで承認された。 1.マウス胚性涙腺（LG）：収穫と顕微解剖<ol><li> 、科学研究倫理委員会のための制度の動物によって承認された手順に従うことで、適切な胚の日に収穫の胚に頸椎脱臼による確認に続いて、上昇したCO 2吸入を妊娠CD-1（またはトランスジェニック）メスを安楽死させる時限。膣栓発見E0の日を指定します。 注：涙腺（地方自治体）は以降のE14シングル蕾の段階から収穫することができる。しかし、E16（5から10つぼみ）の胚は、ex vivo培養のための最も一貫性のある結果を与える。 </li><li> Vを殺菌70％エタノールを用いてマウスの側entral。皮膚をつまんで、滅菌外科用ハサミで正中線で切開する。腹腔を露出させ、皮膚および腹膜を通って切断。冷PBS中の各子宮角と場所の上部にある子宮間膜に沿って切断することにより2子宮角を取り外し（リン酸緩衝生理食塩水）または100 U / mlペニシリンおよび100μg/ mlストレプトマイシンを添加したDMEM F12培地。 </li><li>鉗子を使用すると（＃5）、羊膜嚢から胚を削除し、冷PBSを含む第二の滅菌ペトリ皿に置く。目や頭部領域に手を触れないように注意してください。 </li><li>透視ベースと解剖顕微鏡上に胚を置きます。 1.7 -胚が&lt;E17の場合は、 手順1.5を実行します。胚は&gt; E17であれば、これらの手順を参考に、必須ではないが、そのため1.8のステップに進むことができる。 </li><li>ちょうど下顎骨下の胴体（ステップ1、 図2）を使うから頭を断つGA滅菌メス（＃10ブレード）。下顎骨は解剖板に今あるように頭を回転させます。 （ステップ2、1 番目のカット、 図2）の約1/4の脳の背側のを除去するために頭とメスを安定させるために鉗子を使用する-軟骨がE15の周りに硬く、一度これは特に重要です。 </li><li>メスでは両目の間の鼻を突き刺すことができるように頭を向けます。目が別々の半分（ステップ2、2 番目のカット、 図2）に今あるように、頭の中心を通ってカットを行います。 </li><li> 2＃5鉗子を使用して、頭の半分を取り、余分な組織を除去する（ステップ3、 図2参照）。 （目の低い後部の角にあります）、LGがこの除去過程で失われないように頭を向けるようにしてください。過剰脳、軟骨、および周囲や目の下、鼻組織を除去する。腺は、この時点でほとんど見えているので、眼が適切に過剰な組織除去tの後に配向されていることを確認O LGの場所を失うことを避ける。 </li><li>両方の鉗子で眼の後、底の隅から肌を注意深くをつかむ。慎重にLG電子が露出するようにピンセットで開いて引っ張って皮膚を取り除きます。 LG電子を可視化を支援する組織の上下に追加の照明を使用してください。 注：LGは、暗く、凝縮された間葉内のダクト上の芽としてその外観により区別、この時点で表示されるはずです。 </li><li>慎重に、LGまたはそれに関連するダクトをつかむないように注意しながら、離れて鉗子を用いて周囲の組織から（図と画像を参照）、LGおよび関連間充織を解剖し始める。腺は、周囲の組織から自由になったら、そっとLGダクトの基部に眼の周囲の上皮を把持することによって全体の腺を取り除く。凝縮された間葉への上皮陥入として観察することができる上皮を、周囲の間充織を維持するために注意してください。 注：TISSUのいくつかの追加の除去電子メール（任意の小さな骨片、筋肉、および結合組織）は、隣接する組織からのシグナリング/成長因子を回避する必要があるかもしれない。 </li><li>培養のため、収穫4から5腺およびプレート当たりに関連する間葉（自治体が解剖直後にめっきされる）; RT-PCR 6 RNA溶解緩衝液100μl当たり14腺の最小値を収集する。 </li></ol> 2. エクスビボ培養プレートを準備しますこの手順は、発展途上唾液腺7,8のために使用されるものに基づいています。 <ol><li>ガラス底の直径50mmのマイクロウェル皿7に50μg/ mlのアスコルビン酸および50μg/ mlのホロトランスフェリンとの培養培地（100U / mlペニシリンおよび100μg/ mlストレプトマイシンを補充）したDMEM F12200μlのを追加します。 注：我々は、以前にそれが胚性唾液腺に形態形成を最大化するために見られるような、DMEM F12を選択した。 </li><li> 13mmのPOLYCARBフロートメディアの上に0.1μmの細孔を有するonateメンブレンフィルター。 </li><li>オプションのステップ：（穏やかに混合するために、200μlのピペットを使用して、泡が現れた場合、1分間遠心分離）を3mg / mlの最終濃度となるようにDMEM / F12で1：3-Dラミニン1を希釈する。この15μlのを追加しフィルタリングする3次元ラミニンを希釈した。 注：この希釈は、上皮分岐を損なうことなく、その三次元状態でLGを維持するために最適であることが見出された。しかし、LGの培養に必須ではない。 </li><li>フィルター上またはラミニンに5 LG - 4を配置します。文化自治体ex vivoで 24 - 48時間（ 図3）、または定量PCRまたは免疫染色（ 図4）によるさらなる分析のために20分間、4％PFAで固定します。胚腺組織のqPCR分析のために、Rebustini らをご参照ください。2011年。ホフマンら、（2002）、スタインバーグE：胚腺組織の免疫蛍光分析のために、以下の引用を参照してください。Tら、（2005）。 </li></ol> 3.マウス生後および成体涙腺（LG）：解剖<ol><li>適切な動物倫理委員会の基準に従って出生後、マウスを安楽死させる。生後8日目の仔歳以下の場合は、滅菌したハサミで頭部を切断することによって安楽死させる。生後8日以上の場合は、70％エタノールで毛皮をスプレー。 </li><li> LG電子の位置を確認するには、マウスを上から照明で解剖顕微鏡の下に左右横横たわっていた。注：産後と大人のLGは、頸動脈、咬筋と真皮（ 図5を参照）に隣接している。 </li><li>小さな解剖ハサミを使用して、LGが配置されるべき場所から横方向に表皮に小さな切開を行います。 </li><li>鉗子を使用して、LGを露出するために、耳に向かって表皮を開く引っ張る。 </li><li>優しく周囲の組織からのLGを緩め鉗子を使用してください。 </li><li> LGは、周囲の組織から解放されると、慎重に、ダクトによってLGを削除どこダクトと眼の周囲の上皮（ 図1）を接続することによりグリップ。 </li><li> RT-PCRのためのRNA溶解緩衝液中で自治体を置き、または免疫染色のために20分間、4％PFAで固定します。 </li></ol>

<ol>
	<li>Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+the+pathogenesis+and+treatment+of+keratoconjunctivitis+sicca.">Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.</a> Current allergy and asthma reports. 14, 403 (2014).</li><li>Makarenkova, H. P., 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=FGF10+is+an+inducer+and+Pax6+a+competence+factor+for+lacrimal+gland+development.">FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.</a> Development. 127, 2563-2572 (2000).</li><li>Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regulation+of+lacrimal+gland+secretory+processes:+relevance+in+dry+eye+diseases.">Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.</a> Progress in retinal and eye research. 28, 155-177 (2009).</li><li>Nigam, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+can+the+intrinsic+power+of+branching+morphogenesis+be+used+for+engineering+epithelial+tissues+and+organs.">Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs.</a> Stem cells translational medicine. 2, 993-1000 (2013).</li><li>Qu, X., 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=Glycosaminoglycan-dependent+restriction+of+FGF+diffusion+is+necessary+for+lacrimal+gland+development.">Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development.</a> Development. 139, 2730-2739 (2012).</li><li>Rebustini, I. T., 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=MT2-MMP-dependent+release+of+collagen+IV+NC1+domains+regulates+submandibular+gland+branching+morphogenesis.">MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.</a> Developmental cell. 17, 482-493 (2009).</li><li>Hoffman, M. P., 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=Gene+Expression+Profiles+of+Mouse+Submandibular+Gland+Development:+FGFR1+Regulates+Branching+Morphogenesis+in+Vitro+Through+BMP-+and+FGF-Dependent+Mechanisms.">Gene Expression Profiles of Mouse Submandibular Gland Development: FGFR1 Regulates Branching Morphogenesis in Vitro Through BMP- and FGF-Dependent Mechanisms.</a> Development. 129, 24-5778 (2002).</li><li>Steinberg, 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=FGFR2b+signaling+regulates+ex+vivo+submandibular+gland+epithelial+cell+proliferation+and+branching+morphogenesis.">FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.</a> Development. 132, 1223-1234 (2005).</li><li>Knox, S. M., 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> Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. 329, 1645-1647 (2010).</li><li>Magenheim, 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=Blood+vessels+restrain+pancreas+branching,+differentiation+and+growth.">Blood vessels restrain pancreas branching, differentiation and growth.</a> Development. 138, 4743-4752 (2011).</li><li>Jaskoll, T., 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=FGF10/FGFR2b+signaling+plays+essential+roles+during+in+vivo+embryonic+submandibular+salivary+gland+morphogenesis.">FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis.</a> BMC developmental biology. 5, 11 (2005).</li><li>Takasato, M., 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=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature cell biology. 16, 118-126 (2014).</li><li>Knox, S. M., 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=Parasympathetic+stimulation+improves+epithelial+organ+regeneration.">Parasympathetic stimulation improves epithelial organ regeneration.</a> Nature communications. 4, 1494 (2013).</li></ol>

著者らは、開示することは何もない。

文化への汎用性とex vivoで 、LGを操作は、その開発を研究するための重要な利点を提供します。これは、研究者の仮説と、神経、内皮およびmesencyhmal細胞は器官を形成するように相互作用する方法を、上皮評価することができる多数の摂動をテストすることができる速度を含む。このモデルを利用する場合しかし、警告の数が存在する。まず、その分離によって腺は、それらが神経や血?...

涙腺（LG）は、視力や健康、保守、および眼表面の細胞の保護のために重要なの水性涙液分泌を担当しています。眼の刺激、光感受性によって特徴づけられるとビジョン1を減少させ、水性欠乏ドライアイ病、：最も一般的な衰弱性眼疾患の一つでLGの機能不全をもたらす。眼表面上に5排泄ダクト預金涙 - 人間ではLGが3目の側端より上の軌道に存在します。マウスは、主要な眼の腺の3ペアを持って、そのほとんどが研究され、単一の排泄ダクトを経由して、目に涙を浮かべて旅行する耳（眼窩）へ涙腺（LG）に位置し、前方および腹です。他の腺の器官と同様に、LG電子は、凝縮された間葉内の単一の上皮芽が複数の芽のラウンドとするために、ダクトの形成を受けている上皮の分岐形態形成のプロセスを経て開発分泌腺房とダクトの複雑な相互接続されたネットワーク（ 図1）2メートル。開発中は上皮は血管新生になるだけでなく、頻繁に翼口蓋神経節の副交感神経によって上頸神経節3から交感神経によってより少ない程度に神経支配。これらの細胞の種類すなわち神経細胞、上皮細胞、内皮細胞および間葉細胞のそれぞれとの間の相互作用は、成体組織の機能と維持に不可欠である。しかし、セル間タイプの通信は、これらのプロセスをガイドする方法と同様にLGの発達と再生を調整する基礎となる分子機構は依然として不明である。 胚のエクスビボで培養技術の出現は、複数の分岐臓器4の発達と再生経路の同定を可能にした。 ex vivoで培養する研究者を（私に臓器を操作する機能を提供しますリアルタイムでの臓器の発達および細胞 - 細胞相互作用を特徴付けるために、遺伝的または化学的）規定された条件下で同様に機械的、。マウスの眼窩LGがこの技術に非常に適していると最近の研究では、その開発2,5を規制するシグナル伝達系を定義しました。しかし、LG電子の開発と再生を支える分子手がかりを理解する必要にもかかわらず、それは現在、原因臓器を単離する技術的困難に思わ代役のまま。本稿では、分離して発生プログラムを定義するために、胚ネズミLG のex vivo文化を実行する方法について説明します。

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Name</td>
			<td style="width:129px;">Catalog #.</td>
			<td style="width:213px;">Company</td>
			<td style="width:997px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 without HEPES</td>
			<td>SH3027101</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>P0781</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin solution from bovine serum</td>
			<td>A9576</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% Solution</td>
			<td>15710</td>
			<td>Electron Microscopy Sciences</td>
			<td>Diluted to 4% in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 10X</td>
			<td>BP665-1</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 1X</td>
			<td></td>
			<td></td>
			<td>Prepared from 10X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">holo-Transferrin bovine</td>
			<td>T1283</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L- Ascorbic acid (Vitamin C)</td>
			<td>A4544</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite 100mm Tissue Culture Treated Dishes</td>
			<td>130182</td>
			<td>Thermo Scientific</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope with substage iluminator</td>
			<td>Stemi 2000</td>
			<td>Carl Zeiss</td>
			<td rowspan="2">Any stereo dissecting microscope can be used that has a transmitted light base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substage Illuminator Base for Stereo Microscope</td>
			<td>TLB 4000</td>
			<td>Diagnostics Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35mm Tissue Culture Treated Dishes</td>
			<td>353001</td>
			<td>Corning</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mm uncoated glass bottom dishes</td>
			<td>P50G-1.5-14-F</td>
			<td>MatTek Corporation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Widefield fluorescence microscope</td>
			<td>Axio Observer Z1</td>
			<td style="width:213px;">Carl Zeiss</td>
			<td>Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached 
			digital camera.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>TCS SP5</td>
			<td style="width:213px;">Leica Microsystems</td>
			<td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWISS Micro-Fine Forceps, #5 (11.2cm)</td>
			<td>17-305X</td>
			<td>Integra LifeSciences Corporation</td>
			<td>Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Standard Tip Forceps, #5 (11cm)</td>
			<td>91150-20</td>
			<td>Fine Science Tools (USA), Inc.</td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reusable Plastic Surgical Knife Handles, style no. 3.</td>
			<td>4-30</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Sterile Surgical Blades, no. 10</td>
			<td>4-310</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous-Micro Total RNA Isolation Kit</td>
			<td>AM1931</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cultrex 3D Culture Matrix Laminin I</td>
			<td>3446-005-01</td>
			<td>Trevigen</td>
			<td>6 mg/ml stock diluted 1:1 with DMEM/F12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Timed-pregnant Crl:CD1(ICR) mice</td>
			<td></td>
			<td style="width:213px;">Charles River Labs</td>
			<td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nucleopore Track-Etched Hydrophilic Membranes, 0.1 um pre size, 13mm</td>
			<td>110405</td>
			<td>Whatman</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Timed-pregnant Pax6-Cre,GFP mice</td>
			<td>Tg(Pax-Cre, GFP)1Pgr</td>
			<td>The Jackson Laboratory</td>
			<td>Optional mice for learning how to locate the LG.</td>
		</tr>
	</tbody>
</table>

manipulating the murine lacrimal gland

涙腺（LG）は、角膜の構造と機能、視力に不可欠な透明な組織を維持するために必要な水性涙を分泌する。 5ダクト - 人間では、単一のLGは3を通して眼表面に涙を配信するそれぞれの目の横端上記の軌道上に存在します。マウスは、主要な眼の腺の3ペアを持って、そのほとんどが研究され、耳に眼窩外涙腺（LG）に位置し、前方および腹です。他の腺の器官と同様に、LG電子は、凝縮された間葉内の単一の上皮芽が分泌腺房とダクトの複雑な相互接続ネットワークを形成するために、つぼみとダクト形成の複数のラウンドを受けるものでは上皮の分岐形態形成のプロセスを経て開発しています。この精巧なプロセスは、よくそのような膵臓および唾液腺などの多くの他の上皮の器官で文書化されています。しかし、LGはあまり検討されていると形態形成を制御するメカニズムは不完全に下です立っていた。我々は、このアンダー表現モデル系としては、見つけ解剖とLGを培養することに関連する困難の結果であることを疑う。したがって、ここでは解剖収穫胚および出生後のLGのための技術や組織のex vivo培養のための方法が記載されている。

LG電子は、上皮分岐形態形成のプロセスを経て開発しています。 E14、E15、E16、E17、およびP2で解剖胚自治体の明視野画像は、このイベントを説明する。 <img alt="図1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> 図1. LGは分枝形態形成上皮のプロセスを経て開発しています。新鮮にE14、E15、E16、E17およびP2で...

Watch this Scientific Journal Video about Manipulating the Murine Lacrimal Gland at JoVE.com

Manipulating the Murine Lacrimal Gland

涙腺（LG）は、ビジョンと眼の健康を維持するために必要な涙の水性成分を生成し、分岐器官である。ここでは、LG電子の開発に関与するシグナル伝達経路を解読するネズミLG郭清およびex vivo培養技術を説明しています。

マウス涙腺を操作する

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Research

JoVE Journal

Biology

20.7K ビュー.  University of California San Francisco. 涙腺（LG）は、ビジョンと眼の健康を維持するために必要な涙の水性成分を生成し、分岐器官である。ここでは、LG電子の開発に関与するシグナル伝達経路を解読するネズミLG郭清およびex vivo培養技術を説明しています。 

ビデオ: Manipulating the Murine Lacrimal Gland

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

細胞間相互作用を操作するためのシリコンマイクロチップ

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

生物学

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

体外で分離された神経細胞を操作するためのレーザーピンセットを使用して

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

マウス腎移植の手順

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

マウス唾液腺幹細胞の単離

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

唾液腺器官培養の遺伝子改変と再結合

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

マウス乳腺に局在薬物送達のための管内注入

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

ネズミ弁内皮細胞の単離

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

乳腺生検する方法

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成人ネズミ涙腺および顎下腺からの筋上皮細胞の単離

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...