水晶体上皮片の調製は、ドクタージョンマカヴォイ9の実験室で生まれたの方法から適合されています。この作品は、国立眼研究所、学内研究プログラムZ01 - EY000238 - 22によって資金を供給される

パート1：レンズの取り外し材料と試薬： <ol><li>ミクロ解剖ハサミ、湾曲した、鈍のヒント、（そのようなas.RS - 5983、Roboz手術器械株式会社、株式会社、ゲイサーズバーグ、MD）、ミクロ解剖ピンセット、湾​​曲した先端（などRoboz＃RS5137など）。 </li><li>懸濁培地：培地199を含む0.1％ウシ血清アルブミン、100単位/ mlペニシリン、100μg/mlのストレプトマイシンは、2.5μg/ mlのアムホテリシンBの試薬は、Invitrogen、カールスバッド、カリフォルニア州から入手できます。 </li></ol>手順： <ol><li>国立衛生研究所、ベセスダ、MDから提供されたガイドラインに準拠した新生児ラットを（2-4日齢）安楽死させる。 </li><li>手術用ハサミでまぶたを削除します。外側に膨らみに目を強制的に目のソケットの反対側に湾曲したピンセットで軽く押してください。はさみと眼の後部側に小さな切開を加えます。切開反対眼の側面にピンセットで押すことによって、レンズと付属の硝子体の少量は、レンズが曲がったピンセットで拾い上げできるように、破裂を通って強制することができます。ケアは、水晶体嚢を破壊しないように注意してください。 </li><li> 5ミリリットル暖かい、無菌懸濁培地を含む60ミリメートルプラスチック組織培養皿にレンズを転送するために湾曲したピンセットを使用してください。 </li></ol>第2部：片のマイクロダイセクション材料と試薬： <ol><li>ミクロ解剖ピンセット、0.1mmのヒント、（そのようなRoboz RS - 4976など）。我々は、理由の先端の柔軟性Dumostar合金を使用しますが、他の合金鋼で指定することもできます。彼らはこの手順の実行中に折ったり、曲げたりする傾向があるとして、0.1ミリメートルよりも薄いのヒントとピンセットは、推奨されていません。 </li><li>懸濁培地（パート1を参照） </li><li>ハムのF12培地または培地199（Invitrogen社、Carlsburg、CA）はUnsupplemented </li></ol>手順： <ol><li>次の手順は、滅菌解剖ツールと滅菌培地を使用してクリーンな、ドラフトのない環境で実施されるべきである。 </li><li>ステレオ解剖顕微鏡を用いて、0.1mmの先端のピンセットで任意の付着組織のレンズをきれいにし、5ミリリットル滅菌懸濁培地を（このステップは、汚染を減らすために役立ちます）含む第二の60ミリメートル培養皿に移す。ピンセットで、5ミリリットル滅菌、unsupplemented培地を含む35ミリメートル培養皿にレンズの希望数を転送する。 （下の染料の濃度が解離容易にするため私たちはしばしば、このステップのためにハムのF12（Invitrogen）を使用しますが、培地199にも受け入れられるない抗生物質または他の追加がこのステップで必要とされていない。。）12植を行うことができる限り多くの料理この大きさの中心部に位置します。少ない外植片は解剖用のツールが小さい皿の中で簡単に操作することができないため、行われる場合であってもしかし、35mmの皿を使用する必要があります。それらが必要になるまで37℃で組織培養インキュベーターに残っているレンズ° Cを含む料理を転送する。 </li><li>レンズの後側を識別する。これを認識する多くの方法があります：1）後部は若干フラット化されている前方、より丸みである、2）新生児ラットのレンズは、解剖時の室温に彼らは涼しいと寒い白内障を形成する。コー​​ルド白内障が完全にレンズをいっぱいにしていない場合、後側は不透明領域から最も遠い側です。不透明度が37に皿を温めて、レンズをいっぱいにしている場合° C数分間、それが逆になります。後側は、冷却時に白内障の改革として識別することができます。 3） 水晶体血管膜の痕跡は、後方側に見える場合があります。 4）後部縫合糸は、後方側に見える場合があります。これはカプセルが開かれる場所なので、正しく後側を特定することは、不可欠です。前方側にレンズを開くと、上皮を引き裂くだろう。 </li><li>後方側が識別されたら、上方向に回し、左ピンセット（右利きの労働者用）でレンズを把握する。その後、小さな倍を生成するために右ピンセットで後嚢をつまんで。 </li><li>右ピンセットで後嚢を押しながら、左ピンセットでカプセルの倍を把握し、カプセルに小さな裂傷を作るために反対方向にピンセットの2つのペアを引き出します。 </li><li>ピンセットで後退カプセルの端をつかんで、ピンセットで皿のプラスチックにそれを押すと、他のにして、片側で最初に、下方に引っ張る。カプセルがしっかりと多くの点でプレートに接続されるまで、すべてのレンズの赤道の周りに移動し、これを数回繰り返します。左ピンセットで固定カプセルを保持する、静かにレンズの赤道での繊維細胞と上皮細胞間の接続を中断する権利ピンセットで繊維の塊を揺らし。その後の底部に取り付けられたままカプセル/上皮を、それをロールオフ、離れて繊維の塊を押してください皿。ディッシュ内のすべてのレンズで、このプロセスを続けます。繊維の質量を（またはそれらが他の研究のためのレンズ蛋白質の源として凍結保存）を取り外して廃棄します。 </li></ol>パート3：中央の外植片のマイクロダイセクションと文化材料と試薬： <ol><li>滅菌使い捨てメス、＃15ブレード、（シンシナティ外科株式会社シンシナティ、オハイオ州） </li><li>滅菌リン酸塩はカルシウムとマグネシウム（Invitrogen社、カールスバッド、カリフォルニア州）と緩衝生理食塩水</li><li>培地：100ng/mlのFGF - 2（シグマ - アルドリッチ社セントルイス、MO）を含む懸濁培地。 </li></ol>手順： <ol><li>滅菌メスを使用して、のみ（CE）（図の中央の上皮細胞で構成され、側に中央広場では約2.0ミリメートル残して、差別化（図1A）の初期段階で細胞を含む末梢上皮（PE）を、切り落とす1B）。 </li><li>バイオセーフティキャビネットに中央の植片を含んでいる皿を転送する。新鮮な、平衡化（37℃、5％CO 2）懸濁培地を1mlで一度滅菌PBSを含むカルシウムとマグネシウムで3回洗浄し。これらは、汚染の可能性を大幅に削減洗う。 </li><li>分化1を誘導し、37℃加湿、組織培養インキュベーターで外植片を配置するために培養液の2mLの追加° C、5％CO 2を 。文化の期間は数時間な限り短くしたり、研究されているパラメータに応じて、限り2〜3週間として延長することができる。培地は2〜3日ごとに変更する必要があります。 </li></ol>パート4：分化に関連するイベントの分析のための植の収穫 1。タンパク質やRNAの解析のための収穫植材料： <ol><li> Microdissectingピンセット</li><li> SDS溶解バッファーまたはRNA -後</li></ol>手順： <ol><li>潜伏期間の終わりに、培養液を取り除く。 </li><li>ステレオ解剖顕微鏡を使って、静かにそれぞれ植の端を緩める。 </li><li>ピンセットで各植片を持ち上げて、100μlのSDS溶解緩衝液（タンパク質分析用）またはRNA -以降（RNA分析用）についての入ったチューブに移す。外植片はピンセットに固執しないことを保証するために溶液中でピンセットの先端に撹拌。 </li></ol> 2。免疫蛍光のための収穫植材料と試薬： <ol><li>リン酸塩はカルシウムとマグネシウム（Invitrogen社、カールスバッド、カリフォルニア州）と緩衝生理食塩水</li><li>リン酸（Invitrogen社、カールスバッド、カリフォルニア州）（カルシウムおよびマグネシウムを含まない）緩衝生理食塩水</li><li> 4％パラホルムアルデヒド（ボストンのバイオ製品、ウスター、MA） </li><li> Microdissectingピンセット</li><li>疎水性マーキングペン</li><li>顕微鏡用スライドガラス</li><li> 0.25パーセントリン酸塩のトリトンX - 100は、緩衝生理食塩水。 </li></ol>手順： <ol><li> PBSは、カルシウムとマグネシウムを含むで簡単に外植片を洗い流す。 </li><li>室温で30分間、4％パラホルムアルデヒドを添加することにより組織を固定してください。 </li><li>固定液を取り出して、リン酸緩衝生理食塩水と交換してください。固定片は、それらが免疫染色のために持ち上げてスライドガラスに転送できるように、多少堅くなる。 </li><li>ポジション組織を支援し、カーリングを防止するために、スライド上でPBSの小滴を（カルシウムおよびマグネシウムを含まない）に置きます。 microdissectingピンセットを使用して、外植片を持ち上げ、スライド上のドロップに挿入します。外植片に触れることなく、慎重にガラス上に外植片を平らにし、3〜5分間、室温で空気中の組織を乾燥させるために紙の芯に液体を取り除く。 </li></ol><img src="/files/ftp_upload/1519/fig1.jpg" alt="図1" /> 図1 A.、末梢上皮には分化特異的タンパク質を発現する。示すように、外植片はすぐに顕微解剖の後、N -カドヘリンのために免疫染色した。式がこの地域で細胞が分化し始めていることを示し、末梢上皮の細胞のバンドに見られる。B.は末梢上皮が発現するN -カドヘリンと他の分化特異的タンパク質その細胞を除去するために離れてトリミングすることができます。小さな四角が灰色に記載されている間、赤い円環は、急行N -カドヘリンそのセルの位置を表しますパネル1Aに示すように上皮の象限を表しています。末梢上皮はまだ区別するために開始されていないセルだけが含まれている中央広場を残して、4つのメスをカットして除去することができます。

<ol>
	<li>McAvoy, J. W., Chamberlain, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+(FGF)+induces+different+responses+in+lens+epithelial+cells+depending+on+its+concentration.">Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration.</a> Development. 107 (2), 221-228 (1989).</li><li>Campbell, M. T., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+of+fibre+differentiation+in+cultured+rat+lens+epithelium+under+the+influence+of+neural+retina-conditioned+medium.">Onset of fibre differentiation in cultured rat lens epithelium under the influence of neural retina-conditioned medium.</a> Exp Eye Res. 39 (1), 83-94 (1984).</li><li>Lovicu, F. J., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+lens+epithelial+explants+induced+to+differentiate+into+fibres+by+fibroblast+growth+factor+(FGF).">Structural analysis of lens epithelial explants induced to differentiate into fibres by fibroblast growth factor (FGF).</a> Exp Eye Res. 49 (3), 479-494 (1989).</li><li>Golestaneh, N., Fan, J., Fariss, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+major+intrinsic+protein+(MIP)/aquaporin+0+expression+in+rat+lens+epithelia+explants+requires+fibroblast+growth+factor-induced+ERK+and+JNK+signaling.">Lens major intrinsic protein (MIP)/aquaporin 0 expression in rat lens epithelia explants requires fibroblast growth factor-induced ERK and JNK signaling.</a> J Biol Chem. 279 (30), 31813-31822 (2004).</li><li>Saravanamuthu, S. S., Gao, C. Y., Zelenka, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+is+required+for+lateral+induction+of+Jagged1+during+FGF-induced+lens+fiber+differentiation.">Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation.</a> Dev Biol. 332 (1), 166-176 (2009).</li><li>Lovicu, F. J., McAvoy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-induced+lens+cell+proliferation+and+differentiation+is+dependent+on+MAPK+(ERK1/2)+signalling.">FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.</a> Development. 128 (24), 5075-5084 (2001).</li><li>Dirks, R. P., Kraft, H. J., Van Genesen, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cooperation+between+two+silencers+creates+an+enhancer+element+that+controls+both+the+lens-preferred+and+the+differentiation+stage-specific+expression+of+the+rat+beta+B2-crystallin+gene.">The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stage-specific expression of the rat beta B2-crystallin gene.</a> Eur J Biochem. 239 (1), 23-32 (1996).</li><li>Yang, Y., Stopka, T., Golestaneh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+alphaA-crystallin+via+Pax6,+c-Maf,+CREB+and+a+broad+domain+of+lens-specific+chromatin.">Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin.</a> The EMBO journal. 25 (10), 2107-2118 (2006).</li><li>McAvoy, J. W., T, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+retinas+promote+cell+division+and+fibre+differentiation+in+lens+epithelial+explants.">Neural retinas promote cell division and fibre differentiation in lens epithelial explants.</a> Curr Eye Res. 3 (6), 827-834 (1984).</li></ol>

ラットのレンズの外植片のシステムが正常水晶体線維21,3,4,5に水晶体上皮細胞の分化を研究する研究室の数によって使用されています。 100ng/mlの時にFGF - 2にさらされると外植片は、形態と遺伝子発現の変化は数日間にわたって順次3,4,5を表示されると、分6内シグナル伝達に変更を表示するために開始されます。注意が汚染を防止するために取られれば文化は2-3週間生き抜く。 FGF - 2の前に分化マーカーを発現あらゆる細胞は1,5を追加された場合、彼らはいくつか含まれているので、このプロトコルで説明されている中央の外植片は、分化に関連するイベントのシーケンスを研究するために特に便利です。細胞は、それが可能な分化に関連するシグナル伝達および転写イベントの時間的経過を追跡すること、コホートとして、同期的に分化する。時間の長さが異なるため、培養の外植片は、このようにシーケンスのイベントについての正確な時間情報を提供します。特異的阻害剤は、関連するシグナル伝達経路を特定するために、培養培地に添加してもよい。外植片をSDSゲル電気泳動とイムノブロッティングによるタンパク質の発現を分析したり、RT - PCRによる特定のmRNAの発現を分析するために使用されることがあります。培養期間の長さに応じて、600 ngの/植 - 20から50μgの/植とRNAの収量からのタンパク質収量の範囲は約200です。我々は一般的に皿あたり5から6の外植片は、いくつかのアッセイに十分なタンパク質やRNAを提供することを見つける。外植片からのRNAは、分化に重要である可能性新規遺伝子を識別することができるマイクロアレイ解析により遺伝子発現を評価するためのcDNAを調製するために使用することができます。外植片は、トランスフェクトすることができる。トランスフェクション効率は一般的に低いですが、それは、レポーター遺伝子4をアッセイするための十分です、7、8。外植片の免疫蛍光顕微鏡は、目的のタンパク質の細胞内局在を決定することにより、生化学的方法に有用な補助を提供します。従って、ラットのレンズの外植片の準備と文化は、このようなトランスジェニックやノックアウトマウスの作製など、生体のテクニックで補完することができる哺乳類で、端末のレンズの分化を研究するための強力なシステムを、提供します。

preparation and culture of rat lens epithelial explants for studying terminal differentiation

接眼レンズの前面は、毛様体の根底にある環状のゾーンで増殖する上皮細胞の単層によって覆われている。網膜からの拡散FGFは、それらがレンズの大部分を構成する細長い水晶体繊維細胞、の後の配列への分化を誘導する場所分裂に続いて、これらの細胞は、後方に移動する。水晶体線維に水晶体上皮細胞の分化はFGF - 2の存在下で前方上皮の中心領域の培養片でin vitroで誘導することができる。外植片は、目からレンズを取り外すと解剖ピンセットで後部側のレンズカプセルを把握し、新生児ラットのレンズから調製されています。後嚢をそっと開いて引き裂かれ、組織培養皿のプラスチック製の底に押されている。外植片の周辺領域は、メスで除去され、中央のエリアは、検討すべきパラメータに応じて、限り2〜3週間などのため100ng/mlのFGF - 2の存在下で培養される。培養片の上皮細胞は数日から数週間の期間にわたって近似同期して区別するので、シグナル伝達および遺伝子発現のタイムコースは、分子生化学、薬理学的手法を用いて決定することができる。それは興味の蛋白質の細胞内局在を証明し、シグナル伝達経路の実験操作の生理的影響を明らかにすることができるように免疫蛍光顕微鏡観察は、これらのメソッドへの強力な補助です。

Watch this Scientific Journal Video about Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation at JoVE.com

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

FGF - 2の存在下で培養するとラットレンズ上皮の中心領域の外植片は、同期的に分化する。そのような文化の免疫蛍光顕微鏡は、遺伝子発現と分化に関連付けられているシグナリングイベントに関する新たな情報を提供することができます。

最終分化を研究するための準備とラットの水晶体上皮外植片の培養

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

preparation-culture-rat-lens-epithelial-explants-for-studying

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K ビュー.  National Eye Institute (NEI), National Institutes of Health (NIH). FGF - 2の存在下で培養するとラットレンズ上皮の中心領域の外植片は、同期的に分化する。そのような文化の免疫蛍光顕微鏡は、遺伝子発現と分化に関連付けられているシグナリングイベントに関する新たな情報を提供することができます。

ビデオ: Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18ラット脳の器官型スライス培養

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

生物学

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

遺伝子銃の箇条書きとスライス培養における神経細胞の微粒子銃トランスフェクションの準備

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

外植片から成長した初代ヒト気管支上皮細胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

生体力学とMechanobiological研究ラット尾腱の準備

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

イモリのレンズ再生を研究するために培養虹彩色素上皮細胞のためのシステム

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

人間の腸粘膜の外植片の文化と偏刺激する新規な方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

哺乳類の全胚培養に適したラット血清の調製

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

レンズ無料のビデオ顕微鏡、付着性細胞培養の定量的解析

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

ラット骨髄からの破骨細胞前駆体の分離・精製・分化

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

オルガノイド培養のためのヒト肺上皮前駆細胞の単離と濃縮

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...