心から感謝博士ベン ファウラー TEM 実験で彼の技術的な助けを拡張したいと思います。

このプロトコル オクラホマ州健康科学センター大学/機関審査委員会 (IRB #4509) のガイドラインに従います。プロトコルのすべての部分は、ヘルシンキ宣言の原則を会った。国立開発研究所 (NDRI) とオクラホマ州のライオンズ ・ アイバンクから角膜のサンプルを得た。
1. 初代培養細胞の分離
<ol><li>人間の角膜組織サンプルの受領、21.5 cm2シャーレ ダルベッコ滅菌リン酸緩衝生理食塩水 (DPBS) 2 mL を含む組織に転送 (1 x)。</li>
	<li>こすり、角膜ドームの顔アップを配置することで角膜の上皮を取り外します。完全な除去を確実に、45 ° の角度でかみそりの刃を使用して徹底的に上皮層をこすり落とします。次に、角膜ドーム下向きに反転し、徹底的に完全な除去を確実に 45 ° の角度でかみそりの刃と間質から血管内皮細胞をこすり落とします。DPBS と間質組織を洗浄します。</li>
	<li>間質組織を DPBS を使用してすべての時間で濡れている間質の部分を維持 10 番外科ブレードを使用して小さな断片にカットします。メディア (フラスコあたり 2 × 2 mm の組織の 4 または 5 の部分) を使用しないコート t25 フラスコ培養用フラスコに滅菌鉗子と場所を間質部分をピックアップします。</li>
	<li>約 30-40 分の 37 ° C でフラスコの底に付着する外植体を許可します。イーグル最小必須培地 (実装された EMEM) 10% 牛胎児血清 (FBS) および 1% を含む追加ゆっくりと付着、一度抗生物質/Antimycotic 外植片を妨げないでフラスコに。</li>
	<li>37 ° c、5% CO2インキュベーターでフラスコをそっと置きます。セルを分離する開始されるまで外植片を行わないでとフラスコ内で移行します。約 2-4 週間後電池が 100% の合流点に達する必要があります。100% 合流時に培養メディアを削除することによって、セルを通過し、DPBS とセルを洗ってください。</li>
	<li>セルにトリプシンを追加し、37 ° C でそれらを孵化させなさい約 5 分後に彼らが切り離されることを確認を光学顕微鏡下で細胞を表示し、新鮮なメディアを追加することによって、タンパク質の分解を停止します。</li>
	<li>1,000 x g. での遠心分離は、ペレットを乱すことがなく上澄みを慎重に削除の 15 mL コニカル チューブに細胞懸濁液を転送し、中断の新鮮な実装された EMEM のセル 10% FBS 1% 抗生物質。セルをカウントし、種子の新鮮な実装された EMEM のさらなる拡大のためコート t75 フラスコ培養用フラスコに約 1 × 106セル 1% FBS 1% 抗生物質。</li>
</ol>2. プライマリ HCFs 文化と 3 D 構造アセンブリ
<ol><li>3 D をアセンブルするから構造外植片の栽培実装された EMEM 10% FBS 1% 抗生物質/Antimycotic、通過カウント HCFs。</li>
	<li>種子の約 1 × 106細胞/ウェルと共に新鮮な EMEM の 1.5 mL の 3 D 構造から 0.4 μ m の細孔を有するポリカーボネート膜挿入の主な HCFs の 10% FBS 1% 抗生物質/Antimycotic コンス トラクターの先頭に。同じメディア別 1.5 mL を各構成要素の下に追加します。</li>
	<li>細胞播種の 24 h 後に実装された EMEM 細胞の培養 10% FBS 1% 上追加 1.5 mL と 1.5 mL の各構成要素の下に 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic 酸 (ビタミン C) で刺激した抗生物質/Antimycotic。</li>
	<li>37 ° c、3 週間 5% CO2インキュベーターの文化を維持し、全体の研究期間中に毎日新鮮なメディアを供給します。さらに通路は不要です。</li>
</ol>3. 細胞分化と共培養
<ol><li>8 x 103細胞/ウェル以前組み立てられた 3 D の上に SH SY5Y ひと神経芽腫細胞の 10% を含む実装された EMEM の含む 1.5 mL をコンス トラクターに追加 3 週間後 FBS、1% 抗生物質/Antimycotic、および 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic酸 (ビタミン C) です。<ol>
<li>SH SY5Y 神経芽細胞腫細胞分化を開始する前に、少なくとも 24 時間、角膜細胞の上に成長するを許可します。</li>
	</ol>
</li>
	<li>1 %fbs と 10 μ M の 1.5 mL を含む実装された EMEM の細胞を刺激することによって SH SY5Y 細胞分化の開始構造上にレチノイン酸。構成要素の下に実装された EMEM 10% FBS 1% 抗生物質/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic 酸 (ビタミン C) の 1.5 mL を追加します。暗闇の中でレチノイン酸のステップが実行されることに注意してください。<ol>
<li>5 日間この分化メディアで細胞の培養を続けます。</li>
	</ol>
</li>
	<li>細胞が分化段階に達したら、1.5 mL の 2 を含む無血清培にセルを切り替える脳由来神経栄養因子 (BDNF) と 1% の nM 抗生物質/Antimycotic は、コンス トラクターの先頭に追加することによって、実装された EMEM に追加。構成要素の下に実装された EMEM 10% FBS 1% 抗生物質/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic 酸 (ビタミン C) の 1.5 mL を追加します。暗闇の中で BDNF ステップが実行されることに注意してください。</li>
	<li>表 1に示されるようにそれ以上の分析の処理の前に追加 2 日間培養します。</li>
</ol>

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著者は競合する金銭的な利益を宣言しません。

いくつかの研究ができる角膜疾患のよりよい理解の開発を助けると同様の治療法を発見する様々 な動物モデルの開発で注目されています。しかし、これらの研究から人間に重要な価値は確認されていません。日には、様々 な生体外モデルを開発し、広くその顕著な臨床的意義のため調査が。私たち以前に確立された 3 Dの in vitroモデルです新規のシステムで大幅ビジョン研究の医学の進歩に貢献するの体内イベント体外28の概説の真っ白な能力を示しています,29,30しますこのプロトコルの目的は、この培養角膜モデル糖尿病角膜症、円錐角膜などの角膜の疾患による合併症の細胞および分子メカニズムを研究するために使用することができますの次の世代を開発するには。、とフックス。このような進歩は、臨床的に必要な多くの新しい治療法の発見に向けた強固な基盤を提供すること。
このモデルは、DM の場所と他の角膜疾患構造および細胞の異なった変化を識別するに役立つことがあります。これは、潜在的疾患管理の手伝いをする初期の段階でこれらの病気のスクリーニングの可能性を含む、実質的な意味を提供します。ここは簡単ですが非常に革新的なモデルを提案しました。最も重要な利点の 1 つは角膜の間質細胞を分泌し、人工環境にシードされているのではなく、自分の ECM を組み立てることことです。さらに、神経はより正確にモデル角膜間質神経相互作用を区別するために許可されています。したがって、このメソッドは、セル間質環境の中で存在することと同様、人間の角膜実質と神経ネットワークを勉強する能力を提供します。この文化のプラットフォームは、複雑な細胞間や細胞-マトリックス相互作用の角膜の病気にかかった状態に影響を与える将来の調査になります。この 3 Dの in vitroモデルのアプローチは、提供するために改善し、新規疾病メカニズム、特にと比較されたとき現在従来洞察文化システムや動物モデルに予想されます。
この 3 Dの in vitroモデルの説明生成は、未来の治療の発見の道を開くでしょう。

人間の体内では、角膜は、難解な神経組織です。神経は接触、苦痛、温度のような様々 な感覚を担当し、また創傷治癒、反射を点滅、涙の生産と分泌5,6,7で重要な役割を持っています。角膜間質の神経幹は輪部の神経叢から発生し、放射状周辺の角膜実質を入力します。間質の神経組織はコラーゲン ラメラに平行、彼らはさらに小さい線維束彼らは実質5,8に向かって進むように分岐します。神経線維はさらに上皮層に浸透し、したがって、神経支配は広く広がって角膜上皮と実質の間で。したがって、神経支配は、角膜の健康と病気の状態の重要な役割を持ちます。このプロトコルでは、体内の神経間質の相互作用を模倣する、その種の最初である新しい 3 Dの in vitroモデルの進歩を明らかにします。SH SY5Y 細胞株は、神経の成長を観察するために使用も特徴と、最も確立されたラインの 1 つであるこの研究に使われました。SH SY5Y 細胞ラインは両方の基板の付着 (S 型) を生成する記載されているし、neuroblastic (N 型) 細胞を分化転換9を受けることができます。結果として、この細胞ラインは N 型セルのトリプルの連続 subclone 選択から派生するにもかかわらずそれも含まれていますレチノイン酸と脳由来を使用してニューロンへの分化過程を受けることができる S 型セルの数が少ない神経栄養因子の9。これは角膜合併症糖尿病網膜症 (DM) やその他の眼の疾患に関連付けられているのより良い理解につながる可能性がありますツールを提供します。ために取得し、眼疾患の患者から神経細胞の培養に伴う困難、この 3 Dの in vitroモデルはニューロン間相互作用の勉強と角膜実質とシグナル伝達における実質的な意味を提供します。
病的状況しばしば妥協の生活の質につながる、非常に大規模で体のさまざまな組織に影響を与えます。眼ジストロフィーは、全身疾患と視力の損失や永久的な視力低下につながる多くの場合関連付けられている一般的な合併症です。包括的な研究は、基底の細胞レベルでの効果と同様に、病気の状態の理解を深めるために不可欠。このような病気の影響を研究するには、様々 な生体内と体外のモデルはティッシュ エンジニア リング アプリケーションの助けを借りて開発されています。Corneal ティッシュ エンジニア リング アプリケーションは、科学10、11,12,13,14では、様々 な分野で大きな関心を集めているが、時に重大な制限があります。実際のアプリケーションは、拒絶反応、感染症、瘢痕10,11,12,13,14を移植角膜を含みます。正常に開発され、様々 な生体外モデル3,15,16,17,18,を設置するいくつかの研究がある19,,2021,22,23,24,25,26。3 Dの in vitroモデルは、最も有望な偉大な科学的な興味の。3 D モデルより体内の細胞および生理学的イベント線維化に重要であり、創傷治癒15,27,28,29をミラーする知られています。これらの in vitroモデルは、角膜合併症を含む異なる病状を治療するための新しい治療上のアプローチを見つけることに不可欠な役割を果たします。角膜の機能に神経支配の重要な役割にもかかわらず角膜組織設計構造2,3で末梢神経の増殖を促進するために少しの努力をしました。ただし、目的の組織機能を達成するために標的組織を模倣する提案 3 D培養細胞構造。
糖尿病角膜症神経欠陥のためここで説明するモデルのための明白なアプリケーションですが、円錐角膜、Fuchs のジストロフィーを含む人間の in vitroモデルの恩恵を受けることができますいくつかの他の角膜疾患があります。当社の 3 D モデルは、この見通しから出現し、ドラッグデリバリー システムを評価するために角膜組織の体外表現と新しい眼薬の安全性の開発を提案します。

<table>
	<tbody>
		<tr>
			<td>Healthy corneal tissue</td>
			<td>NDRI</td>
			<td></td>
			<td>Samples from donors with no ocular trauma or systemic disease</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Solution (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Fischer Scientific</td>
			<td>13-812-42</td>
			<td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr>
			<td>Single edge razor blades</td>
			<td>Personna</td>
			<td>270100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical scalpel blades No.10</td>
			<td>Feather Surgical Blade</td>
			<td>2976#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#8217;s Minimum Essential Medium</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td>10% FBS is required for media preparation</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco by Life Technologies</td>
			<td>15240-062</td>
			<td>1% Antibiotic-Antimycotic is required for media preparation</td>
		</tr>
		<tr>
			<td>0.05% Trypsin EDTA(1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane inserts with 0.4-&#956;m pores</td>
			<td>Corning Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>2-O-&#945;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td>
			<td>Sigma-Aldrich</td>
			<td>SMB00390-14</td>
			<td>A concentration of 0.5 mM should be used for the study</td>
		</tr>
		<tr>
			<td>Wax block</td>
			<td>VWR</td>
			<td>50-949-027</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cells</td>
			<td>ATCC</td>
			<td>SHSY5YATCC CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3014-10UG</td>
			<td>Final concentration of 10uM needs to be used</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100MG</td>
			<td>Final concentration of 2nM needs to be used</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide(DMSO)</td>
			<td>VWR-Alfa Aesar</td>
			<td>67-68-5</td>
			<td>Ultra Pure Grade-Sterile DMSO to be used</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td>
			<td>Fisher Scientific</td>
			<td>12-565-351</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td>
			<td>Fisher Scientific</td>
			<td>12-565-349</td>
			<td></td>
		</tr>
	</tbody>
</table>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

ティッシュ ・ エンジニア リングは、世界角膜視力損失1に苦しんで推定 1000 万人ひと角膜交換のための需要が高いため実質的な認識を得ています。実行可能な人間の角膜、三次元 (3 D) 組織で重要な進歩のための要求に対処するため工学2,3、4を行われています。これら角膜モデル範囲簡単な単分子膜システムから多層モデルでは、3 D の完全厚さ角膜同等2に 。
ただし、3 D ティッシュ エンジニア リングの角膜疾患モデルの in vitroのコンテキストでの使用を検討多層 3 D 角膜組織構造、機能、および異なる種類の細胞 (すなわち、神経ネットワークに欠けている類似している日付上皮、実質、内皮細胞) の2,3。さらに、医薬品のための動物テストを削減する試みで角膜の in vitro組織モデルの需要が増加しています。したがってより洗練されたモデルは人間の生理的要求するシステムに合わせて必要なより患者の人口に関連するモデルの開発が絶対に必要です。主な角膜線維芽細胞 (HCFs) 健康なドナーからから神経細胞の三次元共培養モデルを搭載、角膜内の複数セル型ジストロフィー、フックス、糖尿病角膜症、円錐角膜などの病気やを受けますことを考えるSH SY5Y セルの行。これにより、初めて人間の角膜組織内の 2 つのセル型間の相互作用を調査します。このモデルが神経損傷を示す角膜疾患の神経間質相互作用に関連付けられている基になるメカニズムを解剖して可能性があると考えています。この 3 D モデルは生体内で角膜組織の基本的な解剖学的および生理学的な性質を反映し、角膜欠損を調査だけでなく、様々 なエージェントの有効性を動物実験の前にスクリーニング ツールとして将来的に使用できます。

図 1は、モデル 3 D培養作業の順を追って代表イメージです。最初のステップでは、細胞は人間の角膜から分離されます。その後、ポリカーボネート膜上に成長した、自己分泌の 3 D マトリックスを組み立てるためビタミン C と刺激します。この 3 D 構築システム、多層細胞の生体内での合成を誘導する-間質マトリックスのような。その後、神経芽細胞腫細胞が間質細胞の神経細胞の分化を開始することによって続いて上にシードされます。これは角膜の間質細胞とモデル 3 D生体外で分化した神経細胞の共培養に します。図 2は、ポリカーボネート膜を示す 3次元自己組織化構造 (図 2 a) の高倍率の透過電子顕微鏡 (TEM) 像と角膜の間質細胞が自己組織化マトリックスを分泌上シード(図 2 b) に続いて分化した神経細胞間質神経細胞配列を示す上に種をまいた。画像の矢印は、神経細胞を示しています。角膜実質と神経細胞との間の直接のクロストークを与え、間質層に存在するキープレーヤー角膜欠損の規制に影響を与えると考えます。したがって、このモデル人間神経の in vitroモデル差別化 SH SY5Y 神経芽細胞にプレゼント持続可能な神経細胞の形態と細胞外マトリックス (ECM)、培養の以前に確立された 3 D モデルを組み合わせることにより様々 な差別化要因です。したがって、我々 はこのモデルが健康と病気の ECM の形態学的および分子の違いの将来の研究を有効にすると思われます。角膜の病理学と関連付けられている角膜実質神経相互作用の重要な側面を分析し、新しい治療法の開発のための道を開く、この新しい 3 Dの in vitroモデルが役立つと考えています。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56308/56308fig1.jpg"> 図 1: 段階的に 3 Dの in vitro培養モデルを示すイメージ。この画像は、人間の角膜と間質細胞ポリカーボネート膜でシード、3 D の構成要素である、実装された EMEM の培養およびビタミン c の刺激から細胞の隔離を示します。さらに、分化した神経細胞の間質細胞の上にシード処理するとおりです。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56308/56308fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56308/56308fig2.jpg"> 図 2:3 D培養システムの TEM 像を高倍率。ポリカーボネート膜上に (A) 間質細胞配置。(B) Seeded 間質細胞の上に神経細胞を区別されます。スケール バー = 500 nm。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56308/56308fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>分析技術</td>
			<td>潜在的な影響</td>
		</tr>
<tr>
<td>量的なリアルタイムのポリメラーゼの連鎖反応</td>
			<td>コラーゲン マーカーを調べる (Col I、Col III および Col V)、線維化マーカー α-平滑筋アクチン (α SMA)、およびキーのメディエーター受容体 (IGF1 R) は、DM インスリン成長因子 1 (IGF-1)、それを含む</td>
		</tr>
<tr>
<td>透過型電子顕微鏡</td>
			<td>コラーゲン線維と細胞外マトリクス相互作用など構造の変化を調べる</td>
		</tr>
<tr>
<td>蛍光抗体法</td>
			<td>特定の抗体の使用は化学的に細胞の抗原を調査する蛍光色素の共役</td>
		</tr>
</tbody></table>表 1: 分析の技術。

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

このプロトコルでは、新規三次元の in vitroモデル、角膜間質細胞と分化した神経細胞が一緒に培養試験と 2 つの細胞のタイプの相互作用の理解を支援するについて説明します。

角膜組織工学: In Vitroのモデル人間の角膜の神経間質相互作用

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

8.7K ビュー.  University of Oklahoma Health Sciences Center. このプロトコルでは、新規三次元の in vitroモデル、角膜間質細胞と分化した神経細胞が一緒に培養試験と 2 つの細胞のタイプの相互作用の理解を支援するについて説明します。

ビデオ: Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

エバンスブルー色素でゼブラフィッシュ幼生骨格筋の整合性の分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

発生生物学

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

SH-SY5Yヒト神経芽腫細胞株の分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

マルチカラー蛍光レポーターマウスを用いた角膜細胞の系譜トレースする方法

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

開発<em&gt;インビトロ</em&gt;アッセイは、間葉細胞受けた上皮間葉移行の収縮機能を評価します

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

ティッシュ・エンジニアリングと疾病調査に使用するための小児のヒト食道上皮細胞の条件付き再プログラミング

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

軟骨細胞分化のDNAメチル化レベルを定量化する5-mCドットブロットアッセイ<em&gt;インビトロ</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模擬微小重力下でのマウス前胞状卵胞の成長

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

マウス二次口蓋融合のライブイメージング

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

セルショウジョウバエ切り込みをトランスとの結合を評価する集計の試金-配位子とCisによるその阻害-配位子

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...