この作業は、イスラエル貿易労働省のKAMINプログラムとQrons Inc.(777 Brickell Avenue Miami、FL 33131、US)によって資金提供されました。

このプロトコルでの動物の使用は、全国動物管理使用委員会によって承認されました。
注:炭酸カルシウムサンゴの骨格は、アラゴナイトの結晶形で使用する必要があります。神経培養のためにこれまでにテストされたサンゴの種類は、 ポリテスルテア、スティロフォラピスティラータ、トラ キフィリアジェフロイです。 スケルトンは全体または地上で購入できます。
1.サンゴの骨格片をきれいにする
注意: 以下の手順は、以下に説明する溶液は危険であり、火傷や刺激を引き起こす可能性があるため、化学薬品フード内で室温で実行する必要があります。
<ol><li>ハンマーを使ってサンゴの骨格を壊し、それを0.5〜2 cmの断片に分けます。有機および非有機残留物を溶解するには、サンゴの骨格片を10%次亜塩素酸ナトリウム溶液に10分間浸した後、二重蒸留水(DDW)で1回洗浄します。</li>
	<li>残りの有機残留物を除去するには、フラグメントを1M NaOH溶液に5分間浸した後、重水素減少水で1回洗浄します。断片を30%H 2 O2溶液に10分間浸すことによって有機堆積物の除去を続け、次いで断片をDDWで3倍洗浄する。</li>
	<li>余分な重水素減少水をできるだけ取り除き、フード内のサンゴの破片を乾燥させます(1〜8時間)。</li>
</ol>2.ガラスカバースリップのクリーニング
<ol><li>ガラスカバーガラスを100mmのガラス製ペトリ皿に移します。10 mLの95%エタノールを15分間加えます。</li>
	<li>エタノールを取り除き、DDW 3xで洗浄し、各洗浄の間に10分間待ちます。DDWが蒸発するまで、80°Cに予熱した加熱プレートに皿を置きます。乾燥中にプレート内のカバーガラスを数回静かにかき混ぜて、互いにくっつかないようにします。</li>
	<li>カバーガラスをオートクレーブします。</li>
</ol>3.サンゴ骨格粒の調製
<ol><li>完全に壊れるまで、乳鉢と乳棒(手動粉砕)を使用してサンゴの骨格の破片を粉砕します。結果は、20μm〜200μmの範囲のサイズの粒子の混合物です。</li>
	<li>または、電気粉砕機を使用して、1,000 rpmの速度で30秒間、サンゴの骨格の破片を粉砕します(ブレードの長さ= 6 cm、幅= 0.5 cm–1.0 cm)。結果として得られる粒径は、手動粉砕によって生成されるサイズ範囲と同様である。</li>
</ol>4.特定のサイズ範囲の穀物の精製
注意: マトリックス内の粒子のサイズを制御する必要がある場合は、次のろ過ベースの粒子精製手順を使用します。
<ol><li>グレインを手動または電気式の40 μmフィルターメッシュストレーナーに移します。</li>
	<li>ストレーナーを通して粒子をふるいにかけて、粒子を2つの特定の範囲に分割します(電気ストレーナーを使用する場合は、次の条件をお勧めします:振とう= 600振幅/分、バウンス= 6 / s)。この手順では、1つは<40 μm、もう1つは>40 μmの2つのグループの粒径を生成します。 注: 2 つのサイズは、さまざまなメッシュのストレーナーを使用して決定できます。より制限された範囲が必要な場合は、ステップ4.2から異なるメッシュのストレーナーを通して各グループを再ふるいにかけます。</li>
	<li>穀物をオートクレーブします。</li>
</ol>5.サンゴの穀物でコーティングされた皿またはカバーガラスの準備
<ol><li>フラスコ、プレート、またはペトリ皿のコーティング用 注意: 次の手順は、無菌条件下で実行する必要があります。<ol><li>サンゴの骨格粒子をハンクス溶液に溶解した20 μg/mLのポリ-D-リジン(PDL)溶液に加えます。推奨される濃度は、1 mL PDL溶液あたり5 mg / 10 mgの穀物です。</li>
		<li>溶液をフラスコとディッシュ(約2 mL/25 cm2)に注ぎ、4°Cで一晩インキュベートします。 穀物は沈み、底に付着します。</li>
		<li>翌日、フラスコと皿を滅菌重水素減少水で一度洗います。フラスコと皿をフードで乾かします。 注:コーティングしたばかりのフラスコと皿を使用することが好ましい。コーティングされたフラスコとディッシュは、4°Cで保存すれば、コーティング後1週間まで使用できます。 ただし、マトリックスの有効性が損なわれる可能性があります。</li>
	</ol></li>
	<li>コーティングガラスカバースリップ(直径12mm、厚さ0.17mm)<ol><li>サンゴの骨格粒子を任意の所望の濃度で重水素減少水に加える。神経細胞に使用される一般的な密度は、5 mg / mLおよび10 mg / mLです。カバーガラスの中央に40 μLの穀物溶液を注ぎます(図4)。</li>
		<li>カバーガラスを80°Cに予熱した加熱プレートに置き、完全に蒸発するのを待ちます(通常15分)。これらの条件下では、穀物はカバーガラスに付着します。コーティングされたカバースリップをオートクレーブします。無菌状態で保管してください。</li>
		<li>培養の前日に、滅菌済みの24ウェルプレートの蓋にカバーガラスを置きます。各カバーガラスに100 μLの20 μg/mL PDL溶液を加えます。先端を使用して、液体が粒子領域全体とカバーガラス表面の残りの部分を覆っていることを確認します。</li>
		<li>プレートの底で蓋をします。プレートの側面をパラフィンフィルムで包みます。4°Cで一晩インキュベートします。</li>
		<li>翌日、重水素減少水で一度洗い、フードで乾かします。 注意: 新しくコーティングされたカバーガラスを使用することをお勧めします。</li>
	</ol></li>
</ol>6. サンゴ骨格粒子被覆ガラスカバーガラス上での海馬解離細胞の培養
注:海馬解離細胞培養の方法は、以前に公開された手順24、27から変更されました。培養物の調製は、4匹のラットの仔について記載されている。各海馬からの期待収量は1〜1.5 x 106 細胞です。
<ol><li>セットアップ<ol><li>空の60 mmペトリ皿を準備します。2 mLの最小必須培地(MEM)を60 mmのペトリ皿に注ぎ、氷の上に置きます。</li>
		<li>2 mLのMEMを35 mmディッシュに注ぎ、37°C、5〜10%CO2のインキュベーターに入れます。2 mLのMEMを15 mLのチューブに注ぎ、4°Cに保ちます。</li>
		<li>1 mLの初日培地を15 mLチューブに注ぎ、37°C、5〜10%CO2のインキュベーターに入れます。 注:初日培地の組成は、 材料の表に記載されています。</li>
		<li>200 μLの2.5%トリプシン溶液を解凍し、37°Cでインキュベートします。</li>
		<li>炎を使用して、直径0.5 mm、0.75 mm、および1.0 mmのヘッドを備えた3つのガラスパスツールピペットを準備します。</li>
		<li>大きなハサミ1本、小さなハサミ2本、大きなピンセット1本、小さなピンセット4本、メス1本など、適切な手術器具を準備します。</li>
		<li>フードに実体顕微鏡を用意します。</li>
	</ol></li>
	<li>大きなはさみを使用して、0〜3日齢のSprague Dawleyラットの子犬を、頭を体から切り離して空の60mmペトリ皿に犠牲にします(ステップ6.1.1)。遺体を処分します。</li>
	<li>大きなピンセットで口から頭を持って頭を持ち上げます。小さなハサミで頭蓋骨を覆っている皮膚を切ります。</li>
	<li>きれいなはさみで頭蓋骨の下(小脳の側面)に入り、頭蓋骨を右と左に切断して除去できるようにします。小さなピンセットを使用して、頭蓋骨を脳から剥がします。</li>
	<li>きれいなピンセットで脳を頭から分離し、60 mLの冷たいMEMを含む2 mmのペトリ皿に入れます(手順6.1.2を参照)。</li>
	<li>2本の小さなピンセットを使用して、実体顕微鏡下で海馬を解剖します。海馬をMEMを含む準備された35 mmディッシュ(ステップ6.1.2から)に移します。 注意: 子犬ごとに手順6.3〜6.6を繰り返す必要があります。</li>
	<li>メスを使って海馬を約1mmスライスに切ります。200μLのトリプシン溶液(最終濃度0.25%に希釈)を加える。穏やかに混合し、37°C、5〜10%CO2 で30分間インキュベートします。</li>
	<li>インキュベーション後、2 mLのコールドMEM(ステップ6.1.2)をディッシュに追加して、トリプシンを失活させます。最大径のガラスパスツールピペットを使用して(ステップ6.1.5)、37°Cに予熱した1 mLの初日培地(ステップ6.1.3)を含む15 mLチューブにトリプシン処理組織を移します。 注:組織のさらなる処理に最適な培地と組織の比率(v:v)は、1 mL / 8海馬です。培地を組織と一緒に移すことはできるだけ避けてください。</li>
	<li>組織を最大直径のガラスピペットに10〜15回通して粉砕します。次に、中径のガラスピペットを使用してこのプロセスを繰り返します。最小直径のピペットで粉砕を続けます。細胞死を減らすために泡立ちを避けてください。</li>
	<li>残りの組織片を2〜5分間沈め、上清を別の15 mLチューブに移します。血球計算盤を使用して細胞をカウントします。 注:好ましい細胞密度は200,000〜400,000細胞/ mLです。First Day 培地を使用して、470 x g で希釈または遠心分離し、細胞を濃縮します。</li>
	<li>各ガラスカバーガラスに100 μLの細胞を播種します。播種中は、カバーガラス全体を細胞で覆うようにしてください。37°C、5〜10%CO2でインキュベートします。</li>
	<li>翌日、500 μLの神経細胞成長培地をウェルに加えます。 注:神経細胞成長培地の組成は、 材料の表に記載されています。</li>
	<li>ピンセットを使用して各カバーガラスを適切なウェルにそっと移し、カバーガラスを傾けて初日培地を取り外します。蓋から残りのメディアを吸引します。</li>
	<li>37°C、5〜10%CO 2でインキュベートします。 インキュベーション中は培地の交換を避けてください。 培養はこれらの条件下で1ヶ月まで維持することができる。主な懸念は湿度です。したがって、インキュベーターを最大限に加湿した状態に維持するようにしてください。</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+hippocampal+mossy+fiber+and+associational-commissural+pathways+in+a+novel+dissociated+cell+culture+system.">Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (10), 4706-4711 (1996).</li><li>Peretz, H., Talpalar, A. E., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+survival+and+durability+of+neurons+and+astrocytes+on+3-dimensional+aragonite+biomatrices.">Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices.</a> Tissue Engineering. 13 (3), 461-472 (2007).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tissue Engineering. 11 (3-4), 585-596 (2005).</li><li>Baranes, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue Engineering. 13 (3), 473-482 (2007).</li><li>Morad, T. I., 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=Gliosis+of+astrocytes+cultivated+on+coral+skeleton+is+regulated+by+the+matrix+surface+topography.">Gliosis of astrocytes cultivated on coral skeleton is regulated by the matrix surface topography.</a> Biomedical Materials. 14 (4), 045005 (2019).</li><li>Green, D. W., 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+Therapeutic+Potential+for+Marine+Skeletal+Proteins+in+Bone+Regeneration.">A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration.</a> Marine Drugs. 11 (4), 1203-1220 (2013).</li><li>Neto, A. S., Ferreira, J. M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+and+Marine-Derived+Porous+Scaffolds+for+Bone+Tissue+Engineering.">Synthetic and Marine-Derived Porous Scaffolds for Bone Tissue Engineering.</a> Materials. 11 (9), (2018).</li><li>Ahmad Khalili, A., Ahmad, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Cell+Adhesion+Studies+for+Biomedical+and+Biological+Applications.">A Review of Cell Adhesion Studies for Biomedical and Biological Applications.</a> International Journal of Molecular Sciences. 16 (8), 18149-18184 (2015).</li><li>. Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19218486">https://www.ncbi.nlm.nih.gov/pubmed/19218486</a> (2019)</li><li>Drake, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+skeletal+organic+matrix+from+the+stony+coral+Stylophora+pistillata.">Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (10), 3788-3793 (2013).</li><li>Ramos-Silva, 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=The+skeletal+proteome+of+the+coral+Acropora+millepora:+the+evolution+of+calcification+by+co-option+and+domain+shuffling.">The skeletal proteome of the coral Acropora millepora: the evolution of calcification by co-option and domain shuffling.</a> Molecular Biology and Evolution. 30 (9), 2099-2112 (2013).</li><li>Peretz, H., Blinder, P., Baranes, D., Vago, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aragonite+crystalline+matrix+as+an+instructive+microenvironment+for+neural+development.">Aragonite crystalline matrix as an instructive microenvironment for neural development.</a> Journal of Tissue Engineering and Regenerative Medicine. 2 (8), 463-471 (2008).</li><li>Morad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CaCO3 Matrix Dictates Astrocytes Transition to Astrogliosis. , (2019).</li><li>Prada, F., 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=Ocean+warming+and+acidification+synergistically+increase+coral+mortality.">Ocean warming and acidification synergistically increase coral mortality.</a> Scientific Reports. 7, (2017).</li></ol>

著者は、競合する経済的利益がないことを宣言しています。

ここで紹介する手法は、培養中の神経細胞の維持と機能を改善する方法を説明しています。これは、細胞を育成し、その成長と活動を促進するサンゴの骨格粒子で作られたマトリックスに細胞を接着することによって達成されます。この手法を使用すると、脳内の細胞の環境を模倣する神経培養モデルの能力が向上します。
培養基質としてのマトリックスの導入は、古?...

解離した神経細胞(この場合は海馬細胞)の培養は、高い細胞分離とアクセス可能性を提供することにより、神経の成長と機能を研究するための貴重な実験モデルです1,2,3。このタイプの培養は、成長速度と生存率、神経毒性、神経突起の成長とネットワーキング、シナプスの接続性と可塑性、形態学的修飾、神経突起の組織と配線など、収集できる大量の情報のために、神経科学、医薬品開発、および組織工学で頻繁に使用されます1,4,5,6,7。
培養の重要性にもかかわらず、培養細胞は通常、2次元単層のガラスカバーガラス上で成長することを余儀なくされる。ガラスカバーガラスは接着強度の低い非育成基板であり、細胞増殖をサポートする能力が低いため、これらの厳密な環境改変は神経細胞の長期生存能力を著しく低下させます8,9,10,11。
培養された神経細胞は困難な条件で成長することを余儀なくされるため、生存率を高めるための不可欠なアプローチは、可能な限り自然環境を模倣することです12,13。これは、マトリックスとして機能し、細胞の細胞外マトリックスを模倣する生体材料を使用して、細胞が組織のような構造を形成し、栄養を助けることを可能にすることによって達成できます14。
生体材料の使用は、生体適合性の足場として機能し、機械的安定性を提供し、接着、生存、増殖、遊走、形態形成、および分化を含むさまざまな細胞特性を強化するため、細胞培養の改善における有望なアプローチです15,16,17。インビトロで細胞の状態を改善するために、いくつかの種類の生体材料が使用されます。それらの中には、生体高分子、または通常細胞の細胞外マトリックスの一部である生物学的成分がある。これらの生体材料は、主に重合コーティング剤またはヒドロゲル18、19、20の形態として使用される。一方では、上記のマトリックスは、細胞に成長するための使い慣れた3D環境を与え、皿への接着を促進し、それらに機械的支持を与える21、22。他方、それらの重合形態およびヒドロゲル内の細胞の閉じ込めは、増殖培地中に存在する育成成分への細胞のアクセスを妨害し、また顕微鏡的方法による細胞のフォローアップをより困難にする23。
サンゴの外骨格は、生物学的海洋起源のマトリックスです。それらは炭酸カルシウムでできており、機械的安定性があり、生分解性です。培養中の神経細胞を成長させるためのマトリックスとしてサンゴ骨格を使用した以前の研究では、ガラスカバーガラスと比較して、はるかに大きな接着性が示されています24,25。さらに、サンゴの骨格上で増殖した神経細胞は、その骨格が構成されているカルシウムを摂取する能力を示し、栄養欠乏の条件下で神経細胞を保護します26。さらに、サンゴの骨格は、神経細胞の生存率を高め、神経ネットワークの形成を促進し、シナプス結合の速度を高め、組織様構造の形成を可能にする支持的で育成的なマトリックスです27,28。最近の研究では、サンゴ骨格マトリックスの表面トポグラフィーがグリア細胞の分布と活性化に重要な役割を果たしていることも示されています8,29。また、サンゴ骨格は、培養中の骨細胞30、31、肝細胞、心筋細胞などの他の細胞種の培養のためのマトリックスとして有効である(未発表データ)。
したがって、サンゴの骨格は、in vitroで細胞を培養するための有望なマトリックスです。したがって、以下に詳述するプロトコルは、既存の方法で達成されたものよりも安定して繁栄した神経培養を生産するために、サンゴの骨格上で神経細胞を培養する技術について説明しています。このプロトコルは、心筋細胞、肝細胞、および他の細胞型の培養にも有用であり得る。

<table>
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Greiner</td>
			<td>#60-662160</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>#17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>#A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>D &#8211; glucose</td>
			<td>Sigma</td>
			<td>#G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Minimal Essential Eagle (DMEM)</td>
			<td>Sigma</td>
			<td>#D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical sieve</td>
			<td>Ari Levy</td>
			<td>#3700</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serun (FBS)</td>
			<td>Biological Industries</td>
			<td>#04-007-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>First Day Medium</td>
			<td></td>
			<td></td>
			<td>85.1% Minimum Essential Eagle&#8217;s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose.</td>
		</tr>
		<tr>
			<td>Flasks</td>
			<td>Greiner</td>
			<td>#60-690160</td>
			<td>25cm^2, Tissue culture treated</td>
		</tr>
		<tr>
			<td>Fluoro-deoxy-uridine</td>
			<td>Sigma</td>
			<td>#F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips</td>
			<td>Menzel-Glaser</td>
			<td>#BNCB00120RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Romical</td>
			<td>#007130-72-19</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Sigma</td>
			<td>#N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>HANK&#39;S solution</td>
			<td>Sigma</td>
			<td>#H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>Sigma</td>
			<td>#K3375</td>
			<td></td>
		</tr>
		<tr>
			<td>L - glutamine</td>
			<td>Sigma</td>
			<td>#G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual strainer (40&#181;m)</td>
			<td>VWR</td>
			<td>#10199-654</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimun Essential Eagle (MEM)</td>
			<td>Sigma</td>
			<td>#M2279</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td>De-Groot</td>
			<td>4-P090</td>
			<td></td>
		</tr>
		<tr>
			<td>NaClO (Sodium Hypochlorite)</td>
			<td>Sigma</td>
			<td>#425044</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>#S8045</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Neuronal Growth Medium</td>
			<td></td>
			<td></td>
			<td>45% MEM, 40% Dulbecco&#39;s modified eagle&#39;s medium (DMEM), 10% Nutrient mixture F-12 Ham, 0.25% (w/v) bovine serum albumin (BSA), 0.75% D-glucose, 0.25% L-Glutamine, 0.5% B-27 supplement, 0.1% kynurenic acid, 0.01% of 70 % uridine and 30% fluoro-deoxy-uridine.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Greiner</td>
			<td>#60-628160, #60-627160</td>
			<td>60mm, 35mm, respectively.</td>
		</tr>
		<tr>
			<td>Poly D &#8211; Lysine</td>
			<td>Sigma</td>
			<td>#P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Smart Dentin Grinder</td>
			<td>KometaBio</td>
			<td>#GR101</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>#15-090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>#U3750</td>
			<td></td>
		</tr>
	</tbody>
</table>

increasing durability of dissociated neural cell cultures using biologically active coralline matrix

解離した海馬の神経細胞とグリア細胞の培養は、高い細胞分離と制御された環境を提供することにより、神経の成長と機能を研究するための貴重な実験モデルです。しかし、in vitroでの海馬細胞の生存は損なわれます:ほとんどの細胞は培養の最初の週に死にます。したがって、培養中の神経細胞の耐久性を高める方法を特定することは非常に重要です。
サンゴの骨格に由来する結晶性アラゴナイトの形の炭酸カルシウムは、神経培養のための優れた活性マトリックスとして使用することができます。グリア細胞を育て、保護し、活性化することにより、サンゴの骨格は他のマトリックスよりもin vitroでこれらの細胞の生存と成長を促進します。
このプロトコルは、サンゴマトリックス上で海馬細胞を培養する方法を記載しています。このマトリックスは、サンゴの骨格の粒を培養皿、フラスコ、ガラスカバーガラスに取り付けることによって生成されます。粒子は、細胞を微細な3次元(3D)環境に導入して成長し、組織のような構造を形成することにより、細胞の環境を改善するのに役立ちます。サンゴの骨格によってもたらされる3D環境は、粉砕によって細胞のために最適化することができ、グリア細胞の活性に影響を与えることがわかっている特性である粒子のサイズと密度(すなわち、マトリックスの粗さ)の制御を可能にします。さらに、穀物を使用すると、特に光学顕微鏡を使用する場合に、培養物の観察と分析が容易になります。したがって、プロトコルには、in vitroで神経細胞の維持と機能を改善するためのツールとして、サンゴマトリックスの生成と最適化の手順が含まれています。

サンゴ骨格マトリックスを調製するために、サンゴ骨格全体(図1A)をハンマーを使用して0.5〜2 cmの断片に分割し(図1B)、10%次亜塩素酸塩溶液、1M NaOH溶液、および30%H2O2溶液(図1C)を使用して3つのステップ(プロトコルのステップ1)を通じて有機残留物から完全に洗浄しました。サンゴの破片は、骨格の色が茶色(図<...

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Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

解離した海馬細胞培養は、神経科学における極めて重要な実験ツールです。サンゴの骨格をマトリックスとして使用すると、神経保護的および神経調節的役割により、培養における神経細胞の生存と機能が強化されます。したがって、サンゴマトリックス上で増殖した神経細胞はより高い耐久性を示し、それによって培養により適しています。

生物学的に活性なサンゴリンマトリックスを用いた解離神経細胞培養の耐久性向上

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

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1.2K ビュー.  Ariel University. 解離した海馬細胞培養は、神経科学における極めて重要な実験ツールです。サンゴの骨格をマトリックスとして使用すると、神経保護的および神経調節的役割により、培養における神経細胞の生存と機能が強化されます。したがって、サンゴマトリックス上で増殖した神経細胞はより高い耐久性を示し、それによって培養により適しています。

ビデオ: Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心筋組織工学のための生物由来注射用材料の作製

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

生物工学

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

アクティブな細胞培養のための形状記憶ポリマー

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

アコースティックMicrostreamingを使用して標準的な実験系逆転写反応でmRNAの単セルの物理量からcDNA収量を増加させる

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

ラマン分光法を用いた細胞と細胞外マトリックスの非接触、ラベルフリーの監視

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

細胞培養物衝撃波アプリケーション

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

プライマリのグリア細胞の in Vitro Neuroinflammation のモデリングのための三次元ハイドロゲル文化を改善

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

大きな腱欠損を修復する組織設計されたコラーゲン移植片を用いた新規テノルラフィー縫合技術

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

いけにえの中空糸膜細胞培養による細胞外マトリックス繊維の生産

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Methods Article

生物学的に活性なサンゴリンマトリックスを用いた解離神経細胞培養の耐久性向上