著者らは、この研究が補助金契約No.760813の下で、欧州連合(EU)のHorizon 2020研究およびイノベーションプログラムから資金を受け取っていることを認めたいと思います

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aflatoxin+B1:+A+review+on+metabolism,+toxicity,+occurrence+in+food,+occupational+exposure,+and+detoxification+methods.">Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods.</a> Food and Chemical Toxicology. 124, 81-100 (2019).</li><li>Kermanizadeh, A., Brown, D. M., Moritz, W., Stone, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+inter-individual+Kupffer+cell+variability+in+the+governance+of+hepatic+toxicity+in+a+3D+primary+human+liver+microtissue+model.">The importance of inter-individual Kupffer cell variability in the governance of hepatic toxicity in a 3D primary human liver microtissue model.</a> Scientific Reports. 9, 7295 (2019).</li><li>Berger, B., 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=Comparison+of+Liver+Cell+Models+Using+the+Basel+Phenotyping+Cocktail.">Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail.</a> Frontiers in Pharmacology. 7, 443 (2016).</li><li>Ramaiahgari, S. C., 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+3D+in+vitro+model+of+differentiated+HepG2+cell+spheroids+with+improved+liver-like+properties+for+repeated+dose+high-throughput+toxicity+studies.">A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies.</a> Archives of Toxicology. , (2014).</li><li>Li, Y., 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=Factors+affecting+the+in+vitro+micronucleus+assay+for+evaluation+of+nanomaterials.">Factors affecting the in vitro micronucleus assay for evaluation of nanomaterials.</a> Mutagenesis. 32 (1), 151-159 (2016).</li><li>Kirkland, D., Reeve, L., Gatehouse, D., Vanparys, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+in+vitro+genotoxicity+battery+comprising+the+Ames+test+plus+the+in+vitro+micronucleus+test+is+sufficient+to+detect+rodent+carcinogens+and+in+vivo+genotoxins.">A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins.</a> Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 721 (1), 27-73 (2011).</li><li>Curcio, E., 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=Mass+transfer+and+metabolic+reactions+in+hepatocyte+spheroids+cultured+in+rotating+wall+gas-permeable+membrane+system.">Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system.</a> Biomaterials. 28, 5487-5497 (2007).</li><li>Glicklis, R., Merchuk, J. C., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+mass+transfer+in+hepatocyte+spheroids+via+cell+viability,+spheroid+size,+and+hepatocellular+functions.">Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions.</a> Biotechnology and Bioengineering. 86, 672-680 (2004).</li><li>Asthana, A., Kisaalita, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtissue+size+and+hypoxia+in+HTS+with+3D+cultures.">Microtissue size and hypoxia in HTS with 3D cultures.</a> Drug Discovery Today. 17, 810-817 (2012).</li><li>Gaskell, H., 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=Characterization+of+a+functional+C3A+liver+spheroid+model.">Characterization of a functional C3A liver spheroid model.</a> Toxicology Research. 5, 1053-1065 (2016).</li><li>Cho, E. C., Zhang, Q., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sedimentation+and+diffusion+on+cellular+uptake+of+gold+nanoparticles.">The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles.</a> Nature Nanotechnology. 6, 385-391 (2011).</li><li>Hinderliter, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISDD:+A+computational+model+of+particle+sedimentation,+diffusion+and+target+cell+dosimetry+for+in+vitro+toxicity+studies.">ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies.</a> Particle and Fiber Toxicology. 7, 36 (2010).</li><li>Kramer, N. I., di Consiglio, E., Blaauboer, B. 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著者らは開示するものは何もない。

3D肝モデルの適用は、標的にされる特定の生化学的エンドポイントまたは有害な結果経路によってかなり異なる。各モデルには、一次ヒト肝細胞(PHH)モデルのドナー間変動から細胞系モデルにおけるチトクロームp450活性の低下まで、その利点と限界があるが、すべて独自の権利6、12、18、19において価値がある。遺伝毒性を評価する場合、モデルには、能動増殖が必要なため、インビトロ小核アッセイなどの規制承認エンドポイントとの適合性が制限される。遺伝子毒性評価は、DNA修復が一過性の病変を矯正する機会がある場合に細胞分裂後に評価される固定DNA損傷の定量化を必要とするため、これが必要である。残念なことに、高度に分化した肝細胞(すなわち、hepaRG)ベースのスフェロイドまたはPHHマイクロ組織は、最も生理学的に関連する肝臓様特性を示すものと考えられるが、これは静的(不増殖)モデル12、19、20を形成する。その結果、ここで提示される3D HepG2スフェロイドモデルは、遺伝毒性試験をサポートできる適切な代替モデルを提供します。HepG2細胞系スフェロイドは、アルブミンや尿素産生などの基本的な肝臓様特性を維持しながら、スフェロイドの外表面に十分な積極的に細胞を分割し、CYP450活性5、12、19を有する。このインビトロ肝モデルは、この小核アッセイを補完するために開発されており、これは、遺伝毒性試験8、10、11、21の電池で推奨される2つのインビトロアッセイのうちの1つである。しかし、このモデルはDNAシーケンシング解析および遺伝子発現(RNA)技術に容易に適用することができ、彗星アッセイのような他のDNA損傷エンドポイントにさらに適応して利用される可能性がある。しかし、一部のエンドポイント解析において、ENM干渉が果たす役割を考慮することが重要です。例えば、フローサイトメトリーベースの解析は、特に粒子干渉22によるENM遺伝子毒性評価には適さない場合がある。
細胞分裂を活発に行うスフェロイドモデルの制限要因の1つは、その大きさです。シード密度の最適化は、モデルが増殖し続ける十分なセルが必要であるため、重要です。しかし、あまりにも高い細胞数, スフェロイドが過度にコンパクトになる結果, 壊死コアの増加につながります.この壊死の原因は、酸素および栄養拡散を制限していると考えられており、この拡散の限界は、組織23、24の約100〜150μmであると考えられている。しかし、これは細胞型、細胞数、足場相互作用および培養条件25に依存する。以来、直径約700μmがC3Aスフェロイドの中心における壊死の早期発症を避けるための限界であることを示されており、スフェロイド当たり4000HepG2細胞を播種すると、露光時のモデルの直径が≤500μm26であることを保証する。さらに、Shah et al. は、ヘプG2細胞が球状体1個当たり5000細胞を超える播種が培養7日後に生存率の25%減少を示したことを確立した。これを克服するために、本プロトコルで考案されたモデルは、スフェロイドの初期形成に続いて、吊り下げ低下がアガロース被覆井戸に移される重要なステップを経る。これにより、スフェロイド内で増え続ける細胞数を維持するために、より多くの培養培地が存在することを保証する。その結果、HepG2スフェロイドモデルは培養で10日後に70%以上生存し続け、インビトロでの長期ハザード評価に利用することができる。
HepG2スフェロイドモデルは急性および長期の曝露レジーレジーをサポートできるが、培地の完全な置換がスフェロイドの潜在的な損失のために推奨されないとして、培養期間中の細胞培養培地のリフレッシュは、このモデルに対して制限される。ENM露光では、均質なENM分散液が凝集・沈殿する傾向が高いと推測されます。しかし、ENM堆積物が粒子パラメータ(例えば、大きさ、形状および密度)に応じて変化し得る速度は、IN体外沈下、拡散および線量測定(ISDD)モデル、またはその最近の誘導体を使用して理論的に決定できることが注目に値する。この心を用いて、細胞培養液の50%のみが細胞培養の表面から慎重に除去されれば、ENM用量の破壊およびその後の除去は理論的には最小限に抑えられるべきである。しかし、ブラウン運動が遊びで、これは厳密にはそうではないかもしれないし、テストされる各特定のENMの堆積および沈下へのさらなる作業は、長期的な暴露体制27を通じて正しい量体測定が保持されることを確実にするために行われるべきである。主に、これは最終的な蓄積された濃度に不可欠である可能性があり、繰り返し投ずる体制を実行する際に考慮すべき潜在的な制限です。一方、化学物質ベースの暴露は、考慮すべき独自の制限がないわけではないが、化学物質が溶液中に残る傾向があり、新たに添加された濃度に加えて元の化学物質濃度を直接交換するという点でより単純なアプローチを提供し、メディアリフレッシュ中に失われた化学物質をそれに応じて29に置き換えることを保証する。将来の応用には、特定の臓器系が異種生物物質の生物蓄積によって誘発される副作用を改善または克服する能力を評価するために、長期培養期間にわたる反復暴露体制に対するモデルの適合性を評価することが含まれる。
結論として、この3Dインビトロ肝モデルは、現実的な暴露シナリオの範囲を評価するために利用される能力を有し、それによってENMと化学的ハザード評価の両方をより良くサポートするための将来のインビトロアプローチをルーチンで容易にアクセス可能な方法で提供する。

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61141/advanced-3d-liver-models-for-vitro-genotoxicity-testing-following">Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure</a>. The Representative Results section&#160;was updated.
Figure 6 in the Representative Results section was updated from:
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61141/61141fig6.jpg" />
to:
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61141/61141fig6v5.jpg" />
The fourth paragraph in the Representative Results section was updated from:
With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain &#62;70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the &#8805;14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be &#8805;0.06 mg/mL whilst urea production should be &#8805;0.4 ng/&#181;L before conducting an in vitro toxicological assessment with this model.
to:
With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain &#62;70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the &#8805;14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be &#8805;50.0&#160;ng/&#956;L whilst urea production should be &#8805;0.25&#160;ng/&#181;L before conducting an in vitro toxicological assessment with this model.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61141/advanced-3d-liver-models-for-vitro-genotoxicity-testing-following">Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure</a>. The Representative Results section&#160;was updated.

Erratum: Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

人間ベースのアプリケーション(食品、化粧品、衣料品、スポーツ用品、電子機器、輸送、医薬品など)を通じて、多様なエンジニアリングナノ材料(ENM)の急速な開発と実装により、人間が定期的にENMにさらされることは避けられません。これにより、これらの材料が多数の用途で有利であると考える新しいサイズ固有のフィジオ化学的特性が、人間の健康と環境に付随する悪影響を引き起こす可能性があるという懸念が高まっている。現在、多くの国際的な活動は、積極的にこれらのENMへのより生理学的に関連する暴露を反映し、急性、長期、および繰り返し低用量暴露シナリオにわたってこれらの材料の潜在的な毒性を評価するために実施されています。
肝毒性学は、ENM曝露を考慮する際に重要であり、肝臓が曝露後のENM蓄積の主要部位であることを広く知られている1,2。また、肝臓は全身循環に入る物質の代謝と解毒のための第一次臓器系である。2D肝細胞モデルは、複雑な多細胞相互作用の複雑さを正確に模倣しない、または生体内で観察される代謝活性を適切に表さないという受け入れられた理解に基づいて、生体内代替技術のための堅牢で生理学的に関連する3D肝モデルの開発に大きく焦点を当てて4,5が確立されている。高度な3D培養技術を利用することで、インビトロ肝モデルの寿命が向上し、長期にわたる反復暴露体制の調査が可能になります。さらに、この高度な培養フォーマットは、胆汁カナリキュリ、活性トランスポータープロセスおよび改良されたCYP450薬物代謝能力などの強化された生理学的、organotypicの特徴の形成を促進し、モデル6の予測性を向上させる。現在の3Dインビトロ肝モデルは、単一培養物(肝細胞のみ)または共培養(非パレンキサイト細胞を有する肝細胞)からなるいくつかの形式で存在し、 超低接着板のマイクロ組織またはスフェロイド、吊り下げドロップスフェロイド、マトリックスおよび/または足場および微小流体細胞培養プラットフォームに埋め込まれた細胞に及ぶ、それらのすべては、肝毒性評価6、7のための効果的な高度なインビトロモデルとみなされる。しかし、これらのモデルシステムの大半は、高いメンテナンス、特殊な機器を必要とし、高価です。さらに、これらのモデルは、固定DNA損傷を定量化する方法を用いた遺伝毒性試験などのハザードエンドポイントの評価における使用を妨げる静的(すなわち、非分割細胞モデル)であることが多い。遺伝毒性は、規制毒性学の中核的前提条件であり、任意の毒性物質のリスク評価の重要な構成要素である8.外因性物質への暴露後に生じる可能性のあるDNA損傷のすべての形態を定量化するために適用できる単一のアッセイはありません。しかし、インビトロ遺伝子毒性試験用電池のコア成分は、微小核アッセイであり、これは総染色体損傷9を測定する信頼性の高い多面的な技術である。これは、OECD試験ガイドライン487によって記述された金標準技術であり、インビトロDNA損傷および遺伝毒性を評価するための、および規制ハザード評価10、11のための試験電池要件の一部である。
ヒト肝細胞癌細胞株HepG2は、細胞が容易に入手可能であるように初期ハザード評価スクリーニングに広く使用され、比較的安価にソース化し、培養が容易で、高スループットスクリーニング12,13に適している。3D球形構造に培養した場合、それらは肝臓微小環境を良好に再現し、微小核アッセイ3を支持するのに十分な増殖能力を有する肝モデルを提供することが示されている。HepG2スフェロイドモデルのさらなる開発は、長期にわたる反復暴露体制(≤14日間)における遺伝毒性ハザード評価をサポートするために、モデルの長寿および肝臓様機能性を向上させるために確立された。したがって、動物実験を置き換え、低減、改良する3Rの原理に沿って、本プロトコルは、複数の毒物学的エンドポイント(例えば、肝臓機能、(炎症マーカー、細胞毒性および遺伝毒性)に続く、急性および反復的な化学的およびENM暴露を確実に評価することができる高度な3Dインビトロ肝モデルを提供するために確立された。
ここでは、急性または長期にわたるENM曝露後の遺伝毒性ハザード評価のためのインビトロモデルシステムに基づく生理的に関連する3D肝細胞細胞株を確立する方法を提示する。プロトコルは6つの重要な段階に分けることができます:凍結保存HepG2細胞を培養する。ヘプG2スフェロイド製剤;ヘプG2スフェロイド転写は、吊り下げからアガロース懸濁液へ。ヘプG2スフェロイド収穫;マイクロ核アッセイとスコアリング;データ分析を行います。

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		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Aflotoxin B1</td>
			<td style="width:168px;">Sigma Aldrich, UK</td>
			<td style="width:344px;">A6636-5MG</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Agarose</td>
			<td>Sigma Aldrich, UK</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Autoclave Tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BCG Albumin Assay</td>
			<td>Sigma Aldrich, UK</td>
			<td>MAK124</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine Serum Albumin Powder</td>
			<td>Sigma Aldrich, UK</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell Freezing Aid</td>
			<td>Thermo Fisher Scientific, UK</td>
			<td>5100-0001 - Mr Frosty</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cytochalasin B</td>
			<td>Merck, UK</td>
			<td>250233</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cytology Metal Clips</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Cytospin 4 Centrifuge</td>
			<td style="width:168px;">ThermoFisher Scientific, UK</td>
			<td style="width:344px;">CM00730202</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">DMEM with 4.5g/L D-Glucose, L-Glutamine</td>
			<td>GIBCO, Paisley, UK</td>
			<td>41965-039</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">DMEM, phenol-red free with 4.5g/L D-Glucose, L-Glutamine with Hepes</td>
			<td>GIBCO, Paisley, UK</td>
			<td>21063-029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPX Mounting Medium</td>
			<td>FisherScientific, UK</td>
			<td>D/5330/05</td>
			<td></td>
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			<td height="40" style="height:40px;">Ethanol</td>
			<td>FisherScientific, UK</td>
			<td>10048291</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS</td>
			<td>GIBCO, Paisley, UK</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Filter Cards for Shandon Cytospin</td>
			<td style="width:168px;">FisherScientific, UK</td>
			<td style="width:344px;">15995742</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Frosted Glass Slides</td>
			<td style="width:168px;">ThermoFisher Scientific, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Giemsa&#39;s Stain Improved R66 Solution, Gurr</td>
			<td>VWR Chemicals, UK</td>
			<td>MFCD00081642</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Glass Coverslips (24 x 60)</td>
			<td style="width:168px;">Deckglaser, VWR</td>
			<td style="width:344px;">ECN631-1575</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Haemocytometer and Coverslip</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Immersion Oil for Microscope</td>
			<td>Zeiss, UK</td>
			<td>518F, ISO8034</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Laminar Class II Tissue Culture Hood</td>
			<td>Scanlaf Mars</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Light Microscope</td>
			<td style="width:168px;">Zeiss, UK</td>
			<td style="width:344px;">Axiovert 40C</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Methanol</td>
			<td>FisherScientific, UK</td>
			<td>10284580</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microwave</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Non-Filtered, Sterile 200&#181;l and 1000&#181;l Pipette tips</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">NuncMicroWell 96-Well Microplates</td>
			<td style="width:168px;">ThermoFisher Scientific, Denmark</td>
			<td style="width:344px;">167008</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">P1000 and P200 micropipettes</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">P300 and P50 multi-channel pipettes</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS pH 7.4 1X, MgCl2 and CaCl2 Free</td>
			<td>GIBCO, Paisley, UK</td>
			<td>14190-094</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Pen/Strep</td>
			<td>GIBCO, Paisley, UK</td>
			<td>15140-122, Penicillin/Strepmyocin 100X or 10,000U/ml</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphatase Buffer Tablets</td>
			<td>GIBCO, Paisley, UK</td>
			<td>10582-013</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pipette Boy</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Simport Scientific CytoSep Funnels for Shandon Cytospin 4 Centrifuges</td>
			<td style="width:168px;">FisherScientific, UK</td>
			<td style="width:344px;">11690581</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sonifier SFX 550 240V CE 1/2&#34; - Probe</td>
			<td>Branson, USA</td>
			<td>101-063-971R</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">T-25 and T-75 Tissue Culture Flask</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;">T-25 (690175) and T-75 (660175)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan Blue Solution</td>
			<td>Sigma Aldrich, UK</td>
			<td>T8154-100mL</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Urea Assay Kit</td>
			<td>Sigma Aldrich, UK</td>
			<td>MAK006</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Virkon Disinfectant</td>
			<td>DuPont, UK</td>
			<td>Rely+On Virkon</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water Bath (37&#730;C)</td>
			<td>Grant JBNova 18</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">Weighing Balance</td>
			<td style="width:168px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylene</td>
			<td>FisherScientific, UK</td>
			<td>10588070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.05% Trypsin-EDTA</td>
			<td>GIBCO, Paisley, UK</td>
			<td>5300-054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.2mL and 1.0mL Eppendorf Tubes</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.45&#181;m Filter Unit</td>
			<td style="width:168px;">Millex HA, MF-Millipore, UK</td>
			<td style="width:344px;">SLHA033SS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1.0mL Syringe</td>
			<td style="width:168px;">BD Plastipak, FisherScientific, UK</td>
			<td style="width:344px;">300185</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">20mL LS Scintillation Glass Vials, 22-400 Foil Lined PP Caps</td>
			<td style="width:168px;">DWK Life Sciences GmbH, Germany</td>
			<td style="width:344px;">WHEA986581</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">37&#730;C and 5% CO2 ISO Class 5 Hepa Filter Incubator</td>
			<td>NUAIRE DHD Autoflow</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:409px;">3mL Pasteur Pipette</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">50mL Conical Falcon Tubes</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">50mL or 100mL Glass Bottles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">50mL Skirted Falcon Tubes</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5mL, 10mL and 25mL Pipettes</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">9.4cm Square, Petri Dish</td>
			<td style="width:168px;">Greiner-Bio-One, UK</td>
			<td style="width:344px;">688161</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

advanced 3d liver models for in vitro genotoxicity testing following long-term nanomaterial exposure

多様なエンジニアリングナノ材料(ENM)の急速な開発と実装により、ENMへの暴露は避けられず、堅牢で予測的なインビトロ試験システムの開発が不可欠です。肝臓は代謝性恒常性および解毒に重要な役割を果たすだけでなく、ENM蓄積後の暴露の主要な部位であるため、肝臓毒性学はENM曝露を考慮する際に重要である。このことと、2D肝細胞モデルが生体内で観察される複雑な多細胞相互作用および代謝活性の複雑さを正確に模倣していないという理解に基づいて、生体外でのENMハザード評価目的に合わせた生理学的に関連する3D肝モデルの開発に大きな焦点が当てられています。動物実験を置き換え、低減、改良する3Rの原理に沿って、3D HepG2細胞株ベースの肝臓モデルが開発され、拡張および繰り返しENM暴露体制(≤14日)の両方をサポートできるユーザーフレンドリーで費用対効果の高いシステムです。これらのスフェロイドモデル(直径500μm≥)は、増殖能力(すなわち、細胞モデルの分割)を保持し、体外での遺伝毒性を効果的に評価するために「ゴールドスタンダード」マイクロ核アッセイと結合することができます。毒性学的エンドポイントの範囲(例えば、肝機能、(プロ)炎症反応、細胞毒性および遺伝毒性)の両方にわたっていくつかのEnMを使用して、急性(24時間)および長期(120時間)曝露療法を報告する能力が特徴付けられている。この3D in vitro肝モデルは、より現実的なENM暴露を評価するために利用される能力を有し、それによってENMハザード評価をより適切にサポートするための将来のin vitroアプローチを提供し、ルーチン的かつ容易にアクセス可能な方法でENMハザード評価を提供する。

この細胞系ベースの3D肝スフェロイドモデルの長期培養および遺伝毒性ハザード評価の適合性を、ベースライン特性評価を行い、培養中の14日間のモデルの生存率および肝臓様機能性および微小核アッセイに対するその適用性を決定することによって評価した。
3D HepG2肝臓回転楕円体モデルのベースライン特性 インビトロ毒物学的評価の前に、アガロース転移または化学/ENM処理を行う前に、3D HepG2スフェロイドが正しく形成されていることを確認することが重要です。吊り下げ液法を用いて製造されたHepG2スフェロイドは、通常、図5 A-5Cに示すように、平均直径495.52 μmのHを有するコンパクトな球形の球状スフェロイドを形成するのに2〜3日の播種後(4000細胞/スフェロイド)を要する。正しく形成され、体外毒性評価に使用できるHepG2スフェロイドは、滑らかな表面と視覚的な投影のないコンパクトな球形の構造を持っている必要があります。図 5に、品質の良さと品質の悪さ (図 5G-I)スフェロイドの例を示します。後者は破棄する必要があります。通常、プレートごとに形成されたスフェロイドの90〜95%が正しく形成され、さらなる実験のために実行可能である。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61141/61141fig5.jpg"> 図5:吊り下げ液法を介して形成されたHepG2スフェロイドの自然形態を示す光顕微鏡画像。(A-C)ショー2日目と(D-I)4日目HepG2肝臓スフェロイドポストシード。(D-F) は良質 HepG2 スフェロイドの例であり、 (G-I) は形成不良のスフェロイドを示しています。すべての画像は、顕微鏡を使用してX20目的で撮影した。スケールバーは20 μmを表<a href="https://www.jove.com/files/ftp_upload/61141/61141fig5large.jpg" target="_blank">します</a>。
HepG2スフェロイド生存率をさらに確認するために、基本的な着色ブロモクレゾールグリーンアルブミン(BCG)アッセイまたはウレアアッセイを行い、肝臓様の機能性を評価することができる。肝臓様の機能性は、14日間の培養期間にわたってトリパンブルー排除アッセイを使用して生存率に沿って評価され、肝臓スフェロイドモデルの寿命を決定し、長期的または繰り返しENM/化学的ハザードアセスメントをサポートできるかどうかを確立した(図6)。アルブミン濃度は培養期間にわたって一貫した状態を保った。尿素生産は、7日目までに高原に到達する前に、培養で1週間にわたってスフェロイド当たりの尿素の濃度の増加を表示します。3D HepG2スフェロイドで産生されるアルブミンおよび尿素のレベルは、2D形式で培養された同じ細胞株で観察されたものよりも実質的に高い点に注意することが重要である。実際、HepG2細胞、ピークアルブミンおよび尿素レベルの2D培養は、それぞれ0.001mg/mLおよび0.010 ng/μLであった。さらに、ほぼ同一のHepG2スフェロイド系を用いてShahらが発表した以前の研究では、2D培養HepG2細胞5と比較した場合の3D HepG2インビトロモデル系における代謝活性(CYP1A1およびCYP1A2)の顕著な改善を強調した。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61141/61141fig6v5.jpg"> 図6: HepG2肝スフェロイドの14日間ベースライン特性データ吊り下げからの移し換えの後、(A)は14日間にわたってHepG2スフェロイドモデルの生存率を強調し、(B)および(C)は肝臓のようなアルブミンおよび尿素の機能をそれぞれ強調する。SEM±提示された平均データ、n = 4。 <a href="https://www.jove.com/files/ftp_upload/61141/61141fig6v5large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
壊死性コアの必然的な発達に伴い、3D肝スフェロイド培養の既知の限界であり、このHepG2ベースのモデルの生存率は、微小核アッセイ5を支持するために必要な増殖能力を維持しながら、長期(5〜10日)の暴露レジームを維持できることを実証するために確立されなければならなかった。実際、この3D肝スフェロイドモデルは、培養で10日間にわたって70%の生存率を保持することが示されています。このことから、≥14日間の培養期間に観察された持続的な肝臓様の機能と組み合わせて、この3D肝スフェロイドモデルは、10日間の長さ(すなわち、スフェロイドの生存率が70%を下回る前)の長期にわたる反復ENM曝露レジームをサポートすることができる。参考までに、4000細胞/スフェロイドに播種されたHepG2スフェロイドのアルブミンレベルは≥20.0 ng/μL で、尿素生産は≥0.25 ng/μLであるべきである。
工学ナノ材料の遺伝毒性評価 遺伝毒性評価のために、微小核アッセイを用いて、急性(24時間)および長期(120時間)の両方のENM曝露に続く小核の存在を決定した。アフラトキシンB1は、既知の肝発がん性物質16、17であり、小核アッセイに推奨される陽性対照である。最適化実験では、0.1 μMのアルファトキシンB1が3D HepG2肝スフェロイドにおいて有意な陽性(≥2.0倍の増加)の遺伝毒性応答を誘導し、このモデルで行われるすべての微小核アッセイに使用することが示されています。HepG2スフェロイドモデルを用いたマイクロ核アッセイ結果の有効性を保証するために、この3Dインビトロモデルで使用されるHepG2細胞のバックグラウンドマイクロ核周波数は0.6%~1.2%の範囲内にある必要があります。その結果、アルファトキシンB1は、陰性対照で見られるものよりも少なくとも2倍高い遺伝子毒性応答を誘導すべきである。したがって、0.1 μMのアルファトキシン B1 は、1.5% ~ 3.0% の間のマイクロ核周波数を誘導する必要があります。これらの制御パラメータを用いて、ENMに関連する遺伝子毒性インビトロを、確実に評価することができる。OECD試験ガイドライン487に基づいて、ENMまたは化学薬品を試験する際に、選択された濃度が5%以上±5%以上の細胞傷害性を誘発してはならないことに注意することが重要である(負の対照に関連してCPBIまたはRVCC値の低下によって示される)11。図7は、アフラトキシンB1および2つのENM(二酸化チタン(TiO2)およびスライバー(Ag))がHepG2スフェロイドにおける急性および長期曝露の両方に続いて評価された場合に生成されたデータを示し、その後の遺伝子毒性電位をミクロ核アッセイを用いて分析した。評価された両方のENMは、急性(24時間)暴露および長期(120時間)曝露体制に対する非細胞毒性、低用量5.00μg/mLで試験された。TiO2およびAg Enmsの両方にわたって遺伝毒性の同様の傾向が観察され、それによって24時間暴露後に生じた遺伝毒性応答の上昇は、長期5日間の暴露後に明らかではなかった。これは、両方の時点でアフラトキシンB1陽性対照によって誘導された持続的な遺伝毒性にもかかわらずであった。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61141/61141fig7.jpg"> 図7: HepG2肝スフェロイドに対するTiO2およびAg ENM曝露に続く遺伝毒性評価微小核アッセイポスト(A)急性(24時間)および(B)を用いた遺伝毒性(微小核頻度)評価(A)および(B)TiO2およびAg ENMの5.00 μg/mLへの長期(120時間)曝露。ネガティブコントロールはメディアのみであり、正のコントロールはAflatoxin B1の0.1 μMです。平均データ(n=2)は、SD±提示され、負のコントロールに関連して示された有意性:* = p≤0.05。<a href="https://www.jove.com/files/ftp_upload/61141/61141fig7large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>

Watch this Scientific Journal Video about Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure at JoVE.com

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

この手順は、急性または長期の反復用量レジーム上のナノ物質暴露に関連する遺伝子毒性有害性のより生理学的に関連する評価を提供することができるインビトロで高度な3D肝培養物を開発するために使用されるように確立された。

長期ナノ材料暴露後のインビトロ遺伝子毒性試験のための高度な3D肝モデル

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

advanced-3d-liver-models-for-vitro-genotoxicity-testing-following

Research

JoVE Journal

Bioengineering

6.6K ビュー.  Swansea University Medical School. この手順は、急性または長期の反復用量レジーム上のナノ物質暴露に関連する遺伝子毒性有害性のより生理学的に関連する評価を提供することができるインビトロで高度な3D肝培養物を開発するために使用されるように確立された。

ビデオ: Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

有毒吸入化学試験のためのin vitroでの細胞培養モデルにおける

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

生物工学

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

全体タバコ煙への器官型3D気管支及び鼻腔組織培養モデルの反復暴露の影響評価

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多臓器チップ - 長期マルチ組織の共培養のためのマイクロ流体プラットフォーム

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

2Dと3Dの肌の改善<em&gt;インビトロ</em分子クラウディングの使用&gt;モデル

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

頭蓋骨の重要な骨欠損修復の多光子顕微鏡In Vivo遺伝子組み換え 3 D-PLGA/nHAp 足場による血管新生を可視化

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

ゼブラフィッシュ幼虫の改良実験操作のための長期のライブ イメージング デバイス

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

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 ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

体外養殖人間の腸内細菌を研究する技術を適用する高度な

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

流れの下で標的とする血管薬を研究するためのin Vitro 3D細胞培養動脈モデル

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Methods Article

長期ナノ材料暴露後のインビトロ遺伝子毒性試験のための高度な3D肝モデル