Name: eye irritation test (eit) for hazard identification of eye irritating chemicals using reconstructed human cornea-like epithelial (rhce) tissue model
Uploaded: 2015-08-23T14:00:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model at JoVE.com

著者は、EITプロジェクト専用の彼の科学的な支援と時間のためにドクター・ジョンHarbellに感謝したいと思います。著者らはまた、バイヤスドルフAG（ドイツ）、IIVS（米国）、メアリーケイ社（米国）、エイボンプロダクツ社（米国）、プロクター・アンド・ギャンブル/ Cosmital（スイス）、ラボラトリーピエール・ファーブル（フランス）に感謝したいと思いますハーランラボラトリーズ（イギリス）、および眼刺激性試験15の多施設国際事前検証と検証研究に参加するためのLVMH Parfume（フランス）。

<ol>
	<li>National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Request+for+Ocular+Irritancy+Test+Data+From+Human,+Rabbit,+and+In+Vitro+Studies+Using+Standardized+Testing+Methods.">Request for Ocular Irritancy Test Data From Human, Rabbit, and In Vitro Studies Using Standardized Testing Methods.</a> Federal Register. 72 (109), 31582-31583 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+(EC)+No+1907/2006+of+the+European+Parliament+and+of+the+Council+of+18+December+2006+concerning+the+Registration,+Evaluation,+Authorisation+and+Restriction+of+Chemicals+(REACH),+establishing+a+European+Chemicals+Agency,+amending+Directive+1999/45/EC+and+repealing+Council+Regulation+(EEC)+No+793/93+and+Commission+Regulation+(EC)+No+1488/94+as+well+as+Council+Directive+76/769/EEC+and+Commission+Directives+91/155/EEC,+93/67/EEC,+93/105/EC+and+2000/21/EC.">Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC.</a> OJ. 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A., Bruner, L. H., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EC/HO+international+validation+study+on+alternatives+to+the+Draize+eye+irritation+test.">The EC/HO international validation study on alternatives to the Draize eye irritation test.</a> Toxicol in Vitro. 9, 871-929 (1995).</li><li>Curren, R. D., Harbell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+safety:+a+silent+(in+vitro)+success+story.">Ocular safety: a silent (in vitro) success story.</a> Altern Lab Anim. 30, 69-74 (2002).</li><li>Wilhelmus, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Draize+Eye+Test.">The Draize Eye Test.</a> Survey of Ophthalmology. 45, 493-515 (2001).</li><li>Curren, R. D., Harbell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+alternatives+for+ocular+irritation.">In vitro alternatives for ocular irritation.</a> Environ health persp. 106, 485-492 (1998).</li><li>McCain, N. E., Binetti, R. R., Gettings, S. D., Jones, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+ocular+irritation+ranges+of+market-leading+cosmetic+and+personal-care+products+using+an+in+vitro+tissue+equivalent.">Assessment of ocular irritation ranges of market-leading cosmetic and personal-care products using an in vitro tissue equivalent.</a> Toxicologist. 66, 243 (2002).</li><li>Niranjan, P., Dang, A. H., January, B. G., Gomez, C., Harbell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+EpiOcular+assay+for+preclinical+qualification+of+formulas+for+human+clinical+studies.">Use of the EpiOcular assay for preclinical qualification of formulas for human clinical studies.</a> Toxicologist. 96, 249 (2007).</li><li>Yin, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+ocular+irritation+potential+of+surfactants-based+formulations+at+different+concentrations+using+the+EpiOcular+model.">Prediction of ocular irritation potential of surfactants-based formulations at different concentrations using the EpiOcular model.</a> Toxicologist. 108, 378 (2009).</li><li>Harbell, J., Curren, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+the+prediction+of+ocular+and+dermal+toxicity.">In vitro methods for the prediction of ocular and dermal toxicity.</a> Handbook of Toxicology. , (2001).</li><li>Kaluzhny, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+EpiOcular(TM)+eye+irritation+test+for+hazard+identification+and+labelling+of+eye+irritating+chemicals+in+response+to+the+requirements+of+the+EU+cosmetics+directive+and+REACH+legislation.">Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation.</a> Altern Lab Anim. 39, 339-364 (2011).</li><li>Pfannenbecker, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cosmetics+Europe+Multi-Laboratory+Pre-Validation+of+the+EpiOcular+Reconstituted+Human+Tissue+Test+Method+for+the+Prediction+of+Eye+Irritation.">Cosmetics Europe Multi-Laboratory Pre-Validation of the EpiOcular Reconstituted Human Tissue Test Method for the Prediction of Eye Irritation.</a> Toxicol in vitro. , (2013).</li><li>MatTek Corporation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals for use with Reconstructed Human EpiOcular Model (OCL-200-EIT). Protocol#MK-24-007-0055. , (2014).</li><li>Kaluzhny, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpiOcular+Eye+Irritation+Test+(EIT)+for+Hazard+Identification+and+Labeling+of+Eye+Irritating+Chemicals:+Protocol+Optimization+for+Solid+Materials+and+Extended+Shipment+Times.">EpiOcular Eye Irritation Test (EIT) for Hazard Identification and Labeling of Eye Irritating Chemicals: Protocol Optimization for Solid Materials and Extended Shipment Times.</a> Altern Lab Anim. 43 (2), 101-127 (2015).</li><li>Kolle, S. N., Kandarova, H., Wareing, B., van Ravenzwaay, B., Landsiedel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-house+validation+of+the+EpiOcular(TM)+eye+irritation+test+and+its+combination+with+the+bovine+corneal+opacity+and+permeability+test+for+the+assessment+of+ocular+irritation.">In-house validation of the EpiOcular(TM) eye irritation test and its combination with the bovine corneal opacity and permeability test for the assessment of ocular irritation.</a> Altern Lab Anim. 39, 365-387 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructed+Human+Cornea-like+Epithelium+(RhCE)+Test+Method+for+Identifying+Chemicals+Not+Requiring+Classification+and+Labelling+for+Eye+Irritation+or+Serious+Eye+Damage.+Draft+proposal+for+a+new+test+guideline..">Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage. Draft proposal for a new test guideline..</a> OECD Guideline for the Testing of Chemicals. , (2014).</li><li>Blazka, M. E., Harbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+ocular+irritation+potential+of+54+test+articles+using+the+EpiOcular+Human+tissue+Construct+Model.">Evaluating the ocular irritation potential of 54 test articles using the EpiOcular Human tissue Construct Model.</a> Toxicol. In Vitro. , (2003).</li><li>Kandarova, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EpiDerm+Test+Protocol+for+the+Upcoming+ECVAM+Validation+Study+on+In+Vitro+Skin+Irritation+Tests+&#8212;+An+Assessment+of+the+Performance+of+the+Optimised+Test.">The EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests &#8212; An Assessment of the Performance of the Optimised Test.</a> Altern Lab Anim. 33, 351-367 (2005).</li></ol>

この記事の発行手数料はマテック社が支払われました。

我々はEpiOcular組織モデルのために開発された眼刺激性試験（ 図1）を提示しています。 GHS区分とを区別しない、本明細書に提示されるようEITは、感度と特異17 EITを度の高いラベル（GHSなしカテゴリ）を必要としない材料から眼刺激物や腐食（GHSカテゴリー1と2を組み合わせ）を分離することができます1カテゴリからの2物質。 EITは、化粧品および医薬成分を含む化学物質の広い範囲の眼刺激性の分類と表示のために検証されました。他のin vitro試験に関連して、EITは、in vivoウサギ眼刺激性試験の代替として機能します。 EITは露光および露光後のインキュベーション期間の長さ（ 図1）に変化する液体および固体材料のための2つの類似するが異なるプロトコルを使用します。 EITで使用するエンドポイント以前に検証されたヒト上皮組織モデル20,21で使用されているMTTアッセイによって決定し、組織の生存率です。このアッセイを行うために、標準的な細胞培養装置の他に特別な装置が必要とされません。高い組織対組織の再現性のレベル、n ​​= 2の組織の代わりに通常推奨されるN = 3が使用されます。それはそれによって個々の組織14の異なる取り扱いをすると生じる可能性がアッセイのばらつきを最小限に抑え、同時に2つの組織を処理するために、経験豊富なオペレータを可能にするため、2組織を使用し、N =能力は、プロトコルの重要な側面です。また、陽性対照および陰性対照と一緒に、被験物質あたりn = 2の組織、同一の物理的状態（液体または固体）の10の試験物質の刺激を用いて、1キット（24組織）を用いて評価することができます。 材料の信頼性の分類を確保する他の重要なポイントは、陽性コントロールのsuの仕様ですbstance（組織生存率≤50.0％）、重複した組織（差&lt;20.0％）、およびネガティブコントロールOD読み取り値（&gt; 0.8 &lt;2.5）との間の再現性。 EIT試験を行う場合、インキュベーション期間中にプロトコルまたは変化の偏差が変化した結果をもたらすことができるので、それは、検証プロトコルと推奨投薬およびすすぎスケジュール（ 表3および4）に接着することが重要です。同様に、MTTインキュベーションのための3時間の時間からの偏差は異なるMTTの読みになり、アッセイの結果に影響を与える可能性があります。 時折、試験化学物質は、MTT組織生存率アッセイ又はMTTの原因の減少を妨害することができる光学的または他の特性を有していてもよいです。例えば、試験化学物質を直接青紫色の反応生成物にMTTを低減することができる、またはMTTホルマザン（〜570ナノメートル）と同じ範囲の光を吸収する着色された物質であってもよいです。しかし、これらの試験の化学物質はPRますMTTアッセイの際に、材料の十分な量は、依然として上に存在する（またはによって吸収）である場合にのみ、組織の問題をESENT。この干渉を回避するために、大規模な洗浄手順は、EITプロトコルに組み込まれています。すすぎはTAを削除しないとTAはMTT還元を妨害した場合、追加のコントロールはそれを検出し、修正するために使用されなければなりません。被験物質の直接MTT還元が疑われる場合に簡単に説明すると、当該化学物質の50μlの（又は固体50 mg）をSCC（NC、滅菌脱イオン水50μlで作業MTT溶液で3時間インキュベートする、であるべきです同時に実行）。 MTT溶液が青紫色になった場合、試験物品を、MTTが低下していると推定されます。この場合、凍結殺し組織コントロールを使用して機能的なチェックは、試験物質は、組織に結合し、偽のMTT低減信号に先行しているかどうかを評価するために行われるべきです。 TA-さらされ、殺された組織制御でかなりのMTT還元がある場合（未処理の生存組織内の量に対して）、試験品の平均組織の生存率は、殺された対照の平均生存率を差し引くことにより補正しなければなりません。 偽陰性分類14,15,18の発生率が低いことによって実証されるように、EITは、安全性の側面に誤差が生じるが。重要なのは、目には腐食性であり、GHS区分1の化学物質のいずれもが、最も深刻な眼の危険性を表していませんこのアッセイ14,15,18,19に非刺激性として分類されました。最後に、 インビトロ試験方法で RHCEの主要な利点の一つは、原液と（2次元、沈められた細胞培養物では不可能である）固体材料を試験する可能性があります。 EITは、国連GHS分類および表示システムによる材料の広い範囲の眼刺激性を決定する際に重要な一因になります。 Oを決定するために、動物の交換cular毒性は、長年にわたって毒物学研究の目標でした。 EIT試験方法は、2014年にEURL ECVAMでサポートされている正式なバリデーション試験を完了したとEpiOcular EITは、2015年にはOECD TG 492としてOECDテストガイドラインに実装されました。

このような化粧品、洗剤、家庭用クリーナーとして消費者製品は、彼らが目に連絡した場合、重大な損傷を誘発する可能性が種々の化学物質が含まれています。そのため、目への刺激のためのこれらの薬剤のテストは、消費者の安全を確保するための1、米国とEUの規制当局によって必要とされます。混合物および製剤の眼刺激性の評価はまた、化学物質の輸送のためのEUの化粧品指令の下で化粧品成分の標識のためにREACH（登録、評価、認可および化学物質の使用制限）法律を遵守する要件である、とのために殺虫剤や家庭用製品2のラベル。現在、規制当局は、化学品の分類およびラベリングに関する世界調和システム（GHS）3を用いて、眼のハザード評価が必要です。 GHSは、主にドレイズ眼刺激性試験に基づいており、最も広く使用されている眼刺激アッセイもので異物とマイルxturesは、ウサギの眼4の結膜嚢に直接導入されています。 GHS分類によると、GHS区分1（眼腐食）は、露光3,5以下の21日以内に眼の組織または完全に可逆的ではない、目と視力に重大な損傷に深刻な最初の損傷の原因となる化学物質をテストすることを指します。 GHS区分2は、露出の21日以内に完全に可逆的で目に有意な変化を生じる化学物質をテストすることを指します。腐食性または刺激性物質ではない試験化学物質はGHSはありませんカテゴリと呼ばれています。 40年以上にわたり、ドレイズウサギの目のテストでは、その再現性の欠如、人間の応答の過大評価、そして生きている動物5-8の使用のために批判されています。これらの懸念は、洗練、縮小、およびin vivo試験 9の交換のための多くの提案を奨励しています。検証非動物代替物の必要性がさらに強化されました（2009年）化粧品（2005年）化粧品の安全性評価における動物の使用を禁止指令、および成分に第七改正の採用により2。 1996年から、再構成された角膜のような組織モデルは、広く目、原料、界面活性剤ベースの製剤、および、または近傍で使用するために設計された配合混合物の刺激性を評価するために化粧品業界で使用されています10-13。 RHCEの組織モデルの使用は、その本来の、希釈しない形で、組織表面への試験物質の直接局所適用することができます。この方法では、非水溶性製剤は溶剤でそれらを希釈することなく試験することができます。 （代替法の検証のための欧州連合（EU）のリファレンスラボラトリーヨーロッパセンター）EURL ECVAMのに対応して広く、該当簡単で、経済法の要求SINGLを利用眼刺激性試験（EIT）電子露光時間とラベリングを必要としない材料から眼刺激物や腐食を分離することができる（図1）14を開発しました。 83化学物質（44液体および39固体）の結果をもとに、EITは95.5 / 68.2 /と81.8パーセントの感度/特異度と精度（SS＆A）液体のため、固体のための100.0 / 68.4 /と84.6パーセントSS＆A、および97.6を/達成しました68.3 /と全体のSS＆Aの83.1パーセント。 2007年には、EURL ECVAMの後援化粧品ヨーロッパ（旧COLIPA）が主催する多実験室前の検証研究では、正式な検証15にそれをもたらすことを目的としたEITの妥当性と信頼性を評価しました。本研究では、298の独立した試験では、7つの独立の研究室で行いました。研究結果は、すべての参加研究室15全体の変動の低い係数の予測で99.7％の契約を実証しました。その結果、2010年にはEITプロトコルは正式なEURL ECVAMに入りました検証プログラム。バリデーション試験は、in vivoでの参照データ（ドレイズ眼刺激性データ）が利用可能であった対象の個々の物質と化学の混合物、を含む104コード化されたテスト化学物質を利用しました。この作品の成功に基づき、OECDドラフトテストガイドラインには、それは、EITは、国連GHS分類および表示システムによる材料の広い範囲の眼刺激性の分類に大きく貢献することが期待される2014年に提出されました。

eye irritation test (eit) for hazard identification of eye irritating chemicals using reconstructed human cornea-like epithelial (rhce) tissue model

EU化粧品指令、欧州REACH法規に第七改正に準拠するために、ヒトの眼毒性の信頼性と正確な評価のための検証非動物代替の方法が必要とされています。このニーズに応えるために、我々は正常なヒト細胞に基づいて3次元再構成人間の角膜のような上皮（RHCE）組織モデルを利用した眼刺激性試験（EIT）を開発しました。 EITは、眼刺激物や腐食（GHSカテゴリー1と2を組み合わせ）とラベリング（GHSなしカテゴリ）を必要としないものを分離することができます。テストは、2つの別々のプロトコル、液体化学物質と第二のために設計された1つ、固体試験物品のための同様のプロトコルを利用しています。 EIT予測モデルは、単一の露光期間（液体で30分間、固形分6時間）、および単一の組織の生存率のカットオフ（MTTアッセイによって決定される60.0％）を使用します。 EITは95.5 / 68.2 / 81.8パーセントとのSENSを達成した（44液体および固体39）83化学物質の結果に基づいて、液体用itivity /特異性と精度（SS＆A）、100.0 / 68.4 / 84.6パーセントとSS＆A固体用、および97.6 / 68.3 /と全体のSS＆Aの83.1パーセント。 EITは、規制テスト要件を満たすために、動物を使用することなく、液体及び固体の化学物質の広範囲の眼刺激性を分類に大きく貢献します。 EpiOcular EIT法は、TG 492としてOECDテストガイドラインに2015年に実施されました。

Watch this Scientific Journal Video about Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model at JoVE.com

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

我々は、3次元再構成人間の角膜のような上皮（RHCE）組織モデルを利用した眼刺激性試験を開発しました。テストは、眼刺激性及び腐食性物質（GHSカテゴリー1と組み合わせた2）とラベル（GHSなしカテゴリ）を必要としないものとを区別することができます。

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 ...

eye-irritation-test-eit-for-hazard-identification-eye-irritating

Research

JoVE Journal

Bioengineering

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

37.5K ビュー.  MatTek Corporation. 我々は、3次元再構成人間の角膜のような上皮（RHCE）組織モデルを利用した眼刺激性試験を開発しました。テストは、眼刺激性及び腐食性物質（GHSカテゴリー1と組み合わせた2）とラベル（GHSなしカテゴリ）を必要としないものとを区別することができます。 

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

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

アンインビトロ</em表皮を使用して>皮膚刺激性試験（SIT）は、ヒト上皮（RHE）モデルを再構築

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

生物学

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

開発<em&gt;インビトロ</em&gt;眼プラットフォームはコンタクトレンズをテストします

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

生物工学

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

3次元の再構築されたヒト表皮を大規模に育成

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

医学

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

乾燥した後のヒト角膜上皮細胞の代謝活性に及ぼす人工涙液製剤の効果

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮の機械的および化学的損傷のためのEx Vivoおよびin vivo動物モデル

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

クロルピクリンとフッ化水素誘起角膜上皮細胞傷害のための高スループット SiRNA スクリーニング

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

マウスの精密アルカリ熱傷のための修正生検パンチ法

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

著者スポットライト:角膜混濁の治療と再生の研究の進歩 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

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）

要約

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アンインビトロ皮膚刺激性試験（SIT）は、ヒト上皮（RHE）モデルを再構築

開発

3次元の再構築されたヒト表皮を大規模に育成

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

乾燥した後のヒト角膜上皮細胞の代謝活性に及ぼす人工涙液製剤の効果

角膜上皮の機械的および化学的損傷のためのEx Vivoおよびin vivo動物モデル

クロルピクリンとフッ化水素誘起角膜上皮細胞傷害のための高スループット SiRNA スクリーニング

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導