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Medicine

生体材料に対する細胞毒性と細胞応答へのアクセス

Published: July 08, 2021
doi:
10.3791/61512
, , , , , , , , ,
1Institute of Integrated Clinical Practice, Faculty of Medicine,University of Coimbra, Coimbra, Portugal, 2Coimbra Institute for Clinical and Biomedical Research (iCBR) area of Environment Genetics and Oncobiology (CIMAGO),University of Coimbra, Coimbra, Portugal, 3Center for Innovative Biomedicine and Biotechnology (CIBB),University of Coimbra, Coimbra, Portugal, 4Clinical Academic Center of Coimbra, CACC, Portugal, 5Institute of Biophysics, Faculty of Medicine,University of Coimbra, Coimbra, Portugal, 6Laboratory of Oncobiology and Hematology, Faculty of Medicine,University of Coimbra, Coimbra, Portugal, 7Institute of Endodontics, Faculty of Medicine,University of Coimbra, Coimbra, Portugal, 8Institute of Experimental Pathology, Faculty of Medicine,University of Coimbra, Coimbra, Portugal

Summary

この方法論は、フローサイトメトリー、RT-PCR、免疫細胞化学、およびその他の細胞および分子生物学的手法を含む生存率アッセイおよび表現型分析を使用して、可溶性抽出物の調製を通じて生体材料の細胞毒性を評価することを目的としています。

Abstract

生体材料はヒト組織と直接的または間接的に接触するため、その細胞毒性を評価することが重要です。この評価はいくつかの方法で実行できますが、使用するアプローチの間に大きな不一致が存在し、再現性と得られた結果の比較が損なわれます。本論文では、歯科用生体材料に使用する可溶性抽出物を用いて生体材料の細胞毒性を評価するためのプロトコルを提案する。抽出物の調製は、ペレットの製造から培養培地での抽出まで、詳細に説明されています。生体材料の細胞毒性評価は、MTTアッセイを使用した代謝活性、スルホルホダミンB(SBR)アッセイを使用した細胞生存率、フローサイトメトリーを使用した細胞死プロファイル、およびMay-Grünwald Giemsaを使用した細胞形態に基づいています。細胞毒性評価に加えて、免疫細胞化学およびPCRによって評価された特異的マーカーの発現に基づいて細胞機能を評価するためのプロトコルが記載されている。このプロトコルは、抽出物の方法論を使用して、再現性のある堅牢な方法で、生体材料の細胞毒性および細胞効果評価のための包括的なガイドを提供します。

Introduction

生体適合性は、局所的および全身的な損傷のない、組織を統合し、好ましい治療反応を誘発する材料の能力として定義することができます1,2,3。生体適合性評価は、医療用材料の開発に不可欠です。したがって、このプロトコルは、新しい生体材料の開発を目指す、または既存の生体材料の新しいアプリケーションの研究を目指すすべての研究者に体系的かつ包括的なアプローチを提供します。

in vitro細胞毒性試験は、初代細胞培養または細胞株を用いた生体適合性評価の第1段階として広く使用されています。結果は、潜在的な臨床応用の最初の指標を構成します。この試験は、生体材料の開発に不可欠であることに加えて、EUAおよびEU規制当局(FDAおよびCE認証)4,5,6,7,8からの市場導入に関する現在の規制に準拠するために必須です。さらに、生物医学研究における標準化された試験は、類似の生体材料またはデバイスに関する異なる研究からの結果の再現性および比較の点で大きな利点を提供する9。

国際標準化機構(ISO)のガイドラインは、正確で再現性のある方法で材料を試験するために、複数の独立した商業、規制、および学術研究所で広く使用されています。ISO 10993-5はin vitro細胞毒性評価を指し、ISO 10993-12はサンプリング調製10,11に報告しています。生体材料試験には、材料の種類、接触組織、および治療目標に応じて選択される3つのカテゴリが提供されます:抽出物、直接接触、および間接接触8,11,12,13。抽出物は、細胞培養培地を生体材料で濃縮することによって得られる。直接接触試験のためには、生体材料を細胞培養物上に直接配置し、そして、間接接触において、細胞とのインキュベーションをアガロースゲル11などの障壁によって分離して行う。適切なコントロールが必須であり、少なくとも3つの独立した実験を実行する必要があります5、8、10、11、14。

細胞毒性の可能性を判断するには、臨床状態をシミュレートまたは誇張することが重要です。抽出物試験の場合、材料の表面積。 中程度の量;培地および材料pH;材料の溶解度、浸透圧および拡散比。攪拌、温度、時間などの抽出条件は、培地濃縮に影響を与える5。

この方法論により、固体および液体の両方のいくつかの医薬製剤の細胞毒性の定量的および定性的評価が可能になります。中性赤色取り込み試験、コロニー形成試験、MTTアッセイ、およびXTTアッセイ5、10、14などのいくつかのアッセイを実施することができる。

発表されたほとんどの細胞毒性評価研究では、より単純なアッセイ、すなわちMTTおよびXTTが使用されており、限られた情報しか提供されない。生体適合性の評価には、細胞毒性の評価だけでなく、このプロトコルが承認しているように、特定の試験材料の生物活性も含まれる必要があります2。正当化され、文書化されている場合は、追加の評価基準を使用する必要があります。したがって、このプロトコルは、生体材料の細胞毒性評価のための一連の方法を詳述した包括的なガイドを提供することを目的としています。さらに、さまざまな細胞プロセス、すなわち細胞死の種類、細胞形態、特定のタンパク質の合成における細胞機能、および特定の組織産生の評価について説明します。

Protocol

1.ペレットの準備 PVCプレートに既知寸法の円形の穴を開けることにより、ポリ塩化ビニル(PVC)型を作製する。注意: PVC成形品はさまざまなサイズで作ることができます。式 A= h(2πr)+2πr2 (r: 円柱の半径、h: 円柱の高さ) を使用して、PVC 金型の接触面を計算します。 製造元の指示に従って、実験の開始にできるだけ近いところで試験する生体材料を準備します。注:ペ…

Representative Results

ここでの代表的な結果は、歯科用生体材料の研究に関するものです。抽出物の方法論により、代謝活性(図2)、細胞生存率、細胞死プロファイルと細胞形態(図3)、および特定のタンパク質発現(図4)への影響に関して、歯科材料に曝露した後の細胞毒性プロファイルと細胞機能を得ることができます。 MTTア?…

Discussion

このプロトコルは、組織と接触する生体材料のin vitro細胞毒性の評価を指すISO 10993-5を考慮して設計され、生体適合性を評価し、研究の再現性に貢献する21。これは科学における懸念の高まりであり、多くの著者は、in vitro研究の実験デザインですでにこれらの推奨事項に従っています15、22、23、24、25、<sup …

Disclosures

The authors have nothing to disclose.

Acknowledgements

GAI 2013 (コインブラ大学医学部);CIBBは、戦略プロジェクトUIDB/04539/2020およびUIDP/04539/2020(CIBB)を通じて、FCT(科学技術財団)を介して国家基金によって資金提供されています。細胞株MDPC-23を提供してくれたミシガン大学デンタルスクールのJacques Nörに感謝します。

Materials

Absolute ethanol Merck Millipore 100983
Accutase Gibco A1110501 StemPro Accutas Cell Dissociation Reagent
ALDH antibody Santa Cruz Biotechnology SC166362
Annexin V FITC BD Biosciences 556547
Antibiotic antimycotic solution Sigma A5955
BCA assay Thermo Scientific 23225 Pierce BCA Protein Assay Kit
Bovine serum albumin Sigma A9418
CaCl2 Sigma 10035-04-8
CD133 antibody Miteny Biotec 293C3-APC Allophycocyanin (APC)
CD24 antibody BD Biosciences 658331 Allophycocyanin-H7 (APC-H7)
CD44 antibody Biolegend 103020 Pacific Blue (PB)
Cell strainer BD Falcon 352340 40 µM
Collagenase, type IV Gibco 17104-019
cOmplete Mini Roche 118 361 700 0
DAB + Chromogen Dako K3468
Dithiothreitol Sigma 43815
DMEM-F12 Sigma D8900
DNAse I Roche 11284932001
DSP (M-20) Antibody, 1: 100 Santa Cruz Biotechnology LS-C20939
ECC-1 ATCC CRL-2923 Human endometrium adenocarcinoma cell line
Epidermal growth factor Sigma E9644
Hepes 0.01 M Sigma MFCD00006158
Fibroblast growth factor basic Sigma F0291
Giemsa Stain, modified GS-500 Sigma MFCD00081642
Glycerol Dako C0563
Haemocytometer VWR HERE1080339
HCC1806 ATCC CRL-2335 Human mammary squamous cell carcinoma cell line
Insulin, transferrin, selenium Solution Gibco 41400045
May-Grünwald Stain MG500 Sigma MFCD00131580
MCF7 ATCC HTB-22 Human mammary adenocarcinoma cell line
Methylcellulose AlfaAesar 45490
NaCl JMGS 37040005002212
Polyclonal Rabbit Anti-goat immunoglobulins / HRP, 1: 100 Dako G-21234
Poly(2-hydroxyethyl-methacrylate Sigma P3932
Putrescine Sigma P7505
RL95-2 ATCC CRL-1671 Human endometrium carcinoma cell line
Sodium deoxycholic acid JMS EINECS 206-132-7
Sodium dodecyl sulfate Sigma 436143
Substrate Buffer Dako 926605
Tris JMGS 20360000BP152112
Triton-X 100 Merck 108603
Trypan blue Sigma T8154
Trypsin-EDTA Sigma T4049
β-actin antibody Sigma A5316
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BiomaterialsCytotoxicityCell ResponseBioactivityBiocompatibilityIn Vitro TestingCell CultureCell ViabilityCell MorphologyMay-Grunwald StainingUV SterilizationMedical Applications

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Paula, A. B., Laranjo, M., Coelho, A. S., Abrantes, A. M., Gonçalves, A. C., Sarmento-Ribeiro, A. B., Ferreira, M. M., Botelho, M. F., Marto, C. M., Carrilho, E. Accessing the Cytotoxicity and Cell Response to Biomaterials. J. Vis. Exp. (173), e61512, doi:10.3791/61512 (2021).
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