JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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의학

생체 재료에 대한 세포 독성 및 세포 반응에 접근

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

이 방법론은 유세포 분석, RT-PCR, 면역세포화학, 기타 세포 및 분자 생물학 기술을 포함한 생존력 분석 및 표현형 분석을 사용하여 가용성 추출물의 제조를 통해 생체 재료 세포 독성을 평가하는 것을 목표로 합니다.

Abstract

생체 재료는 인체 조직과 직간접적으로 접촉하므로 세포 독성을 평가하는 것이 중요합니다. 이 평가는 여러 가지 방법으로 수행 할 수 있지만 사용 된 접근법간에 높은 불일치가 존재하여 재현성과 얻은 결과 간의 비교가 손상됩니다. 본 논문에서는 치과용 생체재료에 사용하는 수용성 추출물을 이용하여 생체재료 세포독성을 평가하는 프로토콜을 제안한다. 추출물 준비는 펠렛 생산에서 배양 배지에서의 추출에 이르기까지 자세히 설명되어 있습니다. 생체 재료 세포 독성 평가는 MTT 분석을 사용한 대사 활성, Sulphorhodamine B(SBR) 분석을 사용한 세포 생존율, 유세포 분석에 의한 세포 사멸 프로파일, May-Grünwald Giemsa를 사용한 세포 형태를 기반으로 합니다. 세포독성 평가에 추가하여, 세포 기능을 평가하기 위한 프로토콜은 면역세포화학 및 PCR에 의해 평가된 특정 마커의 발현을 기반으로 설명된다. 이 프로토콜은 추출물 방법론을 사용하여 재현 가능하고 강력한 방식으로 생체 재료 세포 독성 및 세포 효과 평가를 위한 포괄적인 가이드를 제공합니다.

Introduction

생체적합성은 조직을 통합하고 국소 및 전신 손상이 없는 유리한 치료 반응을 유도하는 물질의 능력으로 정의할 수 있다 1,2,3. 생체 적합성 평가는 의료용 재료의 개발에 매우 중요합니다. 따라서 이 프로토콜은 새로운 생체 재료 개발을 목표로 하거나 기존 생체 재료에 대한 새로운 응용 프로그램을 연구하는 모든 연구자에게 체계적이고 포괄적인 접근 방식을 제공합니다.

시험관 내 세포 독성 시험은 일차 세포 배양 또는 세포주를 사용하여 생체 적합성 평가의 첫 번째 단계로 널리 사용됩니다. 결과는 잠재적인 임상 적용의 첫 번째 지표를 구성합니다. 이 테스트는 생체 재료 개발에 필수적일 뿐만 아니라 EUA 및 EU 규제 기관(FDA 및 CE 인증)4,5,6,7,8의 시장 도입에 대한 현재 규정을 준수해야 합니다. 또한, 생물의학 연구의 표준화된 시험은 유사한 생체 재료 또는 장치에 대한 다양한 연구 결과의 재현성 및 비교 측면에서 상당한 이점을 제공한다9.

ISO(International Organization for Standardization) 지침은 정확하고 재현 가능한 방식으로 재료를 테스트하기 위해 여러 독립적인 상업, 규제 및 학술 실험실에서 널리 사용됩니다. ISO 10993-5는 시험관 내 세포 독성 평가를 의미하며 ISO 10993-12는 샘플링 제제10,11에 보고합니다. 생체 재료 테스트를 위해 재료 유형, 접촉 조직 및 치료 목표에 따라 선택되는 세 가지 범주가 제공됩니다 : 추출물, 직접 접촉 및 간접 접촉 8,11,12,13. 추출물은 세포 배양 배지를 생체 재료로 풍부하게 하여 얻는다. 직접 접촉 시험을 위해, 생체 재료를 세포 배양물 상에 직접 위치시키고, 간접 접촉에서, 세포와의 인큐베이션은 아가로스 겔(11)과 같은 장벽에 의해 분리되어 수행된다. 적절한 통제가 필수적이며 최소 3 개의 독립적 인 실험이 수행되어야합니다 5,8,10,11,14.

세포 독성 가능성을 결정하기 위해 임상 조건을 시뮬레이션하거나 과장하는 것이 중요합니다. 추출물 테스트의 경우 재료의 표면적;  중간 볼륨; 배지 및 물질 pH; 물질 용해도, 삼투압 및 확산 비율; 교반, 온도 및 시간과 같은 추출 조건은 배지 농축제에 영향을 미칩니다5.

이 방법론은 고체 및 액체의 여러 제약 제형의 세포 독성에 대한 정량적 및 정성적 평가를 허용합니다. 중성 적색 흡수 시험, 집락 형성 시험, MTT 검정 및 XTT 검정 5,10,14와 같은 여러 검정을 수행할 수 있습니다.

발표된 대부분의 세포독성 평가 연구는 제한된 정보를 제공하는 MTT 및 XTT와 같은 더 간단한 분석을 사용합니다. 생체적합성 평가는 세포독성의 평가뿐만 아니라 주어진 시험 물질2의 생체활성의 평가를 포함해야 한다. 정당화되고 문서화된 경우 추가 평가 기준을 사용해야 합니다. 따라서 이 프로토콜은 생체 재료 세포 독성 평가를 위한 일련의 방법을 자세히 설명하는 포괄적인 가이드를 제공하는 것을 목표로 합니다. 게다가, 다양한 세포 과정, 즉 세포 사멸의 유형, 세포 형태, 특정 단백질 합성에서의 세포 기능 및 특정 조직 생산에 대한 평가가 설명됩니다.

Protocol

1. 펠렛 준비 PVC 플레이트에 알려진 치수의 원형 구멍을 수행하여 폴리염화비닐(PVC) 몰드를 준비합니다.알림: PVC 몰딩은 다양한 크기로 만들 수 있습니다. 공식 A= h(2πr)+2πr2 (r: 원통의 반지름, h: 원통의 높이)를 사용하여 PVC 몰드의 접촉면을 계산합니다. 제조업체의 지침에 따라 테스트 할 생체 재료를 준비하십시오.참고: 페이스트/페이스트 제형 바이오 재료의 준비를…

Representative Results

여기서 대표적인 결과는 치과 용 생체 재료 연구를 나타냅니다. 추출물 방법론을 통해 대사 활성(그림 2), 세포 생존율, 세포 사멸 프로필 및 세포 형태(그림 3), 특정 단백질 발현(그림 4)에 대한 영향과 관련하여 치과 재료에 노출한 후 세포 독성 프로필 및 세포 기능을 얻을 수 있습니다. MTT 분석은 간단한 방?…

Discussion

이 프로토콜은 조직과 접촉하는 생체 재료의 시험관 내 세포 독성 평가를 지칭하는 ISO 10993-5를 고려하여 설계되었으며, 생체 적합성을 평가하고 연구 재현성에 기여합니다21. 이것은 과학에서 점점 더 많은 관심사이며, 많은 저자들은 이미 시험관 내 연구 15,22,23,24,25,26,27,28의 실험 설계에서 이러한 권장 사항을 따르고 있습니다.…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank the following for support: GAI 2013 (Faculdade de Medicina da Universidade de Coimbra); CIBB는 전략 프로젝트 UIDB/04539/2020 및 UIDP/04539/2020(CIBB)을 통해 FCT(Foundation for Science and Technology)를 통해 국가 기금으로 자금을 지원받습니다. MDPC-23 세포주를 제공한 University of Michigan Dental School의 Jacques Nör에게 감사드립니다.

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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