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

Adgang til cytotoksicitet og cellerespons på biomaterialer

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

Denne metode sigter mod at evaluere biomateriale cytotoksicitet gennem fremstilling af opløselige ekstrakter ved anvendelse af levedygtighedsanalyser og fænotypisk analyse, herunder flowcytometri, RT-PCR, immunocytokemi og andre cellulære og molekylærbiologiske teknikker.

Abstract

Biomaterialer kommer i direkte eller indirekte kontakt med det humane væv, hvilket gør det vigtigt at evaluere dets cytotoksicitet. Denne evaluering kan udføres ved flere metoder, men der er en stor uoverensstemmelse mellem de anvendte tilgange, hvilket kompromitterer reproducerbarheden og sammenligningen mellem de opnåede resultater. I dette papir foreslår vi en protokol til evaluering af biomaterialer cytotoksicitet ved hjælp af opløselige ekstrakter, som vi bruger til dental biomaterialer. Ekstraktpræparatet er detaljeret, fra pelletproduktion til dets ekstraktion i et dyrkningsmedium. Biomaterialernes cytotoksicitetsevaluering er baseret på metabolisk aktivitet ved anvendelse af MTT-assayet, cellelevedygtighed ved anvendelse af Sulphorhodamine B (SBR) assay, celledødsprofil ved flowcytometri og cellemorfologi ved anvendelse af May-Grünwald Giemsa. Ud over cytotoksicitetsevaluering beskrives en protokol til evaluering af cellefunktion baseret på ekspression af specifikke markører vurderet ved immunocytokemi og PCR. Denne protokol giver en omfattende vejledning til evaluering af cytotoksicitet og cellulære virkninger af biomaterialer ved hjælp af ekstraktmetoden på en reproducerbar og robust måde.

Introduction

Biokompatibilitet kan defineres som et materiales evne til at integrere væv og fremkalde et gunstigt terapeutisk respons, fri for lokale og systemiske skader 1,2,3. Biokompatibilitetsevaluering er afgørende for udviklingen af ethvert materiale beregnet til medicinsk brug. Derfor giver denne protokol en systematisk og omfattende tilgang til enhver forsker, der sigter mod at udvikle nye biomaterialer eller studere nye anvendelser af eksisterende biomaterialer.

In vitro cytotoksicitetstest anvendes i vid udstrækning som den første fase til evaluering af biokompatibilitet ved anvendelse af primære cellekulturer eller cellelinjer. Resultaterne udgør en første indikator for potentiel klinisk anvendelse. Udover at være afgørende for udviklingen af biomateriale, er denne test obligatorisk for at overholde gældende regler for markedsintroduktion fra EUA og EU-regulatorer (FDA og CE-certificering)4,5,6,7,8. Desuden giver standardiseret test inden for biomedicinsk forskning en betydelig fordel med hensyn til reproducerbarhed og sammenligning af resultater fra forskellige undersøgelser af lignende biomaterialer eller udstyr9.

International Organization for Standardization (ISO) retningslinjer bruges i vid udstrækning af flere uafhængige kommercielle, lovgivningsmæssige og akademiske laboratorier til test af materialer på en nøjagtig og reproducerbar måde. ISO 10993-5 henviser til in vitro cytotoksicitetsvurderingen og ISO 10993-12 rapporterer til prøvetagningsforberedelse10,11. Til biomaterialetest leveres tre kategorier, der skal vælges i henhold til materialetype, kontaktvæv og behandlingsmålet: ekstrakter, direkte kontakt og indirekte kontakt 8,11,12,13. Ekstrakter opnås ved at berige et cellekulturmedium med biomaterialet. Til test af direkte kontakt placeres biomaterialet direkte på cellekulturerne, og ved indirekte kontakt udføres inkubation med cellerne adskilt af en barriere, såsom en agarosegel11. Passende kontrol er obligatorisk, og der skal udføres mindst tre uafhængige forsøg 5,8,10,11,14.

Det er afgørende at simulere eller overdrive kliniske tilstande for at bestemme det cytotoksiske potentiale. I tilfælde af ekstraktafprøvning, materialets overfladeareal;  det mellemstore volumen; substratet og materialets pH-værdi forholdet mellem materialeopløselighed, osmolaritet og diffusion og ekstraktionsbetingelserne som omrøring, temperatur og tid påvirker medieberigere5.

Metoden tillader kvantitativ og kvalitativ evaluering af cytotoksicitet af flere farmaceutiske formuleringer, både faste og flydende. Flere assays kan udføres, såsom neutral rød optagelsestest, kolonidannelsestest, MTT-assay og XTT-assay 5,10,14.

De fleste offentliggjorte cytotoksicitetsvurderingsundersøgelser bruger enklere assays, nemlig MTT og XTT, som giver begrænset information. Evaluering af biokompatibilitet bør ikke kun omfatte vurdering af cytotoksicitet, men også bioaktivitet af et givet testmateriale2, som denne protokol godkender. Der bør anvendes yderligere evalueringskriterier, når det er begrundet og dokumenteret. Denne protokol har således til formål at give en omfattende vejledning, der beskriver et sæt metoder til evaluering af biomaterialets cytotoksicitet. Desuden beskrives evalueringen af forskellige cellulære processer, nemlig typen af celledød, cellemorfologi, cellefunktion i syntesen af specifikke proteiner og specifik vævsproduktion.

Protocol

1. Pellets forberedelse Forbered polyvinylchloridformene (PVC) ved at udføre cirkelformede huller med kendte dimensioner i PVC-plader.BEMÆRK: PVC-lister kan laves i forskellige størrelser. Beregn kontaktfladen for PVC-forme ved hjælp af formlen A = h (2πr) + 2πr2 (r: cylinderens radius; h: cylinderens højde). Forbered det biomateriale, der skal testes i henhold til producentens anvisninger og så tæt som muligt på eksperimentets begyndelse.BEMÆRK: Til fremstilling af bioma…

Representative Results

De repræsentative resultater her henviser til undersøgelsen af dentale biomaterialer. Ekstraktmetoden gør det muligt at opnå en cytotoksicitetsprofil og cellefunktion efter eksponering for dentalmaterialerne med hensyn til virkninger på metabolisk aktivitet (figur 2), cellelevedygtighed, celledødsprofil og cellemorfologi (figur 3) og specifik proteinekspression (figur 4). MTT-analysen bruges til at …

Discussion

Denne protokol blev udformet under hensyntagen til ISO 10993-5, som henviser til evaluering af in vitro cytotoksicitet af biomaterialer, der kommer i kontakt med vævene, for at evaluere biokompatibiliteten og bidrage til undersøgelser af reproducerbarhed21. Dette er en voksende bekymring inden for videnskab, og mange forfattere følger allerede disse anbefalinger i det eksperimentelle design af deres in vitro-undersøgelser 15,22,23,24,25,26,27,28.<sup…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker følgende for støtte: GAI 2013 (Faculdade de Medicina da Universidade de Coimbra); CIBB finansieres af nationale midler via FCT (Foundation for Science and Technology) gennem det strategiske projekt UIDB/04539/2020 og UIDP/04539/2020 (CIBB). Vi takker Jacques Nör, University of Michigan Dental School, for at levere cellelinjen MDPC-23.

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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