Accessing the Cytotoxicity and Cell Response to Biomaterials

Summary

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Abstract

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Grünwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

Introduction

Biocompatibility can be defined as the capacity of a material to integrate tissue and induce a favorable therapeutic response, free of local and systemic damages^1,2,3. Biocompatibility evaluation is crucial for the development of any material intended for medical use. Therefore, this protocol provides a systematic and comprehensive approach for every researcher aiming to develop new biomaterials or studying new applications for existing biomaterials.

In vitro cytotoxicity tests are widely used as the first phase for biocompatibility evaluation, usi....

Protocol

1. Pellets preparation

1. Prepare the polyvinyl chloride (PVC) molds by performing circular-shaped holes of known dimensions in PVC plates.
   NOTE: PVC moldings can be made of different sizes. Calculate the contact surface of PVC molds, using the formula A= h(2πr)+2πr² (r: radius of the cylinder; h: height of the cylinder).
2. Prepare the biomaterial to be tested according to the manufacturer's instructions and as close as possible to the beginning of the experiment.
   NOTE: For the preparation of paste/paste formulation biomaterials, an adequate amount of base paste and catalyst are mixed manually with a mixing ....

Results

The representative results here refer to the study of dental biomaterials. The extract methodology allows to obtain a cytotoxicity profile and cell function after exposition to the dental materials, regarding effects on metabolic activity (Figure 2), cell viability, cell death profile and cell morphology (Figure 3), and specific proteins expression (Figure 4).

The MTT assay is used to obtain a quick overv.......

Discussion

This protocol was designed taking into consideration the ISO 10993-5, which refers to the evaluation of in vitro cytotoxicity of biomaterials that contact with the tissues, to evaluate the biocompatibility and to contribute to studies reproducibility²¹. This is a growing concern in science, and many authors are already following these recommendations in the experimental design of their in vitro studies^15,22,

Materials

Name	Company	Catalog Number	Comments
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