Name: accessing the cytotoxicity and cell response to biomaterials
Uploaded: 2021-07-08T13:00:00
Description: Watch this Scientific Journal Video about Accessing the Cytotoxicity and Cell Response to Biomaterials at JoVE.com

We thank the following for support: GAI 2013 (Faculdade de Medicina da Universidade de Coimbra); CIBB is funded by National Funds via FCT (Foundation for Science and Technology) through the Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB). We thank to Jacques N&#246;r, University of Michigan Dental School, for providing the cell line MDPC-23.

1. Pellets preparation
<ol>
	<li>Prepare the polyvinyl chloride (PVC) molds by performing&#160;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(2&#960;r)+2&#960;r2 (r: radius of the cylinder; h: height of the cylinder).</li>
	<li>Prepare the biomaterial to be tested according to the manufacturer&#39;s instructions and as close as possible to the beginning of the experiment.<br ...

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 studies15,22,<sup class="...

Biocompatibility can be defined as the capacity of a material to integrate tissue and induce a favorable therapeutic response, free of local and systemic damages1,2,3. 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, using primary cell cultures or cell lines. The results constitute a first indicator of potential clinical application. Besides being vital for the biomaterial development, this testing is mandatory to comply with current regulations for market introduction, from EUA and EU regulators (FDA and CE certification)4,5,6,7,8. Moreover, standardized testing in biomedical research provides a significant advantage in terms of reproducibility and comparison of results from different studies on similar biomaterials or devices9.
International Organization for Standardization (ISO) guidelines are widely used by multiple independent commercial, regulatory, and academic laboratories for testing materials in an accurate and reproducible manner. The ISO 10993-5 refers to the in vitro cytotoxicity assessment and the ISO 10993-12 reports to sampling preparation10,11. For biomaterial testing&#160;three categories are provided, to be selected according to the material type, contacting tissues, and the treatment goal: extracts, direct contact, and indirect contact8,11,12,13. Extracts are obtained by enriching a cell culture medium with the biomaterial. For the direct contact tests, the biomaterial is placed directly on the cell cultures, and, in indirect contact, incubation with the cells is performed separated by a barrier, such as an agarose gel11. Appropriate controls are mandatory, and a minimum of three independent experiments should be performed5,8,10,11,14.
It is critical to simulate or exaggerate clinical conditions to determine the cytotoxic potential. In the case of extracts testing, the material&#39;s surface area;&#160; the medium volume; the medium and the material pH; the material solubility, osmolarity and diffusion ratio; and the extraction conditions such as agitation, temperature, and time influence media enrichmen5.
The methodology allows the quantitative and qualitative evaluation of cytotoxicity of several pharmaceutical formulations, both solid and liquid. Several assays can be performed, such as neutral red uptake test, colony formation test, MTT assay, and XTT assay5,10,14.
Most cytotoxicity assessment studies published use simpler assays, namely MTT and XTT, which provide limited information. Evaluating biocompatibility should not only involve the assessment of cytotoxicity but also bioactivity of a given test material2, as this protocol endorses. Additional evaluation criteria should be used when justified and documented. Thus, this protocol aims to provide a comprehensive guide, detailing a set of methods for the biomaterial cytotoxicity evaluation. Besides, the evaluation of different cellular processes, namely the type of cell death, cell morphology, cell function in the synthesis of specific proteins, and specific tissue production, are described.

<table>
	<tbody>
		<tr>
			<td>Absolute ethanol</td>
			<td>Merck Millipore</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>StemPro Accutas Cell Dissociation Reagent</td>
		</tr>
		<tr>
			<td>ALDH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC166362</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V FITC</td>
			<td>BD Biosciences</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic antimycotic solution</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>Pierce BCA Protein Assay Kit</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CD133 antibody</td>
			<td>Miteny Biotec</td>
			<td>293C3-APC</td>
			<td>Allophycocyanin (APC)</td>
		</tr>
		<tr>
			<td>CD24 antibody</td>
			<td>BD Biosciences</td>
			<td>658331</td>
			<td>Allophycocyanin-H7 (APC-H7)</td>
		</tr>
		<tr>
			<td>CD44 antibody</td>
			<td>Biolegend</td>
			<td>103020</td>
			<td>Pacific Blue (PB)</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>40 &#181;M</td>
		</tr>
		<tr>
			<td>Collagenase, type IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini</td>
			<td>Roche</td>
			<td>118 361 700 0</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB + Chromogen</td>
			<td>Dako</td>
			<td>K3468</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Sigma</td>
			<td>D8900</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>DSP (M-20) Antibody, 1: 100</td>
			<td>Santa Cruz Biotechnology</td>
			<td>LS-C20939</td>
			<td></td>
		</tr>
		<tr>
			<td>ECC-1</td>
			<td>ATCC</td>
			<td>CRL-2923</td>
			<td>Human endometrium adenocarcinoma cell line</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes 0.01 M</td>
			<td>Sigma</td>
			<td>MFCD00006158</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth factor basic</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain, modified GS-500</td>
			<td>Sigma</td>
			<td>MFCD00081642</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Dako</td>
			<td>C0563</td>
			<td></td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td>VWR</td>
			<td>HERE1080339</td>
			<td></td>
		</tr>
		<tr>
			<td>HCC1806</td>
			<td>ATCC</td>
			<td>CRL-2335</td>
			<td>Human mammary squamous cell carcinoma cell line</td>
		</tr>
		<tr>
			<td>Insulin, transferrin, selenium Solution</td>
			<td>Gibco</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>May-Gr&#252;nwald Stain MG500</td>
			<td>Sigma</td>
			<td>MFCD00131580</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF7</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td>Human mammary adenocarcinoma cell line</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>AlfaAesar</td>
			<td>45490</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>JMGS</td>
			<td>37040005002212</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-goat immunoglobulins / HRP, 1: 100</td>
			<td>Dako</td>
			<td>G-21234</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl-methacrylate</td>
			<td>Sigma</td>
			<td>P3932</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P7505</td>
			<td></td>
		</tr>
		<tr>
			<td>RL95-2</td>
			<td>ATCC</td>
			<td>CRL-1671</td>
			<td>Human endometrium carcinoma cell line</td>
		</tr>
		<tr>
			<td>Sodium deoxycholic acid</td>
			<td>JMS</td>
			<td>EINECS 206-132-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate Buffer</td>
			<td>Dako</td>
			<td>926605</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>JMGS</td>
			<td>20360000BP152112</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Merck</td>
			<td>108603</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin antibody</td>
			<td>Sigma</td>
			<td>A5316</td>
			<td></td>
		</tr>
	</tbody>
</table>

accessing the cytotoxicity and cell response to biomaterials

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-Gr&#252;nwald 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.

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...

Watch this Scientific Journal Video about Accessing the Cytotoxicity and Cell Response to Biomaterials at JoVE.com

Accessing the Cytotoxicity and Cell Response to Biomaterials

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.

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

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