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. 
	NOTE: For the preparation of paste/paste formulation biomaterials, an adequate amount of base paste and catalyst are mixed manually with a mixing spatula. For other materials based on liquid and powder formulations, manual spatulation or mechanical mixing with vibration should be performed, following the manufacturer&#39;s instructions or the adequate for new materials. For liquid materials, this step is not necessary. Start the protocol in step 2.</li>
	<li>Place the biomaterial on the molds with a spatula and let them set for the appropriate time. 
	NOTE: The setting time and setting conditions of the biomaterials must follow the manufacturer&#39;s instructions or the adequate for new materials.</li>
	<li>After setting, remove the biomaterial&#8217; pellets from the PVC molds and place them in a container (a 6 well plate or a Petri dish can be used).</li>
	<li>Sterilize the pellets by placing them under an ultraviolet light (UV) lamp for 20 minutes for each side.</li>
</ol>
2. Obtaining the&#160;biomaterials&#39; extracts
NOTE: All procedures should be performed under strict sterile conditions.
<ol>
	<li>Determine the necessary number of pellets by calculating the pellet surface area based on the formula described in 1.1. 
	NOTE: As a reference value, the contact surface area of 250 mm2/mL11,15 is achieved by adding 9 pellets (r 3 mm x h 1.5 mm) per mL of the medium.</li>
	<li>Prepare the soluble extracts (extract enriched with the biomaterial).
	<ol>
		<li>Place the pellets in a 50 mL tube and add the corresponding of the cell culture medium. Place the tubes for 24 hours in the incubator at 37&#176;, in constant rotation. 
		NOTE: Use the cell culture medium appropriate for the cell cultures.</li>
		<li>After 24 hours, remove the tubes from the incubator. At this point, the extracts correspond to a concentration of 1/1 or 100%.</li>
		<li>Make dilutions of the extract by sequential addition of equal volumes of conditioned medium to cell culture medium. 
		NOTE: No pH adjustment should be made to the media.
		<ol>
			<li>Add 1 mL of culture media to 1 mL of 100% extract to obtain a 50% extract. Add 1 mL of culture media to 1 mL of 50% extract to obtain a 25% extract, and so on (Figure 1). 
			NOTE: Use the concentrations found relevant for each compound.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61512/61512fig1v2.jpg" /> 
Figure 1: Scheme of the preparation and dilutions of soluble extracts. <a href="https://www.jove.com/files/ftp_upload/61512/61512fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Cell incubation with the biomaterials&#8217; extracts
<ol>
	<li>Prepare a cellular suspension and plate it in an adequate cell container, such as a multiwell plate, according to the number of cells needed for the experiments.
	<ol>
		<li>Start with a flask of the desired cells with 80% to 90% confluence.</li>
		<li>Discard the cell culture media, wash with phosphate-buffered saline solution (PBS) and detach the cells with trypsin-EDTA (1 to 2 mL for a 75 cm2 cell culture flask).</li>
		<li>Add the cell culture media (2 to 4 mL for a 75 cm2 cell culture flask), transfer the cell suspension to a tube and centrifuge at 200 x g for 5 min.</li>
		<li>Suspend the pellet in a known volume of cell culture media. 
		NOTE: This protocol is designed for the use of adherent cell cultures; however, simple adaptations can be made to work with suspension cell cultures.</li>
		<li>Count the cells in the hemocytometer and calculate the cell concentration of the cell suspension.</li>
		<li>Suspend the determined amount of cell suspension in culture medium and transfer to multiwell dishes. As a reference value for seeding density, consider 5 &#8211; 20 x 105 cells/cm2. 
		NOTE: The appropriated number of cells must be calculated according to the cell type and cell characteristics, namely cell doubling time.</li>
	</ol>
	</li>
	<li>Incubate the cells for 24 hours to allow cell adhesion.</li>
	<li>After this period, administer the soluble extracts into the culture plates.
	<ol>
		<li>Aspirate the cell culture medium.</li>
		<li>Add the biomaterials&#8217; extracts to each well, according to the sequence of concentrations, as described previously. Add fresh cell culture medium to the control wells.</li>
		<li>Incubate the plates for 24 h or longer. 
		NOTE: Negative controls must be performed in each assay, corresponding to untreated cells, maintained in the culture medium. The incubation times can be selected accordingly to the study goals.</li>
	</ol>
	</li>
</ol>
4. Evaluation of the metabolic activity
<ol>
	<li>After the cell incubation with the biomaterials&#8217; extracts, aspirate the medium from the plates and wash each well PBS.</li>
	<li>Place, in each well, the adequate volume of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) prepared in PBS, pH 7.4.</li>
	<li>Incubate the plates for 4 h or overnight in the dark at 37 &#176; C.</li>
	<li>To solubilize the obtained formazan crystals, add the adequate volume of 0.04 M solution of hydrochloric acid in isopropanol to each well and stir plates for 30 minutes. 
	NOTE: Adjust the amount of MTT and isopropanol according to the size of the wells.</li>
	<li>Stir and homogenize the contents of each well, if necessary, by pipetting up and down until no crystals are seen.</li>
	<li>Quantify the absorbance at a wavelength of 570 nm with a 620 nm reference filter, in the spectrophotometer.</li>
	<li>To calculate the metabolic activity, divide the absorbance of the treated cells by the absorbance of the control cultures. To obtain percentage values multiply by 100.</li>
</ol>
5. Cell death evaluation
NOTE: To perform this evaluation a minimum of 106 cells per condition should be used.
<ol>
	<li>Use centrifuge tubes properly identified, accordingly to the conditions being evaluated.</li>
	<li>After the cell incubation with the biomaterials&#8217; extracts, collect the culture media to the respective tube.</li>
	<li>Detach the cells and add the cell suspension to the respective tubes.</li>
	<li>Concentrate the cell suspensions by centrifugation at 120 x g for 5 minutes.</li>
	<li>Wash the pellets with PBS. Remove the PBS by centrifugation at 1,000 x g for 5 minutes.</li>
	<li>Add 1 mL of PBS and transfer the cell pellets to identified cytometry tubes.</li>
	<li>Remove the PBS by centrifugation at 1,000 x g for 5 minutes.</li>
	<li>Incubate with 100 &#181;L of binding buffer (0.01 M HEPES, 0.14 mM NaCl and 0.25 mM CaCl2)16, and allow the cells to rest for about 15 minutes for cell membrane recovery.</li>
	<li>Add 2.5 &#956;L of fluorescent labelled Annexin-V and 1 &#956;L of propidium iodide for 15 minutes at room temperature in the dark.</li>
	<li>After incubation, add 400 &#956;L of PBS and analyze on the cytometer. For the analysis and quantification of the information use appropriate software.</li>
	<li>Present results as a percentage of live&#160;cells, apoptosis, late apoptosis/necrosis, and necrosis.</li>
</ol>
6. Morphology evaluation
<ol>
	<li>Select the appropriate size of sterilized glass coverslips that fit inside the multiwell plate.</li>
	<li>Place each slide in a well using sterile tweezers.</li>
	<li>Distribute a cellular suspension at an adequate concentration into the wells and let overnight in an incubator at 37 &#176;C in a humidified atmosphere with 95% air and 5% CO2.</li>
	<li>Expose the cell cultures to the extracts, as previously described.</li>
	<li>Aspirate the media and wash with PBS.</li>
	<li>Let the coverslips dry at room temperature and then add a sufficient volume of May-Gr&#252;nwald solution to cover the coverslips; incubate for 3 minutes.</li>
	<li>Remove the dye and wash with distilled water for 1 minute.</li>
	<li>Remove the water and add a sufficient volume of Giemsa solution to cover the coverslips; incubate for 15 minutes.</li>
	<li>Wash the coverslips in running water.</li>
	<li>Transfer the coverslips to a slide.</li>
	<li>Look under a microscope. Take the photographs with the chosen magnification.</li>
</ol>
7. Cell function assessment through reverse transcription polymerase chain reaction (RT-PCR)
NOTE: To perform this evaluation a minimum of 2x106 cells per condition should be used. As an example, alkaline phosphatase is presented as a gene of interest for odontoblasts activity evaluation. Other genes of interest can be seen in Table 1.
<ol>
	<li>Plate the cells as described above. 
	NOTE: The concentration of cells plated might need to be adjusted, accordingly to the cell type and cytotoxicity of the biomaterials being studied.</li>
	<li>Incubate with soluble extracts, as described above.</li>
	<li>Detach the cells to obtain a suspension as described before.</li>
	<li>Wash the cells twice with PBS; for this centrifuge at 200 x g for 5 minutes at room temperature.</li>
	<li>Lyse the cells by suspending the pellet in 1 mL of RNA purification solution (e.g., NZYol), intense stirring, and successive pipetting.</li>
	<li>Incubate the samples for 5 minutes at room temperature.</li>
	<li>Add 200 &#181;L of chloroform and shake the tubes by hand for 15 seconds.</li>
	<li>Incubate for 3 minutes at room temperature.</li>
	<li>Centrifuge lysates at 4 &#176; C for 15 min at 12,000 x g. During this centrifugation, two phases originate in the sample, leaving the RNA in the aqueous (upper) phase.</li>
	<li>Remove the aqueous phase to a new tube and add 500 &#181;L of cold isopropanol to precipitate RNA.</li>
	<li>Incubate samples at room temperature for 10 minutes and centrifuge at 12,000 x g for 10 minutes at 4 &#176; C.</li>
	<li>Remove the supernatant and wash the pellet with 1 mL of 75% ethanol by centrifugation at 7,500 x g for 5 minutes at 4 &#176; C.</li>
	<li>Dry the pellet at room temperature until ethanol evaporation.</li>
	<li>Suspend in RNase-free water.</li>
	<li>Quantify and determine the degree of purity of the samples using absorption spectrophotometry, at the wavelengths 260 nm and 280 nm. Determine the RNA purity and use samples with a purity ratio (A260/280) around 2.0.</li>
	<li>Store samples at -80 &#176; C.</li>
	<li>Proceed to perform RT-PCR following manufacturer&#39;s protocol17. 
	NOTE: Accordingly to the study goal, select the specific markers to be evaluated.</li>
</ol>
8. Cell function assessment through protein identification
NOTE: According to the study goal, select the specific proteins to be evaluated. As an example, dentin sialoprotein (DSP) is presented as a protein of interest for odontoblasts activity evaluation. Other proteins of interest can be seen in Table 1.
<ol>
	<li>Culture cells in coverslips and expose to the extracts, as described before.</li>
	<li>Wash the cell cultures with PBS.</li>
	<li>Fix with 3.7% paraformaldehyde for 30 minutes at room temperature.</li>
	<li>Wash twice with PBS.</li>
	<li>Permeabilize with 0.5% Triton in PBS for 15 minutes.</li>
	<li>Block the peroxidase with 0.3% hydrogen peroxide in PBS for 5 minutes.</li>
	<li>Wash twice with PBS.</li>
	<li>Wash twice with 0.5% bovine serum albumin (BSA).</li>
	<li>Block cell cultures with 2% BSA for 45 minutes.</li>
	<li>Wash with 0.5% BSA in PBS.</li>
	<li>Incubate cultures with the primary antibody according to the selected protein for 60 minutes at room temperature. 
	NOTE: This protocol uses the primary antibody DSP(M20) Antibody (1:100) and the secondary antibody Polyclonal Rabbit Anti-goat immunoglobulins/HRP (1:100).</li>
	<li>Wash five times with 0.5% BSA in PBS.</li>
	<li>Incubate with secondary antibody for 90 minutes at room temperature. 
	NOTE: Make the antibody dilutions using 0.5% BSA&#160;in PBS.</li>
	<li>Wash five times with 0.5% BSA in PBS for 1 minute in each wash.</li>
	<li>Incubate cultures with a substrate and chromogen mixture at a concentration of 20 &#181;L chromogen/mL substrate for 25 minutes.</li>
	<li>Wash twice with 0.5% BSA in PBS.</li>
	<li>Counterstain with Hematoxylin for 15 minutes.</li>
	<li>Wash with a sequence of 0.037 mol/L ammonia and distilled water for 5 minutes to remove excess dye.</li>
	<li>Mount the coverslips on the slides. Use glycerol as the mounting medium.</li>
	<li>Allow drying overnight.</li>
	<li>Look under a microscope. Take the photographs with the chosen magnification.</li>
</ol>
9. Mineralization assessment through Alizarin Red S assay
<ol>
	<li>Prepare an Alizarin Red S solution at a concentration of 40 mM18. Stir the solution for homogenization for 12 hours in the dark. 
	NOTE: To prepare 100 mL of Alizarin Red S solution, solubilize 1.44 g of alizarin powder (Molecular weight: 360 g/mol) in ultrapure water, protected from light. For this solution, the pH value is critical and should be between 4.1 and 4.3.</li>
	<li>Incubate cell culture with soluble extracts, as described above.</li>
	<li>Wash cell cultures three times with PBS.</li>
	<li>Fix with 4% paraformaldehyde for 15 minutes at room temperature.</li>
	<li>Wash three times with PBS.</li>
	<li>Stain with Alizarin Red Staining solution for 20 minutes at 37 &#176;C in the dark.</li>
	<li>After staining, wash the plates with PBS to remove the excess dye.</li>
	<li>Look under a microscope. Take the photographs with the chosen magnification.</li>
	<li>Add an extraction solution, composed by 10% (w/v) acetic acid and 20% (w/v) methanol, to each well, and let stirring for 40 minutes at room temperature.</li>
	<li>Measure the absorbance at 490 nm wavelength on a spectrophotometer19.</li>
</ol>

The authors have no competing financial interests or other conflicts of interest.

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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JoVE Journal

Medicine

3.4K Views.  University of Coimbra, Coimbra, Portugal. 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.

Video: Accessing the Cytotoxicity and Cell Response to Biomaterials

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...