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 &amp;micro;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&amp;uuml;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>&lt;strong&gt;&amp;beta;&lt;/strong&gt;-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 ...

accessing-the-cytotoxicity-and-cell-response-to-biomaterials

Research

JoVE Journal

Medicine

3.6K Views.  University of Coimbra, Coimbra, Portugal. Met de explosie van nieuwe materialen voor medische toepassingen, moet de eerste screening systematisch worden gemaakt, zodat dit protocol een fundamenteel hulpmiddel kan zijn voor onderzoekers die nieuwe biomaterialen willen ontwikkelen en karakteriseren. De methodologie maakt de kwantitatieve en kwalitatieve evaluatie mogelijk van cytotoxiciteit en bioactiviteit van biomaterialen en farmaceutische formuleringen, zowel vast als vloeibaar. Na celblootstelling kunnen verschillende complementai...

Video: Accessing the Cytotoxicity and Cell Response to Biomaterials

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Immunology and Infection

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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 ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Bioengineering

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. 
This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. 
This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

We introduce three methods of direct culture, direct exposure culture, and exposure culture for evaluating the in vitro cytocompatibility of biodegradable implant materials. These in vitro methods mimic different in vivo cell-implant interactions and can be applied to study various biodegradable materials.

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and ...

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells' Response to Metal-Organic Frameworks 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

Thyroid Cancer Cells as Effectors in an ADCC Bioassay for Cancer Immunotherapy

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

Author Spotlight: Optimizing Antibody-Based Cancer Treatments via Antibody-Dependent, Cell-Mediated Cytotoxicity Assay

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
One of the main functions of the cells of the immune system is to remove target cells that have been infected with viruses or have undergone transformation into a tumor cell. In vitro assays for measuring the cytotoxic capacity of immune cells have been a staple in laboratories for many years. These assays have been used to determine the ability of T cells, NK cells, or any other immune cell to kill target cells in an antigen-specific or -nonspecific manner. Death ligands (e.g., Fas ligand or TRAIL), cytokines (e.g., IFNg or TNF), or cytotoxic granules (i.e., perforin/granzyme B) expressed by effector cells are some ways in which target cell death can be induced. With the explosion in tumor immunotherapy research in recent years, there is growing interest in finding agents to increase the cytotoxic activity of immune cells to improve patient outcomes. Conversely, some diseases are hallmarked by the overexuberant activity of immune cell cytotoxic activity, resulting in efforts to identify agents to temper these responses. Thus, having an assay in which the user can easily integrate any number of different effector cells, target cells, and/or response modifiers into the experimental design can serve as a valuable means of quickly assessing the cytotoxic capacity of effector cells and/or the responsiveness of the target cell.
These in vitro assays involve the mixing of different cell populations, as well as using a relatively low number of both effector and target cells. Thus, one necessity of the assay is to label the target cells in a manner that can easily be detected and quantitated, allowing the user to then determine the &#39;percent specific lysis&#39; mediated by the effector cells. Radioactivity - especially, chromium 51 (51Cr) in the form of Na251CrO4- is an inexpensive way to rapidly and nonspecifically label cellular proteins within the target cells (1). The short labeling and total assay times reduces the potential for significant changes in the number and/or phenotype of the target cells, which could influence the outcome of the assay. Upon the loss of membrane integrity of the target cells as a result of the cytotoxic activity of the effector cells, the 51Cr-labelled cellular proteins within the target cells are released into the culture supernatant, becoming available for quantitation. As with any assay examining the function of immune cells in vitro, there are a number of important considerations to consider improving the performance of the experiment. One of the most critical features is to use healthy effector (for maximal cytotoxic activity) and target (for maximal responsiveness and minimal spontaneous death/51Cr release) cells. Effector and target cell contact is required (leading to the common use of round-bottom 96-well plates to encourage cell-cell contact) (2). Finally, data analysis is dependent on the inclusion of positive and negative control target cell populations.
The following protocol will outline the steps for performing a standard 51Cr release assay to measure the cytotoxic ability of a population of effector cells, though a nonradioactive version using Europium has recently been developed. 51Cr is a powerful &#947;-radiation emitter. Consequently, the use of this assay requires proper radiation safety training, dedicated laboratory space, a gamma counter, and disposal of radioactive samples.
The general sequence of events in this assay are: 1) prepare 51Cr-labeled targets; 2) prepare effector cells and add to plate while target cells are labeling; 3) add labeled targets to plate; 4) incubate plate; 5) harvest supernatants; and 6) analyze data after running samples on counter. Samples are commonly prepared in triplicate, and then averaged to account for any subtle pipetting differences.
Proper PPE is important for this assay. Specifically, the user should wear a lab coat and gloves. Safety glasses may be required based on the laboratory or institution. There should be ample lead shielding for safe storage and use of the 51Cr during all steps. Finally, there should be dedicated lab space and equipment set aside for using 51Cr, including all the proper signage to indicate where samples with 51Cr are being kept and a Geiger counter equipped with gamma probe to survey the space for possible contamination.
In this lab exercise, we will determine the ability human peripheral blood mononuclear cells (PBMCs), (CpG stimulated vs. unstimulated) to kill melanoma cells, using human melanoma cell line WM793 as model and the chromium release assay.

Accessing the Cytotoxicity and Cell Response to Biomaterials

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