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

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

In vitro cytotoxicity tests are widely used as the first phase for biocompatibility evaluation, 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 preparation^10,11. For biomaterial testing three categories are provided, to be selected according to the material type, contacting tissues, and the treatment goal: extracts, direct contact, and indirect contact^8,11,12,13. 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 performed^5,8,10,11,14.

It is critical to simulate or exaggerate clinical conditions to determine the cytotoxic potential. In the case of extracts testing, the material's surface area;  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 assay^5,10,14.

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.

1. Pellets preparation

1. Prepare the polyvinyl chloride (PVC) molds by performing circular-shaped holes of known dimensions in PVC plates.
   NOTE: PVC moldings can be made of different sizes. Calculate the contact surface of PVC molds, using the formula A= h(2πr)+2πr² (r: radius of the cylinder; h: height of the cylinder).
2. Prepare the biomaterial to be tested according to the manufacturer's instructions and as close as possible to the beginning of the experiment.
   NOTE: For the preparation of paste/paste formulation biomaterials, an adequate amount of base paste and catalyst are mixed manually with a mixing spatula. For other materials based on liquid and powder formulations, manual spatulation or mechanical mixing with vibration should be performed, following the manufacturer's instructions or the adequate for new materials. For liquid materials, this step is not necessary. Start the protocol in step 2.
3. 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's instructions or the adequate for new materials.
4. After setting, remove the biomaterial’ pellets from the PVC molds and place them in a container (a 6 well plate or a Petri dish can be used).
5. Sterilize the pellets by placing them under an ultraviolet light (UV) lamp for 20 minutes for each side.

2. Obtaining the biomaterials' extracts

NOTE: All procedures should be performed under strict sterile conditions.

1. 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/mL^11,15 is achieved by adding 9 pellets (r 3 mm x h 1.5 mm) per mL of the medium.
2. Prepare the soluble extracts (extract enriched with the biomaterial).
   1. 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°, in constant rotation.
      NOTE: Use the cell culture medium appropriate for the cell cultures.
   2. After 24 hours, remove the tubes from the incubator. At this point, the extracts correspond to a concentration of 1/1 or 100%.
   3. 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.
      1. 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.

Figure 1: Scheme of the preparation and dilutions of soluble extracts. Please click here to view a larger version of this figure.

3. Cell incubation with the biomaterials’ extracts

1. 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.
   1. Start with a flask of the desired cells with 80% to 90% confluence.
   2. 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).
   3. 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.
   4. 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.
   5. Count the cells in the hemocytometer and calculate the cell concentration of the cell suspension.
   6. Suspend the determined amount of cell suspension in culture medium and transfer to multiwell dishes. As a reference value for seeding density, consider 5 – 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.
2. Incubate the cells for 24 hours to allow cell adhesion.
3. After this period, administer the soluble extracts into the culture plates.
   1. Aspirate the cell culture medium.
   2. Add the biomaterials’ extracts to each well, according to the sequence of concentrations, as described previously. Add fresh cell culture medium to the control wells.
   3. 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.

4. Evaluation of the metabolic activity

1. After the cell incubation with the biomaterials’ extracts, aspirate the medium from the plates and wash each well PBS.
2. 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.
3. Incubate the plates for 4 h or overnight in the dark at 37 ° C.
4. 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.
5. Stir and homogenize the contents of each well, if necessary, by pipetting up and down until no crystals are seen.
6. Quantify the absorbance at a wavelength of 570 nm with a 620 nm reference filter, in the spectrophotometer.
7. 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.

5. Cell death evaluation

NOTE: To perform this evaluation a minimum of 10⁶ cells per condition should be used.

1. Use centrifuge tubes properly identified, accordingly to the conditions being evaluated.
2. After the cell incubation with the biomaterials’ extracts, collect the culture media to the respective tube.
3. Detach the cells and add the cell suspension to the respective tubes.
4. Concentrate the cell suspensions by centrifugation at 120 x g for 5 minutes.
5. Wash the pellets with PBS. Remove the PBS by centrifugation at 1,000 x g for 5 minutes.
6. Add 1 mL of PBS and transfer the cell pellets to identified cytometry tubes.
7. Remove the PBS by centrifugation at 1,000 x g for 5 minutes.
8. Incubate with 100 µL 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.
9. Add 2.5 μL of fluorescent labelled Annexin-V and 1 μL of propidium iodide for 15 minutes at room temperature in the dark.
10. After incubation, add 400 μL of PBS and analyze on the cytometer. For the analysis and quantification of the information use appropriate software.
11. Present results as a percentage of live cells, apoptosis, late apoptosis/necrosis, and necrosis.

6. Morphology evaluation

1. Select the appropriate size of sterilized glass coverslips that fit inside the multiwell plate.
2. Place each slide in a well using sterile tweezers.
3. Distribute a cellular suspension at an adequate concentration into the wells and let overnight in an incubator at 37 °C in a humidified atmosphere with 95% air and 5% CO_2.
4. Expose the cell cultures to the extracts, as previously described.
5. Aspirate the media and wash with PBS.
6. Let the coverslips dry at room temperature and then add a sufficient volume of May-Grünwald solution to cover the coverslips; incubate for 3 minutes.
7. Remove the dye and wash with distilled water for 1 minute.
8. Remove the water and add a sufficient volume of Giemsa solution to cover the coverslips; incubate for 15 minutes.
9. Wash the coverslips in running water.
10. Transfer the coverslips to a slide.
11. Look under a microscope. Take the photographs with the chosen magnification.

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.

1. 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.
2. Incubate with soluble extracts, as described above.
3. Detach the cells to obtain a suspension as described before.
4. Wash the cells twice with PBS; for this centrifuge at 200 x g for 5 minutes at room temperature.
5. Lyse the cells by suspending the pellet in 1 mL of RNA purification solution (e.g., NZYol), intense stirring, and successive pipetting.
6. Incubate the samples for 5 minutes at room temperature.
7. Add 200 µL of chloroform and shake the tubes by hand for 15 seconds.
8. Incubate for 3 minutes at room temperature.
9. Centrifuge lysates at 4 ° 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.
10. Remove the aqueous phase to a new tube and add 500 µL of cold isopropanol to precipitate RNA.
11. Incubate samples at room temperature for 10 minutes and centrifuge at 12,000 x g for 10 minutes at 4 ° C.
12. 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 ° C.
13. Dry the pellet at room temperature until ethanol evaporation.
14. Suspend in RNase-free water.
15. 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.
16. Store samples at -80 ° C.
17. Proceed to perform RT-PCR following manufacturer's protocol¹⁷.
    NOTE: Accordingly to the study goal, select the specific markers to be evaluated.

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.

1. Culture cells in coverslips and expose to the extracts, as described before.
2. Wash the cell cultures with PBS.
3. Fix with 3.7% paraformaldehyde for 30 minutes at room temperature.
4. Wash twice with PBS.
5. Permeabilize with 0.5% Triton in PBS for 15 minutes.
6. Block the peroxidase with 0.3% hydrogen peroxide in PBS for 5 minutes.
7. Wash twice with PBS.
8. Wash twice with 0.5% bovine serum albumin (BSA).
9. Block cell cultures with 2% BSA for 45 minutes.
10. Wash with 0.5% BSA in PBS.
11. 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).
12. Wash five times with 0.5% BSA in PBS.
13. Incubate with secondary antibody for 90 minutes at room temperature.
    NOTE: Make the antibody dilutions using 0.5% BSA in PBS.
14. Wash five times with 0.5% BSA in PBS for 1 minute in each wash.
15. Incubate cultures with a substrate and chromogen mixture at a concentration of 20 µL chromogen/mL substrate for 25 minutes.
16. Wash twice with 0.5% BSA in PBS.
17. Counterstain with Hematoxylin for 15 minutes.
18. Wash with a sequence of 0.037 mol/L ammonia and distilled water for 5 minutes to remove excess dye.
19. Mount the coverslips on the slides. Use glycerol as the mounting medium.
20. Allow drying overnight.
21. Look under a microscope. Take the photographs with the chosen magnification.

9. Mineralization assessment through Alizarin Red S assay

1. 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.
2. Incubate cell culture with soluble extracts, as described above.
3. Wash cell cultures three times with PBS.
4. Fix with 4% paraformaldehyde for 15 minutes at room temperature.
5. Wash three times with PBS.
6. Stain with Alizarin Red Staining solution for 20 minutes at 37 °C in the dark.
7. After staining, wash the plates with PBS to remove the excess dye.
8. Look under a microscope. Take the photographs with the chosen magnification.
9. 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.
10. Measure the absorbance at 490 nm wavelength on a spectrophotometer¹⁹.

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...

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

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

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 Nör, University of Michigan Dental School, for providing the cell line MDPC-23.