In memory of Dr. M&#225;rcio Lorencini, a coauthor of this work, an excellent researcher in the &#64257;eld of cosmetics and devoted to promoting cosmetic research in Brazil. The authors are grateful to the Multi-user Laboratory in the Physiology Department (UFPR) for equipment availability and for the &#64257;nancial support of the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) (Finance Code 001) and the Grupo Botic&#225;rio.

The key steps to generate 3D spheroids of ZFL and ZEM2S cell lines in a round-bottom 96-well plate are presented in Figure 1.
NOTE: See the Table of Materials for details related to all materials used in this protocol and Table 1 for solutions and culture media used in this protocol.
1. Cell culture medium and monolayer cultures
<ol>
	<li>Grow both cell lines (ZFL, ZEM2S) as monolayer...

<ol>
	<li>Elje, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+applied+to+HepG2+liver+spheroids.">The comet assay applied to HepG2 liver spheroids.</a> Mutation Research. Genetic Toxicology and Environmental Mutagenesis. 845, 403033 (2019).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnology and Bioengineering. 83 (2), 173-180 (2003).</li><li>Baron, M. G., Purcell, W. M., Jackson, S. K., Owen, S. F., Jha, A. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+size+does+not+impact+metabolism+of+the+&#946;-blocker+propranolol+in+3D+intestinal+fish+model.">Spheroid size does not impact metabolism of the &#946;-blocker propranolol in 3D intestinal fish model.</a> Frontiers in Pharmacology. 9, 947 (2018).</li><li>Lammel, T., Tsoukatou, G., Jellinek, J., Sturve, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+three-dimensional+(3D)+spheroid+cultures+of+the+continuous+rainbow+trout+liver+cell+line+RTL-W1.">Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1.</a> Ecotoxicology and Environmental Safety. 167, 250-258 (2019).</li><li>Jeong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+CBZ-induced+catalysis+and+cytochrome+gene+expression+in+three+dimensional+zebrafish+liver+cellculture.">Differential effects of CBZ-induced catalysis and cytochrome gene expression in three dimensional zebrafish liver cellculture.</a> Journal of Environmental and Analytical Toxicology. 6, 2161 (2016).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> Journal of Visualized Experiments. (51), e2720 (2011).</li><li>Lee, W. G., Ortmann, D., Hancock, M. J., Bae, H., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hollow+sphere+soft+lithography+approach+for+long-term+hanging+drop+methods.">A hollow sphere soft lithography approach for long-term hanging drop methods.</a> Tissue Engineering. Part C, Methods. 16 (2), 249-259 (2010).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods in Molecular Medicine. 140, 141-151 (2007).</li><li>de Souza, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+3D+cultures+of+zebrafish+liver+and+embryo+cell+lines:+a+comparison+of+different+spheroid+formation+methods.">Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods.</a> Ecotoxicology. 30 (9), 1893-1909 (2021).</li><li>Ferreira, T., Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+user+guide.">ImageJ user guide.</a> ImageJ/Fiji. 1, 151-161 (2012).</li><li>Guidony, N. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+goethite+in+the+effects+of+glyphosate+and+Roundup&#174;+on+ZFL+cell+line.">Interference of goethite in the effects of glyphosate and Roundup&#174; on ZFL cell line.</a> Toxicology In Vitro. 65, 104755 (2020).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+is+a+key+factor+influencing+the+invasion+and+proliferation+of+Toxoplasma+gondii+in+fish+cells.">Temperature is a key factor influencing the invasion and proliferation of Toxoplasma gondii in fish cells.</a> Experimental Parasitology. 217, 107966 (2020).</li><li>Lopes, F. M., Sandrini, J. Z., Souza, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+induced+by+glyphosate+and+glyphosate-based+herbicides+in+the+zebrafish+hepatocyte+cell+line+(ZF-L).">Toxicity induced by glyphosate and glyphosate-based herbicides in the zebrafish hepatocyte cell line (ZF-L).</a> Ecotoxicology and Environmental Safety. 162, 201-207 (2018).</li><li>Lachner, D., Oliveira, L. F., Martinez, C. B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+water+soluble+fraction+of+gasoline+on+ZFL+cell+line:+Cytotoxicity,+genotoxicity+and+oxidative+stress.">Effects of the water soluble fraction of gasoline on ZFL cell line: Cytotoxicity, genotoxicity and oxidative stress.</a> Toxicology In Vitro. 30, 225-230 (2015).</li><li>Morozesk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+multiwalled+carbon+nanotubes+co-exposure+with+cadmium+on+zebrafish+cell+line:+Metal+uptake+and+accumulation,+oxidative+stress,+genotoxicity+and+cell+cycle.">Effects of multiwalled carbon nanotubes co-exposure with cadmium on zebrafish cell line: Metal uptake and accumulation, oxidative stress, genotoxicity and cell cycle.</a> Ecotoxicology and Environmental Safety. 202, 110892 (2020).</li><li>Dognani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrous+membranes+for+low-concentration+Cr+VI+adsorption:+kinetic,+thermodynamic+and+the+influence+on+ZFL+cells+viability.">Nanofibrous membranes for low-concentration Cr VI adsorption: kinetic, thermodynamic and the influence on ZFL cells viability.</a> Materials Research. , 24 (2021).</li><li>ZEM2S (ATCC&#174;CRL-2147&#8482;). American Type Culture Collection Available from: <a target="_blank" href="https://www.atcc.org/products/crl-2147">https://www.atcc.org/products/crl-2147</a> (2022)</li><li>Bell, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+human+hepatocyte+spheroids+as+a+model+system+for+drug-induced+liver+injury,+liver+function+and+disease.">Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease.</a> Scientific Reports. 6, 25187 (2016).</li><li>Gajski, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxic+potential+of+selected+cytostatic+drugs+in+human+and+zebrafish+cells.">Genotoxic potential of selected cytostatic drugs in human and zebrafish cells.</a> Environmental Science and Pollution Research International. 23 (15), 14739-14750 (2016).</li><li>Meng, Q., Yeung, K., Chan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+effects+of+octocrylene+on+zebrafish+larvae+and+liver+cell+line+(ZFL).">Toxic effects of octocrylene on zebrafish larvae and liver cell line (ZFL).</a> Aquatic Toxicology. 236, 105843 (2021).</li><li>Mueller-Klieser, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+determination+of+oxygen+consumption+rates+and+diffusion+coefficients+in+multicellular+spheroids.">Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids.</a> Biophysical Journal. 46 (3), 343-348 (1984).</li><li>Glicklis, R., Merchuk, J. 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The authors declare no conflict of interest.

This is a simple, easy, and fast method for generating spheroids of zebrafish liver and embryo cell lines. This method was developed by this group based on modifications of existing 3D spheroid methods to overcome problems reported in scientific studies related to spheroid formation, as well as uncertainties in data accuracy from 3D spheroid assays. For instance, the problems reported lie in difficulties of handling, the time-consuming nature of generating spheroids, the necessity of selecting spheroids of a similar size...

Spheroids are small spheres of cells formed when cells are cultured in close cell-to-cell contact in 3D culture. The capacity of spheroids to mimic the in vivo tissue environment has already been studied in a variety of cell lines and primary cells1,2. However, although spheroids are well developed for mammalian toxicity studies, the development of spheroids for toxicity studies with non-mammalian vertebrates (e.g., fish) is still in progress3. For fish cell lines, spheroids have been developed by a variety of different methods, such as orbital shaking (OS) using different types of well-plates3,4,5,6,7, and the method of magnetic levitation using magnetic nanoparticles8. However, some of these culture methods for spheroids may have more disadvantages than others.
For example, gyratory methods in large microplates (24-well plates) may generate a high number of spheroids differing in size and shape; indeed, multi-spheroid structure formation has been demonstrated7. This requires intense efforts to handpick spheroids with a similar size and shape for an experiment. The hanging drop 3D culture method is commonly used for generating spheroids of mammalian cell lines1,2,9,10,11, whereby single spheroids per drop can be generated, avoiding the problems described above. However, although a modified hanging drop method (hanging drop + orbital shaking) is able to generate ZFL spheroids using an inexpensive method, it has its disadvantages12. The cellular aggregates formed cannot be maintained for long periods in the drops; thus, they need to be transferred to well plates. This process requires intense handling and long periods of work in a laminar flow hood, since it is performed dropwise using a micropipette12. In addition, this method requires 10 days to fully form the ZFL spheroids (5 days in hanging drop + 5 days in OS)12. These disadvantages can limit the application of 3D fish spheroids for toxicity testing, especially considering potential applications for chemical prioritization and product sustainability.
Thus, this article describes a 3D culture protocol able to generate single spheroids of ZFL (D. rerio normal hepatocyte) and ZEM2S (D. rerio blastula phase embryo) cell lines based on the combined use of 96-well, ultra-low attachment plates (ULA-plates) and an orbital shaker (22 mm rotational diameter). The method applied is simple and reproducible, and can generate high numbers of spheroids of similar size and shape in a short period (5 days). The advantages of this method can support the application of fish 3D models for aquatic toxicity studies in both industry and academia, as well as the progress of implementing alternative methods for ecotoxicity testing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well Clear Round Bottom Ultra-Low Attachment Microplate, Individually Wrapped, with Lid, Sterile</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, powder, high glucose, pyruvate</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix, powder</td>
			<td>Gibco</td>
			<td>21700026</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Processing and analysis in Java (ImageJ) 1.52p software</td>
			<td>&#160;National 
			Institutes of Health, USA</td>
			<td></td>
			<td>Available at: https://imagej.nih.gov/ij/index.html</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium, powder</td>
			<td>Gibco</td>
			<td>41300021</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker&#160;</td>
			<td>Warmnest</td>
			<td>KLD-350-BI</td>
			<td>22 mm rotation diameter</td>
		</tr>
		<tr>
			<td>Dulbeccos PBS (10x) with calcium and magnesium</td>
			<td>Invitrogen</td>
			<td>14080055</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>31800-014</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS - Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, powder,&#160; bioreagent for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain (0,4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEM2S cell line</td>
			<td>ATCC</td>
			<td>CRL-2147</td>
			<td>This cell line was kindly donated by Professor Dr. Michael J. 
			Carvan (University of Wisconsin, Milwaukee, USA)</td>
		</tr>
		<tr>
			<td>ZFL cell line</td>
			<td>BCRJ</td>
			<td>256</td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3d culture method of spheroids of embryonic and liver zebrafish cell lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Single spheroids per well with a stable size and shape are formed by this method. Figure 2 illustrates the formation process of single spheroids of ZFL and ZEM2S cells in a well of a ULA-plate under orbital shaking (70 rpm). The ZFL and ZEM2S cell lines have different behaviors in 3D culture. The ZEM2S cell line presents features that confer the ability to readily form a spheroidal shape since the first day of the orbital shaking (Figure 2E), while the ZFL cell ...

Watch this Scientific Journal Video about A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines at JoVE.com

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

This protocol can easily generate zebrafish spheroids, a 3D in vitro model with different potential application in fish toxicity testing. This is a simple and fast method to generate single spheroids of zebrafish cell lines very similar in size and shape. If you follow all the described steps correctly, one should not encounter any problems.Zebrafish spheroids provides may greatly benefit the mechanism, exploration of chemicals'effects on fish, which helps to improve the environmental safety assessment of products such as cosmetics. Demonstrating the procedure will be Irisdoris Rodrigues de Souza, a doctor in genetics from my laboratory. To begin, take a T75 flask of zebrafish cells at around 80%confluence.Remove the complete medium, and wash the cells by adding 0.01 molar PBS to the culture flask with a pipet. Wash the culture flask with PBS by gently shaking the flask and then remove the PBS. Add three milliliters of trypsin 0.5 millimolar EDTA to the culture flask, and incubate at 28 degrees Celsius for three minutes.For the detachment of cells from the flask, gently tap the flask to release the cells. And then stop the trypsin digestion by adding three milliliters of complete culture medium to the flask. Using a pipet, transfer the cell suspension to a 15 milliliter conical centrifuge tube and centrifuge at 100 G for five minutes.Then carefully remove the supernatant. Add one milliliter of the complete medium for the respective cell line in use and resuspend the pellet using a micropipet. Add 10 microliters of the cell suspension and 10 microliters of Trypan blue dye to a micro tube to count the cells and evaluate their viability.Mix the cell suspension and dye using a pipet, and transfer 10 microliters of this mixture to a Neubauer chamber. Count the cells in the four large squares or quadrants placed at the corners of the chamber, considering cells that do not take up Trypan blue as viable. Then calculate the number of viable cells using the equation shown on the screen.Multiply this cell number by two to calculate the final cell number in the cell suspension. After calculating the cell number, adjust the cell suspension to plate 200 microliters of this suspension per well of a 96-well round bottom ULA plate with the number of cells required for each cell line. Prepare the suspension with the adjusted concentration of cells in a medium reservoir and mix it using a multi-channel micropipet without forming foam or bubbles.Add 200 microliters of the adjusted cell suspension to each well of the round bottom ULA plate. using a multi-channel pipette. The plate must be sealed with Parafilm or adhesive sealing foil to avoid culture medium evaporation from the 96-well plate.Incubate the plate on an orbital shaker at 70 RPM for five days in a 28 degrees Celsius incubator without carbon dioxide. After five days, observe the spheroids under an inverted light microscope to evaluate the generated spheroids. To measure their shape and size, obtain pictures of the spheroids, and use software to process and analyze the images.After setting the software to measure a selected area based on a known scale, select the outer side of the spheroid to determine its area and circularity. The spheroid's area is used to determine its size by calculating the diameter, d, and the software provides the spheroid's circularity by a formula that uses the area and perimeter selected. A ZFL cell aggregate is formed on the first day of orbital shaking.The cell aggregate increases its circularity on the third day, and reaches an adequate spheroidal shape on day five. A 16-day-old ZFL spheroid in ULA plate is shown here. The ZEM2S cell line forms spheroids from the first day of orbital shaking.The spheroid maintains its shape and increases in size on the third day, and its maximum circularity is reached on day five. A 10-day-old ZEM2S spheroid in a ULA plate is shown here. The measured size of spheroids of the ZFL and ZEM2S cell lines and the measured circularity index of ZFL and ZEM2S spheroids are shown in this figure.This graphical image represents the growth pattern of ZFL and ZEM2S spheroids. A ZFL spheroid and ZEM2S spheroid formed after five days of orbital shaking at 100 RPM are shown here. The growth pattern and circularity index of the spheroids formed at 70 and 100 RPM rotational speeds are presented in this figure.70 RPM is the most adequate speed to form ZFL and ZEM2S spheroids by this method. Staining of cell membranes by Lectin Alexa Fluor 594 and nuclei by DAPI demonstrate the cellular integrity in the core of the spheroids. Fluorescence of resorufin formed by a reduction of AlamarBlue demonstrates viable cells in the core of both the spheroids.MTT viability assay in 3D culture and 2D culture of the ZEM2S and ZFL cell line is shown here, demonstrating no significant differences. When attempting this procedure, make sure you seal the plates to avoid changing the pH of the culture media and evaporation in the wells. The spheroids can be applied in a variety of in vitro assays to evaluate toxic effects in the fish liver or developmental studies using the embryo ZEM2S cell line.This protocol can help push forward the development of in vitro fish test, contributing to the progress of no-animal-based ecotoxicity studies.

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JoVE Journal

Environment

2.1K visualizações.  Federal University of Paraná (UFPR). Este protocolo pode facilmente gerar esferoides de zebrafish, um modelo 3D in vitro com diferentes potenciais aplicações em testes de toxicidade em peixes. Este é um método simples e rápido para gerar esferoides únicos de linhagens celulares de peixe-zebra muito semelhantes em tamanho e forma. Se você seguir todas as etapas descritas corretamente, não se deve encontrar problemas.Os esferoides do peixe-zebra podem beneficiar muito o mecanismo, a exploração dos efeitos dos prod...