Name: a 3d culture method of spheroids of embryonic and liver zebrafish cell lines
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Description: Watch this Scientific Journal Video about A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines at JoVE.com

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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+more+representative+in+vitro+method+for+fish+ecotoxicology:+morphological+and+biochemical+characterisation+of+three-dimensional+spheroidal+hepatocytes.">Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes.</a> Ecotoxicology. 21 (8), 2419-2429 (2012).</li><li>Alves, R. F., Rocha, E., Madureira, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+hepatocyte+spheroids+-+A+powerful+(though+underexplored)+alternative+in+vitro+model+to+study+hepatotoxicity.">Fish hepatocyte spheroids - A powerful (though underexplored) alternative in vitro model to study hepatotoxicity.</a> Comparative Biochemistry and Physiology. Toxicology &amp; Pharmacology. 262, 109470 (2022).</li><li>Baron, M. 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=Pharmaceutical+metabolism+in+fish:+using+a+3-D+hepatic+in+vitro+model+to+assess+clearance.">Pharmaceutical metabolism in fish: using a 3-D hepatic in vitro model to assess clearance.</a> PloS One. 12 (1), 0168837 (2017).</li><li>Langan, L. 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. S., 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=ABC+proteins+activity+and+cytotoxicity+in+zebrafish+hepatocytes+exposed+to+triclosan.">ABC proteins activity and cytotoxicity in zebrafish hepatocytes exposed to triclosan.</a> Environmental Pollution. 271, 116368 (2021).</li><li>da Silva, N. D. 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. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocadherin-19+and+N-cadherin+interact+to+control+cell+movements+during+anterior+neurulation.">Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation.</a> The Journal of Cell Biology. 191 (5), 1029-1041 (2010).</li><li>Bradford, C. S., Sun, L., Collodi, P., Barnes, D. 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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 ...

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