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 monolayers in an incubator at 28 &#176;C without CO2, and subculture them at a subcultivation ratio of 1:3 when they reach ~80% confluence.
	<ol>
		<li>Start with a T75 flask of zebrafish cells at ~80% confluence, cultured as described above.</li>
		<li>Remove the complete medium and wash the cells by adding 1x phosphate-buffered saline (PBS) (0.01 M) to the culture flask with the aid of a pipette.</li>
		<li>With the aid of a pipette, add 3 mL of 1x trypsin-0.5 mM EDTA (0.05% [v/v]) to the culture flasks, and incubate at 28 &#176;C for 3 min for the detachment of cells from the flask.</li>
		<li>Gently tap the flask to release the cells, and then stop the trypsin digestion by adding 3 mL of complete culture medium to the flask.</li>
		<li>Using a pipette, transfer the cell suspension to a 15 mL conical centrifuge tube, and centrifuge at 100 &#215; g for 5 min.</li>
		<li>After the pellet formation, carefully remove the supernatant, add 1 mL of the complete medium for the respective cell line in use (ZFL or ZEM2S), and resuspend using a micropipette. Take an aliquot for cell counting.</li>
	</ol>
	</li>
</ol>
2. Cell counting with trypan blue dye exclusion test
<ol>
	<li>Add 10 &#181;L of the cell suspension and 10 &#181;L of trypan blue dye to a microtube to count the cells and evaluate their viability. Mix the cell suspension and dye using a pipette.</li>
	<li>Then, transfer 10 &#181;L of this mixture (cell suspension + trypan blue) to a Neubauer chamber and count the cells in the four large squares (quadrants: Q) placed at the corners of the chamber, considering cells that do not take up trypan blue as viable. Calculate the number of viable cells using equation (1): 
	<img alt="Equation 1" src="/files/ftp_upload/64859/64859eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1)</li>
	<li>To calculate the final cell number in the cell suspension, multiply the cell number determined using equation (1) by 2 (the dilution factor due to the use of trypan blue). 
	NOTE: Alternatively, an automated cell counting system can be used.</li>
</ol>
3. Cell plating in ULA-plates
<ol>
	<li>After calculating the cell number, adjust the cell suspension to plate 200 &#181;L of this suspension per well of a 96-well round-bottom ULA-plate with the number of cells required for each cell line, as indicated below:
	<ol>
		<li>Plate 7,000 viable ZFL cells/well; thus, for the entire ULA-plate, use 700,000 cells in 20 mL of complete medium.</li>
		<li>Plate 3,500 viable ZEM2S cells/well; thus, for the entire ULA-plate, use 350,000 cells in 20 mL of complete medium.</li>
	</ol>
	</li>
	<li>Prepare the cell suspension with the adjusted concentration of cells in a medium reservoir and mix it using a multichannel micropipette, taking care not to form foam or bubbles. Using the multichannel micropipette, add 200 &#181;L of the adjusted cell suspension to each well of the ULA-plate. 
	NOTE: The plate must be sealed with parafilm or adhesive sealing foil to avoid culture medium evaporation from the 96-well plate.</li>
</ol>
4. Spheroid formation
<ol>
	<li>Incubate the ULA-plate on an orbital shaker at 70 rpm for 5 days in a 28 &#176;C incubator. Allow the spheroids to form over 5 days of orbital shaking (Figure 2), reaching an average size of ~225 &#181;m in diameter (ZFL spheroids) and ~226 &#181;m in diameter (ZEM2S spheroids)12. 
	NOTE: After 5 days of incubation (maximum circularity), the spheroids are ready to be used.</li>
	<li>To maintain the spheroids in culture for more than 5 days, remove 100 &#181;L of the spent medium every 5 days, and add 100 &#181;L of fresh complete culture medium using a multichannel micropipette. 
	&#8203;NOTE: Take care not to aspirate the spheroids during this process.</li>
</ol>
5. Measuring size (diameter) and shape (circularity index) of spheroids
<ol>
	<li>Acquire the images.
	<ol>
		<li>Under an inverted light microscope with an imaging capture system, obtain an image of a defined scale. 
		NOTE: Use a microscope stage calibration slide or a Neubauer slide (in which the quadrant sizes are known) to obtain the scale.</li>
		<li>Under the microscope and using the same objective lens used to obtain the scale&#39;s picture, obtain images of the fully formed spheroids (i.e., 5-day-old spheroids). 
		NOTE: All images must be taken using the same imaging capture system, as image resolution is important to determine the spheroids&#39; size and shape, and it may differ among types of systems.</li>
	</ol>
	</li>
	<li>Set the scale.
	<ol>
		<li>Using ImageJ software, open the image of the defined scale (click File | open).</li>
		<li>Select the straight-line selector from the toolbar and, using the mouse, drag out a line across the extension of the defined scale in the image.</li>
		<li>Set the scale by selecting Analyze | Set scale, and wait for the Set Scale window to open.</li>
		<li>In the Set Scale window, fill the blank of Known distance with the known distance (&#181;m) corresponding to the straight line; fill the Unit of length with um for &#181;m. Click OK. 
		&#8203;NOTE: The scale in pixels/&#181;m is displayed at the bottom of the window.</li>
	</ol>
	</li>
	<li>Set the measurement parameters.
	<ol>
		<li>In the ImageJ software, select Analyze | Set measurements to open the Set measurements window.</li>
		<li>In the Set measurements window, select the boxes for the desired measurements (i.e., Area and Shape descriptors). Click OK.</li>
	</ol>
	</li>
	<li>Obtain the spheroids&#39; diameter and circularity.
	<ol>
		<li>Open the picture of a spheroid (File | Open).</li>
		<li>Select the freehand selection tool in the toolbar and, using the mouse, select the outer side of the spheroid, as demonstrated in Figure 3A. 
		NOTE: To zoom in or out on the image, press the Ctrl key and use the mouse to scroll down or up, or press the Ctrl key and use the up or down arrow keys on the keyboard.</li>
		<li>Select Analyze | Measure to open the Results window, in which the measured values are displayed.</li>
		<li>Using the Area value, calculate the spheroids&#39; size (diameter) using equation (2): 
		<img alt="Equation 2" src="/files/ftp_upload/64859/64859eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;(2)</li>
		<li>The circularity index is given in the Results window as &#34;Circ.&#34;, and is automatically calculated by the software using equation (3): 
		<img alt="Equation 3" src="/files/ftp_upload/64859/64859eq03.jpg" style="margin: auto;" />&#160; &#160; &#160;(3) 
		NOTE: The circularity index of 1.0 represents a perfect spheroidal shape, while a value close to 0.0 indicates an elongated shape13.</li>
	</ol>
	</li>
</ol>

<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. 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. C., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+mass+transfer+in+hepatocyte+spheroids+via+cell+viability,+spheroid+size,+and+hepatocellular+functions.">Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions.</a> Biotechnology and Bioengineering. 86 (6), 672-680 (2004).</li><li>Ho, R. K., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commitment+of+cell+fate+in+the+early+zebrafish+embryo.">Commitment of cell fate in the early zebrafish embryo.</a> Science. 261 (5117), 109-111 (1993).</li><li>Biswas, S., Emond, M. R., Jontes, J. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cultures+from+zebrafish+embryos+and+adult+tissues.">Cell cultures from zebrafish embryos and adult tissues.</a> Journal of Tissue Culture Methods. 16 (2), 99-107 (1994).</li><li>He, 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=Genetic+and+transcriptome+characterization+of+model+zebrafish+cell+lines.">Genetic and transcriptome characterization of model zebrafish cell lines.</a> Zebrafish. 3 (4), 441-453 (2006).</li></ol>

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 ...

a-3d-culture-method-spheroids-embryonic-liver-zebrafish-cell

Research

JoVE Journal

Environment

2.3K Views.  Federal University of Paraná (UFPR). 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).

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

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Alternative Method of Removing Otoliths from Sturgeon

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a transverse vertical incision on the top of the skull with a hand or electrical saw may damage the otolith if not performed correctly. Sturgeons (Acipenseridae) are one family in particular that have a very large and thick skull. A new laboratory method entering the brain cavity from the ventral side of the fish to expose the otoliths was easier than other otolith extraction methods found in the literature. Methods reviewed in the literature are designed for the field and are more efficient at processing large quantities of fish quickly. However, this new technique was designed to be more suited for a laboratory setting when time is not pressing and successful extraction from each specimen is critical. The success of finding and removing otoliths using this technique is very high and does not compromise the structure in any manner. This alternative technique is applicable to other similar fish species for extracting the otoliths.

The goal of the protocol is to show an effective method to extract the otoliths from sturgeon carcasses.

Extracting the otoliths (ear bones) from fish that have very thick skulls can be difficult and very time consuming. The common practice of making a ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Tracking Infiltration Front Depth Using Time-lapse Multi-offset Gathers Collected with Array Antenna Ground Penetrating Radar

A Ground Penetrating Radar (GPR) system based on a ground-coupled, densely populated antenna array was used to collect data during an infiltration experiment conducted at a test site near the Tottori Sand Dune, Japan. The antenna array used in this study consists of 10 transmitting antennas (Tx) and 11 receiving antennas (Rx). For this experiment, the system was configured to use all possible Tx-Rx pairings, resulting in a Multi-Offset Gather (MOG) consisting of 110 Tx-Rx combinations. The array was left stationary at a position directly above the infiltration area and data were collected every 1.5 seconds using a time-based trigger. Common-Offset Gather (COG) and Common Mid-Point (CMP) data cubes were reconstructed from the MOG data during post-processing. There have been few studies that used time-lapse CMP data to estimate changes in velocity of propagation. In this study, electromagnetic (EM) wave velocity was estimated heuristically at 1-minute intervals from the reconstructed CMP data through curve fitting, using the hyperbola equation. We then proceeded to calculate the depth of the wetting front. The evolution of the wetting front over time obtain through this method is consistent with the observations from a soil moisture sensor which was placed at a depth below 20 cm. The results obtained in this study demonstrate the ability of such array GPR system to monitor a subsurface dynamic process like water infiltration accurately and quantitatively.

Here we present a Ground Penetrating Radar (GPR) system based on a ground-coupled, densely populated antenna array for monitoring the dynamic process of subsurface water infiltration. A time-lapse radar image of the infiltration process allowed estimating the depth of the wetting front during the course of the infiltration process.

A Ground Penetrating Radar (GPR) system based on a ground-coupled, densely populated antenna array was used to collect data during an infiltration ...

On the Preparation and Testing of Fuel Cell Catalysts Using the Thin Film Rotating Disk Electrode Method

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a high surface area carbon, and to test their performance in thin film rotating disk electrode (TF-RDE) measurements. The TF-RDE methodology is widely used for catalyst screening; nevertheless, the measured performance sometimes considerably differs among research groups. These uncertainties impede the advancement of new catalyst materials and, consequently, several authors discussed possible best practice methods and the importance of benchmarking.
The visual tutorial highlights possible pitfalls in the TF-RDE testing of Pt/C catalysts. A synthesis and testing protocol to assess standard Pt/C catalysts is introduced that can be used together with polycrystalline Pt disks as benchmark catalysts. In particular, this study highlights how the properties of the catalyst film on the glassy carbon (GC) electrode influence the measured performance in TF-RDE testing. To obtain thin, homogeneous catalyst films, not only the catalyst preparation, but also the ink deposition and drying procedures are essential. It is demonstrated that an adjustment of the ink&#39;s pH might be necessary, and how simple control measurements can be used to check film quality. Once reproducible TF-RDE measurements are obtained, determining the Pt loading on the catalyst support (expressed as Pt wt%) and the electrochemical surface area is necessary to normalize the determined reaction rates to either surface area or Pt mass. For the surface area determination, so-called CO stripping, or the determination of the hydrogen underpotential deposition (Hupd) charge, are standard. For the determination of the Pt loading, a straightforward and cheap procedure using digestion in aqua regia with subsequent conversion of Pt(IV) to Pt(II) and UV-vis measurements is introduced.

Preparing and testing Pt/C fuel cell catalysts is subject to continuous discussion in the scientific community with respect to reproducibility and best practice. With the presented work, we intend to present a step-by-step tutorial to make and test Pt/C catalysts, which can serve as benchmark for novel catalyst systems.

We present a step-by-step tutorial to prepare proton exchange membrane fuel cell (PEMFC) catalysts, consisting of Pt nanoparticles (NPs) supported on a ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...