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>

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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 and shape to perform an assay, as well as difficulties in the process of transferring spheroids from the drops to microplates in the hanging drop method3,7,12. Further, this protocol recommends the culture of ZFL and ZEM2S cell lines in a CO2-free condition. The culture medium composition and the CO2-free environment proposed in this protocol for both cell lines are widely reported in the literature. The ZFL cell line is usually cultured in L-15 and RPMI media with or without the addition of sodium bicarbonate and without CO214,15,16,17,18,19,20. The ZEM2S cell line is cultured according to instructions of the bioresource center, and its culture media was formulated for CO2-free cultures; thus, CO2 and air mixture can be detrimental to cells when using this type of culture media21.
The present fish spheroid protocol was developed after evaluating several parameters (i.e., different cell densities, speed of orbital shaking [70 or 100 rpm], and the use of different 96-well plates [flat bottom or round-bottom]) to determine the best parameters to form ZFL spheroids. Additionally, a comparison with other 3D culture methods (i.e., hanging drop method and hanging drop method combined with orbital shaking method) was made, indicating that the orbital shaking of round-bottom ULA-plates had the best performance (i.e., easy, fast, and reproducible method). Table 2 shows the parameters and performance of other 3D culture methods evaluated to form ZFL spheroids.
Round-bottom ULA-plates have already been reported to form single spheroids of human cells followed by centrifugation22 or orbital shaking23,24. In this protocol, we are proposing the use of the same plate, except under orbital shaking to generate single spheroids of zebrafish cell lines (i.e., one spheroid per well of a 96-well plate). This is particularly advantageous because it can easily and rapidly form a good quantity of spheroids, and the formed spheroids can be maintained in culture on the same plate. Thus, chemical exposure can be done directly on the ULA-plate to perform different types of toxicity assays.
In order to determine the adequate orbital shaking speed to form ZFL and ZEM2S spheroids, we compared the spheroids&#39; formation at 70 and 100 rpm. The results show that this method performs better at 70 rpm, and a higher rotational speed can significantly impact ZEM2S spheroids&#39; size and shape (Figure 5D,F). ZFL spheroids did not show significant differences in size and shape at these rotational speeds; however, lower circularity indexes were obtained at 100 rpm, showing a negative impact in spheroidal shape at a higher rotational speed (Figure 5C,E).
The initial cell density of 3,500 ZEM2S cells and 7,000 ZFL cells generate spheroids with similar sizes on the fifth day in culture (226.63 and 225.62 &#181;m, respectively). Given that oxygen and nutrients are transported by diffusion in spheroids25, their diameter may strongly influence the nutrition and cellular integrity in spheroids&#39; core; generally, spheroids up to 100 &#181;m are recommended to avoid necrotic core26. To verify the cellular integrity in the spheroids&#39; core, we evaluated the nuclear and membrane integrity by immunostaining with DAPI and Lectin Alexa Fluor 594 by confocal microscopy (Figure 6A,B), and the spheroids&#39; integrity was demonstrated. The cellular viability in the spheroids&#39; core was also verified by exposing them to resazurin (Alamarblue) and analyzing the fluorescence of resorufin by a confocal microscope (Figure 6C,D). When the cells are viable, the resazurin is reduced to resorufin (fluorescent substance) by cellular metabolic function. The performance of the MTT assay also demonstrated no significant difference in cell viability comparing ZFL and ZEM2S spheroids with monolayer culture (2D culture) (Figure 6E,F). Although the spheroids are greater than 100 &#181;m, the results show that this protocol generates viable spheroids of ZFL and ZEM2S cell lines with adequate nutrition even in their inner portion.
The cell densities of ZFL and ZEM2S cells and the speed of orbital shaking applied in this protocol allowed the formation of very stable spheroids in size and shape, with low variability among the wells (Figure 4), avoiding the need to select adequate spheroids as a previous step of performing an assay. If the assay procedure requires transferring the spheroids to other plates or tube/microtubes, they can be easily transferred with a micropipette without disaggregation problems.
Here, we demonstrated a protocol developed based on the best parameters to form viable ZFL and ZEM2S spheroids. The ZFL and ZEM2S cell lines may be useful in aquatic toxicity testing to estimate chemical effects on fish23,24,27,28,29,30. Additionally, developmental endpoints may also be studied using fish embryo cell lines, such as the ZEM2S cell line (&#64257;broblast cells of the blastula phase), which retain a degree of pluripotency28. These make these zebrafish spheroids potentially useful for fish toxicity testing. Furthermore, as an easy fish spheroid method, it can be used in high-throughput ecotoxicity testing, supporting its application in industry and academia.

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><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&#039;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>&amp;nbsp;National&lt;br/&gt; Institutes of Health, USA</td><td></td><td>Available at: https://imagej.nih.gov/ij/index.html</td></tr><tr><td>Leibovitz&#039;s L-15 Medium, powder</td><td>Gibco</td><td>41300021</td><td></td></tr><tr><td>Orbital shaker&amp;nbsp;</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,&amp;nbsp; 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.&lt;br/&gt; 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 line requires 5 days to reach the desirable spheroidal shape (Figure 2A-C). The spheroids generated by this method also showed stability in terms of shape in longer periods of culture (periods &#62;5 days), as demonstrated in Figure 2D (16-day-old ZFL spheroid) and Figure 2H (10-day-old-ZEM2S spheroid). The spheroids&#39; diameter and circularity were determined by measuring their projected area in the obtained images using software for processing and analyzing scientific images13 (Figure 3). The Table of Materials indicates the software used for image processing and analysis. ZFL cells tend to form large cell aggregates (average size 319 &#177; 23.33 &#181;m) on the first day of orbital shaking, which decreases over time until day 5 (225.62 &#177; 19.20 &#181;m) (Figure 4A), enhancing the circularity over time in culture (Figure 4B). Unlike ZFL cells, the ZEM2S cell line forms small spheroids from the first day (202.64 &#177; 5.78 &#181;m), which progressively increase until day 5 (226.63 &#177; 4.80 &#181;m) (Figure 4C). Figure 4E shows the different growth patterns in the ZFL and ZEM2S cell lines in 3D culture. We recommend using the ZFL and ZEM2S spheroids on the fifth day because they reach a higher spheroidal shape (circularity index of 0.80 &#177; 0.01 and 0.83 &#177; 0.00, respectively) (Figure 4B,D), which indicates a greater stability of this 3D model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64859/64859fig01.jpg" /> 
Figure 1: The key steps to generate 3D spheroids of ZFL and ZEM2S cell lines in a round-bottom 96-well plate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64859/64859fig02.jpg" /> 
Figure 2: Single spheroids of ZFL and ZEM2S cell lines formed in round-bottom 96-well ULA-plates under orbital shaking. A ZFL cell aggregate is formed on the first day of OS (A). The cell aggregate increases its circularity on the third day (B) and reaches an adequate spheroidal shape on day 5 (C). (D) A 16-day-old ZFL spheroid in ULA-plate. The ZEM2S cell line forms spheroids from the first day of OS (E). The ZEM2S spheroid maintains its shape and increases in size on the third day (F), and its maximum circularity is reached on day 5 (G). (H) A 10-day-old ZEM2S spheroid in a ULA-plate. Scale bars = 100 &#181;m. Abbreviations: OS = orbital shaking; ULA = ultra-low attachment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64859/64859fig03.jpg" /> 
Figure 3: Determining spheroids&#39; size and circularity using software for processing and analyzing scientific 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 (A). The spheroid&#39;s area is used to determine its size by calculating the diameter d, and the software provides the spheroid&#39;s circularity by a formula that uses the area and perimeter selected (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64859/64859fig04.jpg" /> 
Figure 4:&#160;Reproducibility of the 3D spheroid method showed by uniform-sized spheroids (diameter) and circularity (spheroidal shape). The measured size of spheroids of the ZFL (A) and ZEM2S (B) cell lines. The measured circularity index of ZFL (C) and ZEM2S (D) spheroids. The growth pattern of ZFL and ZEM2S spheroids (E). Data are representative of three technical replicates from different cell batches. The numbers above each point indicate the mean of the triplicates for days 1, 3, and 5. Data showed as mean &#177; standard deviation (n = 10). This figure has been modified from Souza et al.12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64859/64859fig05.jpg" /> 
Figure 5: Comparison between the formation of ZFL and ZEM2S spheroids performed at two rotational speeds (70 and 100 rpm). A ZFL spheroid (A) and ZEM2S spheroid (B) formed after 5 days of orbital shaking at 100 rpm (scale bars represent 100 &#181;m). The growth pattern of ZFL spheroids (C) and ZEM2S spheroids (D) formed at 70 and 100 rpm. The circularity index of ZFL spheroids (E) and ZEM2S spheroids (F) formed at 70 and 100 rpm. Data are shown as mean &#177; standard deviation (n = 10). *indicates statistical significance (p &#60; 0.05). n.s.: non-significant difference (t-test) between spheroids formed on day 5 at 70 and 100 rpm. This figure has been modified from Souza et al.12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64859/64859fig06.jpg" /> 
Figure 6: The cellular integrity and viability of ZFL and ZEM2S spheroids (5 days old) formed by orbital shaking in round-bottom ultra-low attachment well-plate. Staining of cell membranes by Lectin Alexa Fluor 594 (red) and nuclei by DAPI (blue) demonstrate the cellular integrity in the core of a ZEM2S spheroid (A) and ZFL spheroid (B). Fluorescence of resorufin (red) formed by a reduction of Alamarblue demonstrates viable cells in the core of both the ZEM2S (C) and ZFL (D) spheroids. The images represent orthogonal planes captured using a confocal microscope. MTT viability assay in 3D culture (spheroids) and 2D culture of the ZEM2S (E) and ZFL cell line (F). Data showed as mean of absorbance measured at 570 nm &#177; standard deviation. ns: non-significant difference by Welch&#39;s t-test. This figure has been modified from Souza et al. (2021)12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Solutions for ZFL and ZEM2S cultivation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859_Table1.zip" target="_blank">Please click here to download this Table.</a>
Table 2: Comparison of the 3D spheroid methods used to form single ZFL spheroids. * The best result for a tested parameter. a The selected parameter from the best results verified in the conventional hanging drop method. b The selected parameter from&#160;Baron et al.3 for forming a fish spheroid with orbital shaking. c Variations equally suitable to a tested parameter.&#160;Abbreviations: HD = hanging drop; OS = orbital shaking; ULA = ultra-low attachment; poly-HEMA = poly(2-hydroxyethyl methacrylate). This table has been modified from Souza et al.12.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64859/64859_Table 2 (1).xlsx" target="_blank">Please click here to download this Table.</a>

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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Nanyang Technological University

Research

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2.4K 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 Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Bioengineering

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Developmental Biology

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Cancer Research

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Biochemistry

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Biology

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.