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

This protocol can easily generate zebrafish spheroids, a 3D in vitro model with different potential application in fish toxicity testing. This is a simple and fast method to generate single spheroids of zebrafish cell lines very similar in size and shape. If you follow all the described steps correctly, one should not encounter any problems.

Zebrafish spheroids provides may greatly benefit the mechanism, exploration of chemicals'effects on fish, which helps to improve the environmental safety assessment of products such as cosmetics. Demonstrating the procedure will be Irisdoris Rodrigues de Souza, a doctor in genetics from my laboratory. To begin, take a T75 flask of zebrafish cells at around 80%confluence.

Remove the complete medium, and wash the cells by adding 0.01 molar PBS to the culture flask with a pipet. Wash the culture flask with PBS by gently shaking the flask and then remove the PBS. Add three milliliters of trypsin 0.5 millimolar EDTA to the culture flask, and incubate at 28 degrees Celsius for three minutes.

For the detachment of cells from the flask, gently tap the flask to release the cells. And then stop the trypsin digestion by adding three milliliters of complete culture medium to the flask. Using a pipet, transfer the cell suspension to a 15 milliliter conical centrifuge tube and centrifuge at 100 G for five minutes.

Then carefully remove the supernatant. Add one milliliter of the complete medium for the respective cell line in use and resuspend the pellet using a micropipet. Add 10 microliters of the cell suspension and 10 microliters of Trypan blue dye to a micro tube to count the cells and evaluate their viability.

Mix the cell suspension and dye using a pipet, and transfer 10 microliters of this mixture to a Neubauer chamber. Count the cells in the four large squares or quadrants placed at the corners of the chamber, considering cells that do not take up Trypan blue as viable. Then calculate the number of viable cells using the equation shown on the screen.

Multiply this cell number by two to calculate the final cell number in the cell suspension. After calculating the cell number, adjust the cell suspension to plate 200 microliters of this suspension per well of a 96-well round bottom ULA plate with the number of cells required for each cell line. Prepare the suspension with the adjusted concentration of cells in a medium reservoir and mix it using a multi-channel micropipet without forming foam or bubbles.

Add 200 microliters of the adjusted cell suspension to each well of the round bottom ULA plate. using a multi-channel pipette. The plate must be sealed with Parafilm or adhesive sealing foil to avoid culture medium evaporation from the 96-well plate.

Incubate the plate on an orbital shaker at 70 RPM for five days in a 28 degrees Celsius incubator without carbon dioxide. After five days, observe the spheroids under an inverted light microscope to evaluate the generated spheroids. To measure their shape and size, obtain pictures of the spheroids, and use software to process and analyze the images.

After setting the software to measure a selected area based on a known scale, select the outer side of the spheroid to determine its area and circularity. The spheroid's area is used to determine its size by calculating the diameter, d, and the software provides the spheroid's circularity by a formula that uses the area and perimeter selected. A ZFL cell aggregate is formed on the first day of orbital shaking.

The cell aggregate increases its circularity on the third day, and reaches an adequate spheroidal shape on day five. A 16-day-old ZFL spheroid in ULA plate is shown here. The ZEM2S cell line forms spheroids from the first day of orbital shaking.

The spheroid maintains its shape and increases in size on the third day, and its maximum circularity is reached on day five. A 10-day-old ZEM2S spheroid in a ULA plate is shown here. The measured size of spheroids of the ZFL and ZEM2S cell lines and the measured circularity index of ZFL and ZEM2S spheroids are shown in this figure.

This graphical image represents the growth pattern of ZFL and ZEM2S spheroids. A ZFL spheroid and ZEM2S spheroid formed after five days of orbital shaking at 100 RPM are shown here. The growth pattern and circularity index of the spheroids formed at 70 and 100 RPM rotational speeds are presented in this figure.

70 RPM is the most adequate speed to form ZFL and ZEM2S spheroids by this method. Staining of cell membranes by Lectin Alexa Fluor 594 and nuclei by DAPI demonstrate the cellular integrity in the core of the spheroids. Fluorescence of resorufin formed by a reduction of AlamarBlue demonstrates viable cells in the core of both the spheroids.

MTT viability assay in 3D culture and 2D culture of the ZEM2S and ZFL cell line is shown here, demonstrating no significant differences. When attempting this procedure, make sure you seal the plates to avoid changing the pH of the culture media and evaporation in the wells. The spheroids can be applied in a variety of in vitro assays to evaluate toxic effects in the fish liver or developmental studies using the embryo ZEM2S cell line.

This protocol can help push forward the development of in vitro fish test, contributing to the progress of no-animal-based ecotoxicity studies.