A Rapid and Simple Procedure for the Isolation of Embryonic Cells from Fish Eggs

Materials and reagents

Biological materials

Fertilized fish eggs in the stages from blastodisc to the late gastrula.

Solutions

1. PBS soluble tablets (Sigma-Aldrich, catalog number: P4417), for cell isolation

2. 70% ethanol solution (Sigma-Aldrich, catalog number: 65348-85), for cell preservation

3. o-safranin ready-to-use solution 0.1% (Scientific Laboratory Supplies, catalog number: TMS-009-C), for protocol verification

4. Propidium iodide ready-to-use solution 1 mg/mL (Thermo Fisher Scientific, catalog number: P3566), for protocol verification

5. RNAse A ready-to-use solution 10 mg/mL (Thermo Fisher Scientific, catalog number: EN0531)

6. Phosphate-saline buffer solution (see Recipes)

7. 70% ethanol (see Recipes)

Recipes

1. Phosphate-saline buffer solution

   Reagent	Final concentration	Quantity
   PBS	150 mM total of all salts: 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4	1 tablet
   H2O	n/a	Up to 100 mL
   Total	n/a	100 mL




2. 70% ethanol

   Reagent	Final concentration	Quantity
   Ethanol (95%)	70%	74 mL
   H2O	n/a	26 mL
   Total	n/a	100 mL

Laboratory supplies

1. Microcentrifuge tube pestles (Sigma-Aldrich, catalog number: BAF199230001-100EA)

2. Conical bottom microcentrifuge tubes (Sigma-Aldrich, catalog number: HS4325-1000EA)

3. Centrifuge tubes (Sigma-Aldrich, catalog number: CLS430790)

Equipment

1. Microcentrifuge (Thermo Fisher Scientific, catalog number: 75004081)

2. Microscope with 8–20× zoom lens for data validation (Thermo Fisher Scientific, catalog number: 4479672)

3. Vortex mixer (VELP Scientifica, catalog number: F202A0173)

4. Dissecting needles (Thermo Fisher Scientific, catalog number: 13-820-024)

5. Flow cytometer for data validation (Beckman Coulter, catalog number: CO9752)

Software and datasets

1. CytExpert software for flow cytometry (used for data validation)

Procedure

1. This protocol has been tested on fertilized nelma eggs of medium size (3–4 mm in diameter) among teleost fish (including zebrafish). Before the procedure, rinse the eggs with PBS: place the eggs in a centrifuge tube (15 mL) and pour 10–20 mL of cool PBS solution into it. Drain the liquid and repeat this step to prevent any contamination that could affect further analysis. This step can also be done in any way available in the laboratory: using a tea strainer, beakers, gauze, etc.

2. Using a pestle, pop the eggs (or single egg) in the tube. It is better to add eggs and process them gradually. Do not homogenize nor strongly push the pestle to avoid damage to the cells: it is enough to break the shell. The sample volume from the processed eggs should not exceed 2/3 of the tube. A 1.5 mL centrifuge tube can be fitted with a different number of eggs, depending on their size. Thus, approximately 30 eggs of 3–4 mm diameter can be sequentially placed in the tube during the process.

3. Fill the tube with eggs with cold PBS and shake it on a vortex for 5 s or mechanically using a pestle or dissecting needle to release the contents of all eggs outside the visible shell.

4. Mechanically remove all shells from the tube using a dissecting needle. It is very important to ensure that there are no residual shells in the resulting translucent medium to avoid cell loss.

5. Centrifuge at 300–500× g for 5 min at 4 °C.

6. Discard the supernatant without affecting the precipitate. Add 1–1.5 mL of cold PBS and centrifuge again.

7. (Optional) Repeat step 6 1–2 times to wash the cells out of small debris and resuspend the pellet in 100 μL of cold PBS.

   Following these steps, we obtain a pellet containing the cells. The volume of PBS can be changed as desired (50–500 μL). This suspension contains a certain amount of small suspended non-cellular particles. After the procedure, the total proportion of cells among other floating non-cellular particles in the suspension is at least 10%.

   The cell suspension is ready to use for any analysis. It can also be fixed for long-term cell storage using standard protocols such as using chilled ethanol (Ciancio et al., 1988).

Data analysis

Flow cytometry and microscopy were used for protocol verification. The CytExpert software was used for all cytometry data.

Validation of protocol

Maturating nelma eggs (Stenodus leucichthys nelma) were used to validate the protocol (Figure 1A).

Figure 1. Maturating Stenodus leucichthys nelma egg. (A) White arrow shows the position of the blastodisc. (B) Isolated cells from the blastodisc stained with O-safranin.

Thirty fish eggs were processed by the protocol described above. Then, the cell-containing pellet was resuspended in 300 μL of PBS and fixated in 70% ethanol (Ciancio et al., 1988) to ensure long-term preservation prior to analysis. A small 10 μL aliquot of the obtained suspension was stained with safranin (Swain and De, 1990). The suspension was air-dried at room temperature on a glass slide causing the cells to adhere to it. Then, the slide was immersed in a solution of safranin for 5 min and washed one time with PBS. The result was checked using a light microscope (Figure 1B ).

In order to verify the results and additionally show the real presence of isolated cells, an alcohol suspension of the obtained cells (300 μL) was stained with propidium iodide (Riccardi and Nicoletti, 2006). The alcohol was washed off the cells twice with PBS by centrifugation at 300× g for 5 min; cells were then resuspended in 100 μL of PBS (+0.1 μg/mL RNase A and 0.01 μg/mL propidium iodide). After 30 min of incubation in the dark at 4 °C, stained cells were analyzed using a flow cytometer (channel PE) (Figure 2). The flow cytometer analyzed 500,000 events. Using a software interface, side scatter, and forward scatter data, only single cells were selected for analysis. The histogram along the PE channel clearly showed peaks corresponding to the cell cycle of isolated cells (Figure 2A). The vertical line on the dot plot (Figure 2B) showed the position corresponding to the G1 peak of the cell cycle in the histogram (Figure 2A). On the dot plot diagram (Figure 2B) for the same channel, there is size cell dispersion (y-axis) that corresponds to the stage of embryogenesis (small cell blastula) where cells acquire different sizes (Figure 2B). According to our data, approximately 100,000 cells were isolated (Figure 2B) from 30 eggs (Figure 1A), which was quite commensurate with other known methods.

Figure 2. Results of flow cytofluorometry (PE channel). Cell cycle histogram (A) and dot plot (B) showing the heterogeneity of the cell analyzed from small cell blastula.

Thus, the protocol was tested in two independent ways: visual (microscopic) and photometric. The procedure efficiency for cell isolation from fish egg blastodiscs was proved. The protocol can be applied to most teleost fish species with middle-sized eggs at early maturating stages (blastula, gastrula); this protocol may be suitable for other representatives of teleost fish with different diameters of eggs [large (salmon) or small (zebrafish)].

General notes and troubleshooting

General notes

Before you start: to isolate cells from the blastodisc, it is enough to have developing eggs at the stage of large cell blastula. Based on specific goals, it is important to mind the number of cells needed for analysis. If it is necessary to analyze each egg individually, it is important to know the total number of required cells in the developing egg. For examining a specific number of eggs (if general statistics on the target analysis is needed), the stage of development of the eggs is not so important.

Troubleshooting

No cells after procedure:

1. Take more eggs for processing and repeat the procedure.

2. Ensure that you have taken the developing eggs. An embryo should be visible under a light microscope.

3. Remember that in the first step of the isolation protocol, after rinsing the eggs, it is sufficient to gently pop the eggs. Do not completely homogenize.

Acknowledgments

This study was supported by the RSF grant 23-26-00257.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

1. Ciancio, G., Pollack, A., Taupier, M. A., Block, N. L. and Irvin, G. L. (1988). Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation. J. Histochem. Cytochem. 36(9): 1147–1152.

2. Durkin, M., Qian, X., Popescu, N. and Lowy, D. (2013). Isolation of Mouse Embryo Fibroblasts. Bio Protoc 3(18): e908.
3. Fetherman, E. R., Lepak, J. M., Brown, B. L. and Harris, D. J. (2015). Optimizing Time of Initiation for Triploid Walleye Production Using Pressure Shock Treatment. North Am. J. Aquacult. 77(4): 471–477.
4. Riccardi, C. and Nicoletti, I. (2006). Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1(3): 1458–1461.
5. Rieger, S. (2019). Dechorionation of zebrafish embryos with Pronase for metronidazole-mediated ß-cell ablation V.2. DiaComp Protocols.
6. Swain, D. and De, D. N. (1990). Differential Staining of the Cell Cycle of Plant Cells Using Safranin and Indigo-Picrocarmine. Stain Technol. 65(4): 197–204.