Development and Characterization of Primary Brain Cultures from Japanese Quail Embryos

Materials and reagents

Biological materials

1. Fertilized Japanese quail eggs (Coturnix japonica). The eggs we employ are obtained from our quail colony. Eggs are collected from cages with male and female quails. Nevertheless, there are multiple commercial providers of fertilized quail eggs; however, the number of farms and thereby commercial availability may vary from one country to another.

1. B-27 serum-free supplement (50×) liquid (Gibco, catalog number: 17504-044)

2. Pen/Strep solution (Sartorius, catalog number: 03-031-1B)

3. L-glutamine (GlutaMAX) (Gibco, catalog number: 35050-038)

4. Neurobasal-A medium (Gibco, catalog number: 10888-022)

5. Papain from papaya latex (Sigma-Aldrich, catalog number: 9001-73-4)

6. Phosphate buffered saline (PBS) (Sartorius, catalog number: 02-023-5A)

7. Poly-D-lysine hydrobromide (PDL) (Sigma-Aldrich, catalog number: 27964-99-4)

8. DNase I set (Geneaid, catalog number: DNS300)

9. Sylgard 184 silicone elastomer (Sigma-Aldrich, catalog number: 761036-5EA)

10. DMEM/F-12 (Ham) (Sartorius, catalog number: 01-170-1A)

1. 0.2% PDL (see Recipes)

2. Quail neurobasal medium (QNBM) (see Recipes)

3. Digestion solution (see Recipes)

4. Papain stock (see Recipes)

Recipes

Note: Prepare all solutions in a laminar flow cell culture hood.

1. 0.2% PDL (store at -20 °C)

   Reagent	Final concentration	Amount
   PDL	2 mg/mL	100 mg
   Double-distilled water (DDW)	n/a	50 mL
   Total	n/a	50 mL




2. Quail neurobasal medium (NBM) (store at 4 °C)

   Reagent	Final concentration	Amount
   B-27	2%	5 mL
   Pen/Strep	1%	2.5 mL
   L-glutamine	0.25%	625 µL
   Neurobasal-A medium	n/a	Complete to 250 mL
   Total	n/a	250 mL




3. Digestion solution (prepare on the day of extraction)

   Reagent	Final concentration	Amount
   Papain stock	33 U/mL	1 mL
   PBS	n/a	2 mL
   DNase I	57 U/mL	85 µL
   Total	n/a	3.085 mL




4. Papain stock

   Reagent	Final concentration	Amount
   Papain	10 U/mg	100 mg
   PBS	n/a	10 mL
   Total	n/a	10 mL

Laboratory supplies

1. Spray bottle with 70% ethanol (Bio-Lab Chemicals, catalog number: 000521020500)

2. Tissue culture dish (60 mm × 15 mm) (Corning, catalog number: 430166)

3. Tissue culture dish (100 mm × 20 mm) (Corning, catalog number: 430167)

4. Microtubes, 1.7 mL (Corning, catalog number: MCT-175-C)

5. Conical bottom tubes, 15 ml (Greiner Bio-One, catalog number: 188261)

6. Conical bottom tubes, 50 ml (Greiner Bio-One, catalog number: 227270)

7. Cell strainer 40 μm (LifeGene, catalog number: LG-CSS010040S)

8. Parafilm (Sigma-Aldrich, catalog number: P7543)

9. Delicate task Kimwipes (Kimberly-Clark professional, catalog number: 34120)

10. Minutien Pins, 1 cm, 0.0175 mm tip (Fisher Scientific, catalog number: 2600215)

Equipment

1. Stereomicroscope system (Zeiss Stemi 2000, Fisher Scientific, catalog number: 10331390)

2. Microbiological safety cabinet class II (Thermo Scientific, catalog number: 51025757)

3. Isotemp water bath (PolyScience, catalog number: E12020004)

4. HERAcell 150i CO₂ incubator (Thermo Fisher Scientific, catalog number: 50116047)

5. Gilson (or equivalent) pipettes and tips (P20/P200/P1000) (Fisher Scientific)

6. Dumont #5 forceps (Roboz surgical store, catalog number: RS-4905) (Figure 1, #1)

7. Curved dissecting scissors (Stoelting, catalog number: 52132-11P) (Figure 1, #2)

8. Plastic Pasteur pipette (blunt tip obtained by trimming pipette ~1 cm from tip) (Sigma-Aldrich, catalog number: 747775 (Figure 1, #3)

9. Vannas spring scissors (Fine Science Tools, catalog number: 15000-08) (Figure 1, #4)

10. Iris forceps (Fine Science Tools, catalog number: 11064-07) (Figure 1, #5)

11. Fine-angled forceps (Stoelting, catalog number: 52102-02P) (Figure 1, #6)

12. Egg incubator, 37 °C (DMP Engineering, model: INCA 100)

    
    

    
    Figure 1. Fine surgical tools required for the removal of eggshell and embryo from egg

Procedure

1. PDL plate coating

   Note: Do this in a sterile laminar flow cell culture hood.

   1. Fill 60 mm Corning dishes with 5 mL of 0.2% PDL solution (see Recipes).

   2. Place the plates in a 37 °C incubator overnight.

      Critical: Non-satisfactory coating of the plates can lead to cell dissociation from the surface of the plate and neuronal death causing unhealthy, nonviable cultures.

   3. Collect the PDL solution using a pipette (the solution can be recycled) until plates are empty.

   4. Wash the plates three times using sterile DDW.

   5. Allow residual DDW to evaporate and the plates to dry (leave in the laminar flow cell culture hood).

   6. Seal each plate with parafilm to keep sterile.

   7. Keep coated plates at 4 °C for later use. Plates can be stored for several weeks.




2. Silicone plate coating

   Note: Do this in a sterile laminar flow cell culture hood.

   1. Prepare a 60 mm Corning dish coated with 7 mm silicone, using the Sylgard 184 silicone elastomer kit. The manufacturer’s instructions are included and simple to follow.

   2. Keep overnight at room temperature.




3. Embryo extraction and brain dissection from quail eggs

   1. Obtain fresh fertilized eggs. Fertilized eggs cannot be distinguished from unfertilized ones at this stage (Figure 2A).

      
      

      
      Figure 2. Egg shell removal and embryo extraction from quail eggs. A. Clean quail eggs post-incubation. B. Eggshell penetration. C. Circular eggshell crack, letting the “cap” loose. D. Quail embryo in dish, together with yolk sac and blood vessels. E. Separating embryo from yolk sac and blood vessels using scissors. F. Embryo picked up for transfer with plastic Pasteur pipette.

   
   

   2. Store eggs in a suitable incubator at 37 °C with 70%–80% humidity for 7–9 days.

   3. Spray eggshells with 70% ethanol and wipe dry (gently, using Kimwipes), before placing inside the laminar hood.

      Note: From this point onward, all procedures are performed in the laminar hood.

   4. Using Dumont #5 forceps (Figure 1, #1), gently penetrate the eggshell in its upper quarter (Figure 2B).

   5. Crack the eggshell with a circular forceps movement, letting the eggshell’s “cap” loose (Figure 2C).

   6. Pour egg content into a 100 mm Corning dish.

   7. Using curved dissection scissors (Figure 1, #2), separate the embryo from the yolk sac and cut the interacting blood vessels (Figure 2D, E).

   8. Transfer the embryo with a blunt plastic Pasteur pipette (Figure 1, #3; Figure 2F) into a 100 mm Corning dish filled with DMEM (Figure 3A).

      
      

      
      Figure 3. Embryo forebrain extraction. A. Isolated embryo in a Petri dish, filled with DMEM. B. Embryo decapitation using dissection scissors. C. Silicone-coated Petri dish and fixation pins (dashed box). D. Isolated embryo head in DMEM, pinned to the silicone surface. Inset: closer view of pins (arrowhead). E. Forebrain dissection using forceps and scissors. Inset: closer view of the right forebrain (dashed region). F. One isolated forebrain (without meninges).

   
   

   9. Decapitate the embryo with curved dissection scissors (Figure 3B).

   10. Transfer the head of the embryo using a plastic Pasteur pipette to a 60 mm Petri dish coated with silicone and filled with DMEM.

   11. Immobilized the head using Minutien Pins inserted in each eye (Figure 3C, D) and place under a light microscope.

   12. With the Vannas spring scissors (Figure 1, #4), create a mid-sagittal incision from the nape to the eye line (Figure 3E). Peel the soft tissues overlaying the brain with Iris forceps (Figure 1, #5).

   13. Slip the Vannas spring scissors under the forebrain and gently cut through the tissues that are connected to it (Figure 3E, inset).

   14. Using Iris forceps and fine-angled forceps (Figure 1, #5 & #6, respectively), separate the forebrain from the head.

   15. Clean the two hemispheres gently from residual connective tissue and meninges by pealing the meningeal layers with the Iris and the fine-angled forceps (Figure 1, #5 & #6).

       Critical: Partial pealing and removal of the meninges can cause fibroblast overgrowth and dominate the culture.

   16. Transfer the clean hemispheres to a 35 mm Petri dish filled with 2 cm of DMEM (Figure 3F) using a plastic Pasteur pipette (Figure 1, #3).

   17. Repeat steps C4–16 until the desired number of hemispheres is obtained. See Troubleshooting.




4. Tissue dissociation

   1. Place the isolated forebrains in a 15 mL conical tube containing the digestion solution (3.085 mL; see Recipes) using a plastic Pasteur pipette (Figure 1, #3).

   2. Incubate at 37 °C for 1 h (no shaking is needed).

      Critical: Incubation of cells for less than 1 h may lead to insufficient dissociation of the tissue, which will result in the appearance of cell clumps in the culture, whereas longer incubations (>1 h) may lead to excessive cell (especially neuronal) death.

   3. Gently remove the solution with a 5 mL pipette without disturbing the precipitated forebrains.

   4. Remove the residual solution with a 1,000 μL plastic tip.

   5. Wash the forebrains by adding PBS to the tube. Repeat three times.

   6. After the third wash, remove the PBS completely using a 1,000 μL plastic tip and replace it with 2 mL of quail neuro-basal medium (NBM) (see Recipes).

   7. Manually dissociate the tissue by gentle trituration with a large, 5 mL plastic pipette tip (×15 times).

   8. Then, triturate the solution gently with a smaller 1,000 μL plastic pipette tip until a homogenous solution is obtained (with no visible tissue fragments).

   9. Place a 40 μm cell strainer on a 50 mL conical tube and prewash it with 1 mL of quail NBM.

      Critical: Prewashing the cell strainer prevents cells from sticking to the strainer.

   10. Transfer the entire solution containing the dissociated brains onto the cell strainer placed with a 1,000 μL plastic tip.

   11. After passing the entire solution through the cell strainer, wash the strainer with additional quail NBM to elute the remaining cells from the strainer. The final volume of the filtered (strained) solution should be ~5 mL of quail NBM per one quail’s hemisphere. The optimal density for seeding is 5 × 10⁵ cells/cm². See Troubleshooting.




5. Plating

   1. Transfer 5 mL of strained solution onto a sterile 60 mm tissue culture dish that has been pre-coated with 2 mg/mL PDL.

      Note: Cells do not adhere to glass coverslips even if the coverslips have been pre-coated with PDL. They clump after several hours and die.

   2. Place the seeded plates in a standard biological cell culture incubator at 37 °C and 5% CO₂ for 1 h to ensure cell attachment to the surface of the plates.

      After 1 h, gently remove the medium by aspiration and replace it with fresh and pre-warmed quail NBM.

      Critical: Aggressive handling and removal of the medium can cause cells to detach from the dish.

   3. Place the plates back in the incubator for 72 h.

   4. After 72 h, replace half the medium with fresh and pre-warmed quail NBM.

   5. Repeat step E4 every other day.

   6. Individual cells should be easily discernible in cultures after one day in vitro (Figure 4A and B, left top and bottom panels). Cells should begin sprouting processes within this time frame. Cellular clumps are an indication of a dying culture (Figure 4A, right panel compared to right panel in B).

      
      

      
      Figure 4. Viability of primary cultures grown on glass or plastic after one week in vitro. A, B. Cells grown in vitro at days 1 and 4 post-dissection, on PDL-coated glass coverslips in Petri dishes (A) or directly on the Petri dish (also coated with PDL) (B). The top-right image in A shows a clumping of cells, which does not appear in B. Insets in B show a closer view of the cells’ morphology in culture. Note the development of processes (bottom left) and the appearance of neurons (bottom right). C. MAP2-immunostaining of quail neurons at 14 days in vitro (DIV).

Validation of protocol

This protocol is robust and reproducible. Number of replicates done is >30. Primary neuronal cultures were employed for electrophysiological recordings (patch clamp), calcium imaging, high-throughput viral infection screening, immunohistochemical staining, and single-cell mRNA sequencing experiments (see Figures 1, 2, 3, 4, 6e–g, Suppl. Figure 2 [3]).

General notes and troubleshooting

General notes

This protocol was also found to be suitable for producing primary brain cultures from the forebrains of age-matched chicken embryos (see suppl. Figure 8 in [3]). We therefore assume it may also be suitable for different bird species.

Sources of variability between different cultures may arise from the initial conditions and age of the embryos. Embryos undergoing healthy development are likely to produce more viable cultures, compared to embryos with delayed development. Cultures prepared from poorly developing embryos may yield sparser cultures.

Troubleshooting

Cell density in culture: We recommend dissociating and plating one hemisphere per plate (i.e., one hemisphere per 5 mL of culture medium) (Figure 4A, B; left panels). We noted that this yields favorable conditions for cellular growth, without excessive density of cells. We find that the optimal seeding density for viability of the culture is 5 × 10⁵ cells/cm². To increase cell density, simply increase the number of hemispheres per plate, for instance plating 1.5 hemispheres per plate. For sparser cultures, dilute the cell suspension in a larger volume of medium. These adjustments may offer strategic means to tailor cell density to experimental requirements, thereby influencing cellular interactions, proliferation rates, and experimental outcomes.

Acknowledgments

Funding: Support was provided by the Rappaport Family Thematic grant (S.B. and Y.G.).

The protocol was first described and validated in Zoabi et al. [3]. A custom-made AAV1 variant (AAV1-T593K) enables efficient transduction of Japanese quail neurons in vitro and in vivo. Communications Biology, 6(1): 337.

Competing interests

The authors declare no competing interests.

Ethical considerations

Animal experiments were approved by the Technion Institutional Animal Care and Use Committee (permit no. IL-157-11-17 and IL-19-10-143) and all experiments strictly followed the approved guidelines.

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