Materials and Reagents

Pipette tips (RIKAKEN, catalog numbers: RST 481SCV, RST 4820YV, RST 4810BV)
1.5 ml tube (WATSON, catalog number: 131-815C)
Cell strainer (Corning, catalog number: 352235)
4-well cell culture plate (Thermo Fisher Scientific, catalog number: 176740)
Syringe-driven filter unit (Millipore, catalog number: SLGV033RS)
Filter paper (ADVANTEC, catalog number: 00011090)
E12.5 Embryo mice (C57BL/6J)
D-MEM/Ham's F-12 (Wako, catalog number: 048-29785)
Dispase (Gibco, catalog number: 17105-041)
Collagenase (Wako, catalog number: 034-22363)
FBS (Sigma, NRE-172012)
L(+)-Ascorbic Acid (Wako, catalog number: 016-04805)
Paraformaldehyde (PFA) (Wako, catalog number: 162-16065)
Sodium Hydroxide (Wako, catalog number: 198-13765)
Acetic acid (Wako, catalog number: 017-00251)
10x PBS(-) (Wako, catalog number: 314-90185)
Alcian Blue 8GX powder (Sigma, catalog number: A5268-25G)
Enzyme solution (see Recipes)
L(+)-Ascorbic acid stock solution (see Recipes)
Chondrogenic medium (see Recipes)
8% PFA stock solution (see Recipes)
1% acetic acid solution (see Recipes)
Alcian blue solution (see Recipes)

Equipment

Pipettes (Glison, catalog numbers: F144802, F123615, F123602)
Microtube rotator (AS ONE, catalog number: MTR-103)
CO₂ incubator (SANYO, model: MCO-17AIC)
High speed refrigerated microcentrifuge (TOMY, model: MX-307)
Automated cell counter (Bio-Rad, model: TC-20)
Microscope (Kyence, model: BZ-9000)

Software

BZ-2 application (Keyence)
ImageJ (NIH, https://imagej.nih.gov/ij/)

Procedure

Figure 1. Summary of experimental procedure. In brief: A-C. Dissect the limb buds from embryonic mice. D-E. Digested them with enzymes (dispase and collagenase). F-I. Seed the cells at high density. J. Add the chondrogenic medium and incubate for 6 days. K. Alcian blue staining.

Preparation of the cells
1. Dissect the limb buds from mouse embryonic day 12.5 (the part surrounded by circle in Figure 1B). One limb bud is enough to induce 2 or 3 wells of cartilage (Figures 1A-1C).
2. Collect the limb buds in a 1.5 ml tube and add 1 ml enzyme solution (0.1% Dispase, 0.1% Collagenase in D-MEM/Ham's F-12) (Figure 1D).
3. Rotate the tube at 60 rpm on a microtube rotator (AS ONE) for 1.5 h at 37 °C (Figure 1E).
4. Centrifuge the tube for 5 min at 300 x g and resuspend the cells in 1 ml D-MEM/Ham's F-12 (Figure 1F).
5. Apply the cell suspension to a 40 μm cell strainer placed in a 5 ml round bottom tube and measure the cell density using an automated cell counter (Bio-Rad) (Figure 1G).
Cartilage formation
1. Centrifuge the tube for 5 min at 300 x g and resuspend the cells in D-MEM/Ham's F-12 containing 10% FBS to adjust the cell density to 1.5 x 10⁷ cells/ml (Figure 1H).
2. Place a 10 μl droplet in 4-well culture plate and incubate the cells in the CO₂ incubator for 1.5 h (Figure 1I).
3. Add the 500 μl chondrogenic medium (D-MEM/Ham's F-12 containing 10% FBS and 50 μg/ml L-ascorbic acid) with care not to disrupt the droplet and incubate in the CO₂ incubator (Figure 1J).
4. Change the medium every 2 days and incubate for 6 days.
Alcian blue staining (Figure 1K)
1. Fix the cells in 500 μl 4% PFA for 10 min and wash the cells twice using Milli-Q water.
2. Add 500 μl 1% acetic acid for 5 min and replace acetic acid by 500 μl Alcian blue solution for 30 min.
3. Wash the cells with 500 μl 1% acetic acid and wash twice using Milli-Q water.
4. Image the stained cells using optical microscope (Figure 2).
  
  Figure 2. Alcian blue staining

Data analysis

Alcian blue stained areas were measured with ImageJ software (Abramoff et al., 2004). Appropriate statistical analysis should be carried out on the obtained data.

Recipes

Enzyme solution
Dissolve the dispase (Gibco) and collagenase (Wako) (0.1% w/v each) in D-MEM/Ham's F-12 (Wako) and filter with a 0.22 μm syringe filter (Millipore)
L(+)-Ascorbic acid stock solution
Dissolve the ascorbic acid (Wako) (50 mg/ml) in PBS(-) and filter with syringe filter (Millipore)
Chondrogenic medium
D-MEM/Ham's F-12 (Wako) containing 10% FBS (Sigma) and 50 μg/ml L-ascorbic acid
8% PFA stock solution
Add the 40 g of PFA (Wako) in 400 ml of hot Milli-Q water (60 °C) and add 50 μl of 10 N NaOH (Wako), continue to stir until PFA dissolves
Add 50 ml of 10x PBS(-) and adjust to final volume of 500 ml
1% acetic acid solution
Add 1 ml of acetic acid (Wako) to 99 ml of Milli-Q water
Alcian blue solution
Dissolve 1 g of Alcian blue 8GX (Sigma) in 100 ml of 3% acetic acid solution (Wako) and filter with filter paper (ADVANTEC)

Acknowledgments

This work was supported in part by the Japan Society for the Promotion of Science (16H05131 and 17KT0051 to E.H.), the Japan Agency for Medical Research and Development (17824969 to E.H.).

Competing interests

The authors declare no conflict of interest.

Ethics

The study protocol meets the guidelines of the Japanese Pharmacological Society and was approved by the Committee for Ethical Use of Experimental Animals at Kanazawa University, Kanazawa, Japan.

References

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