Human NP cell isolation and culture

Nucleus pulposus (NP) cell isolation

Cell culture medium:

Material:

Digestion Preparation

Collagenase XI: stock 800 units/mg x 100mg = 80000 U

• for 1500 U/ml dissolve 100mg Collagenase XI in 53,3mL PBS 

Dispase II: stock 0.8 units/mg x 1000mg (1g) = 800 U

• for 2,4 U/mL dissolve 1g in 333,3mL PBS

Medium Production 

1. Mix 250mL DMEM+ GlutaMAX with 250mL Ham’s F12 Nutrient + GlutaMAX
2. Remove 65,5mL from the mixture and store in two labeled 50 mL tubes at 4°C
3. Add 

• 50mL FBS Superior
• 10mL Pen/Strep
• 5mL Amphotericin B 
• 0,5mL L-Ascorbic Acid Phosphate Magnesium Salt (conc. 100 mg/mL)

Cell Isolation

1. Remove the NP with scalpel and forceps from the disc tissue. Cut the NP into small pieces and sprinkle with a few milliliters cell culture medium. 

2. Collect the NP pieces in a 50 mL tube and wash 3x with DPBS. Discard the DPBS. 

3. Add 4 mL Collagenase XI (1500 U/mL), 2 mL Dispase II (2.4 U/mL) and 6 mL cell culture medium.

4. Incubate at 37 °C, 5% CO₂ (and 95% humidity) for 6-8 h (or overnight) while shaking at 250 rpm. 

Cell Culture

5. After 6 h the NP pieces should be digested by the enzymes. Pipette the cell solution onto a cell strainer with 100 µm pore sizes and transfer to a new 50 mL tube. 

6. Centrifuge at 1400 rpm for 7 min at 7°C (Eppendorf 5810R, rotor A-4-81). 

7. Carefully aspirate the supernatant and add 10 mL cell culture medium to the cell pellet. Mix the cell solution and transfer to a 75 cm² cell culture flask. 

8. Culture the cells in an incubator at 37°C, 5% CO₂ and 95% humidity.

9. Change medium 2x per week. 

10. Split the cells at around 80% confluency.

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