Neurosphere culture

Dissect SC from 5cm animals (GFP) Place SC into L15 medium (Gibco Cat. No: 31415-029) Remove meninges and cut SC in smaller pieces Transfer SC into PAP mix: Ovo mix (1:1), total volume 300µl Incubate at RT for 1.0 h Add 1 volume of additional Ovo mix Mix and incubate at RT for 5 minutes Dissociate the cells with a 1ml tip Add the cell suspension to 9ml of DMEM:F12 medium (Gibco Cat. No: 31331-028) at RT Centrifuge 3min at 700rpm Remove supernatant Add 1ml of Neurosphere medium and dissociate cells with 1ml tip Add an additional 3ml of Neurosphere medium and culture in 25ml flask at 25°C 5% CO2

Keep the cells for 7-10 days until Spheres are forming

Papain Mix:

• 30U/ml Papain (Sigma Cat. No: P3125)
• 0.24mg/ml cysteine (Sigma Cat. No: C7477)
• 40µg/ml Dnase I Type IV (Roche Cat. No: 104 159)

Ovomucoid Mix:

• 45mg trypsin inhibitor (Sigma Cat. No: T6522)
• 21mg BSA (Sigma Cat. No: A3294)
• 390µl Dnase I Type IV (Roche Cat. No: 104 159)
• 39ml L15 medium (Gibco Cat. No: 31415-029)

Sterile filtered!!!

Neurosphere Medium:

• DMEM:F12 + Glutamax (Gibco Cat. No: 31331-028)
• PenStrep (1:100)
• B27 Supplement (1:50) (Gibco Cat. No: 17504-044)
• FGF2 (20ng/ml)

Neurosphere transplantation

WT (white) animals around 3-4cm GFP-Neurospheres

anesthetize animals in 0.01% Bencocain remove skin and muscle with a tungsten needle and dissect a piece of SC from the side (Myotom 5 behind cloaca) add a Neurosphere, wait 5min until wound closure put the animal back in water wait for 7days that the NS can integrate in the SC cut the tail around 500µm behind the integrated NS and let the NS regenerate into the SC (6d Regenerate)