Adhesive cell culture

Material

ECM coating and composition of culture media These steps for preparation were carried out under sterile terms in cell culture room at clean bench.

• Gelatine:

Gelatine (Sigma, Porcine Skin, 300 blom) 500 ml ddH2O

First 3.75 g gelatine was weight and put into a cell culture bottle. Then 500 ml ddH2O were added, mixed and heated to 65° C for 15min until it is complete dissolved. Afterwards the solution was sterile filtered into a 500 ml bottle, aliquot in 40 ml tubes and stored at 4° C. For gelatine coating heating up the gelatine until it is liquid and add 250µl in in vitro fertilization dishes (Corning) and after 2 min remove the gelatine with vacuum pump to get a small film on the plastic surface. Drying time is 30 min at room temperature. Now the coated wells were used for adhesive cell culture.

• Washing Media: DMEM:F12 Glutamax

10µl/ml Glutamin 10µl/ml Pen/Strep

• Culturing Media: DMEM:F12 Glutamax

10µl/ml Glutamin 10µl/ml Pen/Strep 20µl/ml B27 (50x) 20ng/ml FGF2 (20µg/ml) 100ng/ml Shh (Curis)

• 0.03% Benzocaine: 10x TBS

400% Holt fredter 10% Benzocaine di water

Procedure

Spinal cord excision from axolotl tail

The experiments were performed on 6–8cm big wild type animals (d/d) under stereo microscope. All animals were anesthetized in 0.03% ethyl-p-aminobenzoate. Then 1–1.5 cm from the tails were amputated by one single cut with scalpel and blasted in 70% Ethanol for 10s and afterwards two washing steps in PBS/PenStrep. The tail was sliced open from the dorsal side until the region of the spinal cord with tungsten needles and the spinal cord was removed over the length of several segments and kept in Washing Media. Then the spinal cord was separated in circa 500 µm pieces and put into in vitro fertilization dishes containing 250µl of culturing media. After that 100µl media was removed with pipette for better adhesion. The culture was stored in an incubator at 25°C with 2% CO2. Biweekly 100 µl media were changed from the dishes.

Passaging

Remove the Medium and add 1M DPBS/EDTA to the cells Incubate at RT for 60 -90 minutes Coat flasks with Gelatine for 5min, remove the Gelatine and let the surface dry Take the cells out in an 15ml/50ml Falcon tube and Centrifuge 3min at 700rpm Remove DPBS/EDTA Add 1ml of Culture medium and dissociate cells with 1ml tip Add an additional 9ml of Culture medium and count 10µl of the cell mix in a Neugebauer Counter Chamber Add an equal amount of culture medium to the cells to get around 120 000 – 150 000 cells/ml in the flask (5ml/25cm2, 15ml/75cm2, 35ml/175cm2) Keep the cells at 25°C, 5% CO2 and passage every 3-4 weeks

Freezing

Remove the Medium, but keep it in a Falcon Tube and add 1M DPBS/EDTA to the cells Incubate at RT for 60 -90 minutes Take the cells out in an 15ml/50ml Falcon tube and Centrifuge 3min at 700rpm Remove DPBS/EDTA and add DPBS for cell counting Centrifuge again for 3min at 700rpm Add maximum 5 Mio cells in 0.9ml Medium/0.1 ml DMSO in a Cryotube Freeze in -80C in a Cryobox (for slowly freezing use Isopropanol)