46C-ES cells/cyst formation
Thawing of 46C ES cells: Prepare the growth medium:
- 405 mlDMEM + high glucose * Glutamax (Invitrogen: 31966)
- 75 ml FCS
- 5 ml L-Gln 100x (Invitrogen)
- 5 ml P/S 100x
- 5 ml NEAA (Invitrogen: 11140; 100x)
- 5 ml β-Mercaptoethanol (take 7µl of β-Mercaptoethanol and mix it in 10 ml PBS; take 5ml this solution for the medium)
- 50µl LIF (Chemicon, ESGRO-LIF 107 units)
Prewarm the medium to 37°C. Thaw the cells quickly at 37°C in the waterbath and add them to 9 ml of prewarmed medium in a Falcon tube. Mix them and centrifuge (1000rpm, 5’, RT). Resuspend the pellet in 10 ml of growth medium and put them to a 10cm² dish. (culture conditions: 37°C, 5% CO2)
The next day change the medium to get rid off all unattached cells (remove the medium and add prewarmed fresh medium).
Splitting of 46C ES cells: Notes: On d2 you need to split the cells. After thawing there are a lot of differentiated cells growing in the plate next to undifferentiated ES cells. To get rid off the differentiated cells as they would disturb the experiment ES cells are only trypsinized for 1:45 minutes at 37°C. Differentiated cells remain attached whereas ES cells are already floating! If there are a lot of differentiated cells coming off, you could get rid off them by preplating them. For this put the cells for ca. 20-30 minutes in a petri dish in the incubator. Differentiated cells will attach whereas ES cells remain in suspension!
- Discard medium/PBS/Trypsin-EDTA
- Wash once with prewarmed PBS
- Add 1,5 ml 1x Trypsin-EDTA (1,5ml/10cm² dish); incubate 1:45’ at 37°C
- In the meanwhile add 8,5ml prewarmed Medium into a fresh petri dish to stop the reaction
- Have a look at the cells after 1:45’ in Trypsin EDTA (only ES cells (undifferentiated ones) should detach, differentiated cells attach more firmly to the plastic). Take the cell suspension, hold it in the pipet for another 0,5-1 min at RT before stopping the trypsinization reaction by adding the cells into the plate with the fresh medium which was just prepared.
- Pipet cells up and down in order to disrupt the cell clumps. Touch the plastic with the tip of the pipet and empty the pipet – avoid bubbles! Repeat this pipetting step around 10x.
- Centrifuge the cells 3’ 900rpm RT
- Resuspend the cells in fresh medium and put them to culture for 20 minutes (preplating step). After 20’ pipet all unattached cells into a Falcon tube and count the cells.
- Seed 650.000 cells/10cm dish for further culture (10ml of medium is more than enough).
Preparation of neural cysts
- For a differentiation experiment (making cysts) put 1.000.000 ES cells into one Falcon tube and spin them (1000 rpm/ 3’).
- Discard supernatant, flick the pellet, add 3ml PBS, spin 3’ 1000rpm
- Discard supernatant, flick the pellet, add 3ml N2B27, spin 3’ 1000rpm
- Discard supernatant, flick the pellet, add 200µl N2B27
Cells are ready to use for making cysts!
- Add 10 µl of cells into 150µl of thawed Matrigel, mix quickly and prepare drops on your glass bottom dish – flatten the drops. Move the dishes to the incubator (37°C) w/o medium for 15’ to gel the Matrix. After 15’ take the dishes out, add 2ml N2B27 and let the cysts grow. Best results are obtained if the medium is changed every second day.
1. N2B27 Medium: 50 ml neural basal medium (Gibco) 50 ml DMEM/F12 1 ml B27 0,5 ml N2-supplement 100µl β-Mercaptoethanol (prepare a stock solution of 10 µl in 1,4 ml H2O = 0,1M) 250 µl Pyruvate + Glutamate (prepare a solution of 5,5 ml Gln 100mM and 8,25 ml Pyruvate) 100 µl P/S
N2-supplements (5ml): 335µl BSA (75mg/ml in PBS) 625µl Insulin (250mg + 10ml 0,01M HCl o/n 4°C on shaker; next day + 2,5ml 0,01M HCl + some drops HCl 37% until dissolved), stock -20°C 500µl Apo-Transferrin (500mg/ml water) 5 µl Selenite (3mM) 50 µl Putrescine (1,6g/10ml water) 16,5µl Progesterone (0,6 mg/ml EtOH) 3,47 ml DMEM/F12
Freezing of ESCs Pellet 2x106 cells in a falcon tube. Add 1ml growth medium + 10% DMSO to the pellet and transfer the cell suspension to the vial for freezing. Freeze at -80°C immediately.