ES Cell/ Neural Differentiation Protocol

A.  DEFROSTING

1.  Remove vial of ES cells from liquid nitrogen container, and defrost in TC 37o water bath. This takes 1-2 minutes.

2.  Clean tube with 70% ethanol, dry, and place in TC hood.

3.  Using a plugged pasteur pipet, plate on a feeder layer (irradiated MEFs) in a 60mm dish containing 4.5 ml of ES medium.

4.  Next day, look at cells. If grown to 60% confluency, or more, go on to freezing and splitting (passaging) cells. If not, change the medium.

B.  FREEZING

1.  Aspirate medium from dish, and rinse dish with 1ml trypsin or PBS, removing immediately.

2.  Add 1ml 0.25% trypsin and incubate at 37o C. Check cells for dissociation after 2-3 minutes. (Gently rotate/shake dish and observe under inverted microscope).

3.  Using a 5ml pipet, add 1ml ES cell medium and gently pipette up and down to break up aggregates. Check with scope.

4.  Transfer a portion of the cell suspension into a 15ml Falcon tube. Spin tube @1000 rpm (200-250g), for 5 minutes. (In general, we freeze 7-10 cm2/ cryovial. A 60mm dish = 20 cm2.)

5.  Remove medium and resuspend pellet in freeze medium , 0.8ml/vial.

6.  Transfer cell suspension into cryovial(s) and label with cell type, passage number, date, and surface area cm2.

7.  Place vial(s) into a Nalgene isopropanol freezing container or a styrofoam box, and place in –80oc freezer. IMPORTANT: CELLS MUST FREEZE SLOWLY!

8.  Plate remaining cell suspension on a 60mm gelatinized TC dish containing 5ml ES cell medium, total volume.

9.  Next day, check cells. If not 60% confluent, change medium. If 60% or more confluent, aspirate medium and trypsinize using 1ml of 0.25% trypsin.

10.  Add 1ml of ES cell medium and pipette up and down gently to break up aggregates.

11.  Transfer cells to a 100mm gelatinized TC dish containing 8-10ml ES cell medium.

12.  Next day, check cells and change medium. Split if required.

C.  NEURAL DIFFERENTIATION OF ES CELLS

1.  Check cells. If 60% (or more) confluent, aspirate medium and add 2.5ml 0.25% trypsin.

2.  Incubate at 37o C . Check cells for dissociation after 2-3 minutes.

3.  Add 5.5ml differentiation media and gently pipette up and down to break up aggregates. Do NOT dissociate to single-cell suspension – leave small clumps.

4.  Dispense 1ml of cell suspension (about 5 X 106 cells) into each of 8 Fisher petri dishes, 100mm(non-gelatinized) containing 9ml of differentiation medium.

5.  Incubate at 37oC. This is day 0

6.  Day 1-, check cells for aggregate formation.

7.  Day 2-, change medium. (See section D below).

8.  Day 3-, check as on day1-.

9.  Day 4-, change to medium containing 5x10-7 M retinoic acid (RA).

10.  Day 4-/1+, check cultures.

11.  Day 4-/2+, change to fresh RA containing medium.

12.  Day 4-/3+, check cultures.

13.  Day 4-/4+, collect embroid bodies and remove medium as in section D, below.

14.  Add 1 ml 0.25% trypsin, and incubate in 37oC TC H2O bath for 10-15 minutes, gently agitating periodically.

15.  Remove some of the trypsin and add differentiation medium (with no RA) to a total of 5ml. Gently pipette up and down for 5-10 minutes. Do not dissociate completely to single cells.

16.  Aliquot varying amounts of cell suspension into 3 ->12 wells on (gelatinized) 6-well plates, 2ml differentiation medium/well.

17.  Check for differentiation each day for 5 days. Neurons are apparent by day 3, and peak at day 5. To culture beyond five days, use Neurobasal Medium with B27 supplement.

D.  CHANGING MEDIA DURING DIFFERENTIATION

1.  Gently transfer cells to a 15 ml tube, using 10ml pipet. (You reuse the same plates, so you may want to add 2-3ml of medium to each dish so that they do not dry out while pelleting the Embroid Bodies . . . a few EBs are left on the dish. This is optional.)

2.  Allow EBs to settle for 10-15 minutes.

3.  Remove as much medium as possible using a 10 ml pipet.

4.  Resuspend in differentiation medium to a total volume of 10 ml/dish, and return to a Fisher 100mm petri dish.

5.  When adding medium with RA, it is easier to prepare the supplemented medium in a tube/ sterile bottle as the EBs are settling out. Use 5ml of 1mM stock RA /10ml of medium. (1mM stock RA is kept in dark at 4oC)