Human cortical cell culture

2D-differentiation modified Ira’s protocol

• Prepare Matrigel-coated 5cm dishes 1 day before starting following experiment (1 vial of Matrigel in 10ml DMEM/F12, 2ml of matrigel sol./5 cm dish).
• When hESC culture reaches 70-80% confluent.
• Prepare gelatin-coated 5 cm dish (Add 2 ml of the gelatin solution to cover the bottom of the 5-cm dish and incubate it at 37 °C for 10 min. Aspirate the gelatin solution before addition of the medium).
• Check the hESC colonies (i.e. colony shape, cell morphology, confluency).

1. Day –2: After removing hESC medium, wash hESC with warmed DPBS (2 ml/well).

2. Incubate in Accutase (1 ml/well, 6-well plate) for 5 min at 37˚C.

3. Dissociate into single cell suspension by gently pipetting.

4. Add 4 ml of hESC medium to stop enzymatic reaction of Accutase.

5. Transfer to 15 ml tube and centrifuge at 280 xg for 3 min at RT.

6. Resuspend hESC in 5 ml of hESC medium containing 10µM ROCK-inhibitor medium by gently pipetting.

7. Transfer cell suspension to gelatin-coated dish (5 cm dish).

8.  Incubate cell for 40 min at 37˚C.

9.  Transfer medium containing floating ESC to Matrigel-coated 5cm dishes at the density 10x10^4 cells/5 cm dish.

10. Leave for 2 days at 37˚C.

11. Day 0: Change the medium from hESC medium containing ROCK-inhibitor to 5 ml of DDM/B27 medium containing 100 ng/ml Noggin.

12. Change DDM/B27 medium containing Noggin every two days until 8th days after starting differentiation.

13. Day 8: Change DDM/B27 medium containing Noggin everyday until 16th days after starting differentiation.

14.  Change DDM/B27 medium everyday until day 25.

Optional step:

15.  Day 25: After removing DDM/B27 medium, wash NSC with warmed DPBS (5 ml/dish).

16.  Add 1 ml of warmed Accutase and incubate for 10 min at 37˚C.

17.  Dissociate into single cell suspension by gently pipetting.

18.  Add 4 ml of DDM/B27 medium to stop enzymatic reaction.

19.  Transfer to falcon tube and centrifuge at 280 xg for 3 min at RT.

20-1.  For freezing, discard the supernatant and resuspend the cell pellet in mFreSR (2 ml/dish/2 vials) and frozen in vials in a freezing container. The freezing container stays for 1 o/n at –80˚C, the vials are transfer into a liquid nitrogen tank.

20-2.  For culture, discard the supernatant and resuspend the cell pellet in DDM/B27 Nb/B27 medium and count cell number. Plate on Matrigel-coated plates or coverslips.

20-3.  For low density culture, discard the supernatant and resuspend the cell pellet in DDM/B27 Nb/B27 medium. Plate cells into mouse astrocytes coated well (e.g. 6-well) at 5x10^4 cells per cm^2.

Thawing NSC:

1. Thaw the cells in a 37˚C water bath.

2. Transfer the thawed NSC to warmed DDM/B27 Nb/B27 medium (5 ml/vial).

3. Centrifuge the cells at 280 xg for 3min at RT.

4. Discard the supernatant and resuspend the cell pellet in DDM/B27 Nb/B27 medium and plate on Matrigel-coated plates.
 

Material and Reagents for culture:

• Matrigel (Corning, hESC-qualified Matrix, Cat #: 354277)
  Make aliquots (half volume of dilution factor) and keep at –80˚C.
• mFreSR (STEMCELL TECHNOLOGIES, Cat #: 05855)
• Accutase (Thermo Fisher SCIENTIFIC, Cat #: A1110501)
• DDM/B27 medium

• Nb/B27 medium

• DDM/B27 Nb/B27 medium: 1:1 mixture of DDM/B27 and Nb/B27 medium.

Characterization of human PSC-derived Cortical Cells by Immunostaining:

Antibody list:

1. Fixation at 4˚C for 60 min (500 µl of 4% PFA in PBS). 
2. Wash 500 µl of 0.3% Triton-X 100/PBS x 2 times.
3. Incubate in 500 µl of 0.3% Triton-X 100/PBS at r/t for 30 min.
4. Aspirate Triton-X 100/PBS and add 500 µl (12-well plate) of primary antibodies contain blocking buffer. 
5. Incubate at 4˚C for o/n. 
6. Wash 500 µl PBST x 3 times.
7. Aspirate PBST and add 500 µl (12-well plate) of secondary antibodies (dilution: 1/1,000) contain PBST.
8. Incubate at r/t for 2 h.
9. Wash 1 ml PBST x 3 times.
10. Mount using Glycergel Mounting Medium (DAKO).
11. Keep 4 ˚C.