Materials 21 Tissue Culture

2.1.1. Supplies

1. Dulbecco's modified Eagle's medium/Ham's (DMEM) (Invitrogen, Carlsbad, CA; cat. no. 1195073).

2. DMEM/F12 (Invitrogen; cat. no. 11330032).

3. Glasgow minimum essential media (GMEM) (Invitrogen; cat. no. 11710035).

4. Minimum essential medium alpha-medium (Invitrogen; cat. no. 32571036).

5. 1X phosphate-buffered saline (PBS) (Invitrogen; cat. no. 14190144).

6. Fetal bovine serum (FBS) (Hyclone, Logan, UT; cat. no. SH30071.03).

7. Knockout serum replacement (KSR) (Invitrogen; cat. no. 10828028).

8. Penicillin-streptomycin (100X) (Invitrogen; cat. no. 15140122).

9. Nonessential amino acids (Invitrogen; cat. no. 11140050).

10. l-glutamine (Invitrogen; cat. no. 25030081).

11. P-mercaptoethanol (Specialty Media, Phillipsburg, NJ; cat. no. ES-007-E).

12. Sodium pyruvate (Sigma, St. Louis, MO; cat. no. S8636).

13. Basic fibroblast growth factor (bFGF) (Sigma; cat. no. F0291).

14. Trypsin-ethylenediaminetetraacetic acid (EDTA): 0.05% trypsin, 0.53 mM EDTA (Invitrogen; cat. no. 25300054).

15. Cell dissociation buffer (CDB) (Invitrogen; cat. no. 13150016).

16. Dimethylsulfoxide (Sigma; cat. no. D8418).

18. 0.1% gelatin (Specialty Media; cat. no. ES-006-B).

19. Collagen type I-coated Biocoat 25-mm culture dish (Becton Dickinson, Franklin Lakes, NJ; cat. no. 356456).

20. Papain dissociation system (Worthington Biochemical Corp., Lakewood, NJ; cat. no. LK003150).

21. B27 Supplement (Invitrogen; cat. no. 17504-044).

22. Collagenase IV (Invitrogen; cat. no. 17104-019) (see Note 1).

23. Fibronectin, human (Invitrogen; cat. no. 33016-015).

24. Human brain-derived neurotrophic factor (R&D Systems, Minneapolis, MN; cat. no. 248-BD).

25. Human epidermal growth factor, recombinant (R&D Systems; cat. no. 236-EG).

26. Human neurotrophin 3 (R&D Systems; cat. no. 267-N3).

28. N2 Supplement (Invitrogen; cat. no. 17502-048).

29. Neurobasal media (Invitrogen; cat. no. 17504).

30. Poly-l-lysine 30-70 kDa (Sigma; cat. no. P9404).

1. Medium for undifferentiated hES cells: hES cell line BG01 is maintained in DMEM/F12, supplemented with 15% FBS, 5% KSR, 2mM nonessential amino acids, 2 mM l-glutamine, 50 |g/mL Penn-Strep, 0.1 mM P-mercaptoethanol and 4 ng/mL of bFGF. For 100 mL: 75 mL DMEM/F12, 15 mL FBS, 5 mL KSR, 2 mL l-glutamine, 1 mL nonessential amino acids, 0.5 mL penicillin-streptomycin, 1 mL P-mercaptoethanol, and 16 ||L bFGF (25 mg/mL).

2. Medium for mouse embryonic fibroblast (MEF) cells: MEF feeder cells are maintained in DMEM, supplemented with 10% FBS and 50 |g/mL penicillin-streptomycin. For 100 mL: 90 mL DMEM, 10 mL FBS, and 0.5 mL penicillin-streptomycin.

3. Media for PA6 cells: the mouse stromal cell line PA6 is maintained in minimum essential medium, supplemented with 10% FBS and 50 |g/mL penicillin-streptomycin. For 100 mL: 90 mL minimum essential medium, 10 mL FBS, and 0.5 mL penicillin-streptomycin.

4. Medium for differentiation of hES cells on PA6: hES cells are differentiated on a monolayer of PA6 in GMEM, supplemented with 10% KSR, 2 mM l-glutamine, 1 mM sodium pyruvate, and 0.1 mM P-mercaptoethanol. For 100 mL: 87 mL GMEM, 10 mL KSR, 1 mL l-glutamine, 1 mL P-mercaptoethanol, and 1 mL sodium pyruvate.

5. Medium for EB differentiation: hES cell-derived EBs are maintained in DMEM supplemented with 20% FBS, 1% nonessential amino acids, 1 mM l-glutamine and

0.1 mM ß-mercaptoethanol. For 100 mL: 77 mL DMEM, 20 mL FBS, 5 mL KSR, 1 mL l-glutamine, 1 mL nonessential amino acids, and 1 mL ß-mercaptoethanol.

6. Media for neural progenitor proliferation: neural progenitor proliferation media comprises DMEM/F12 with B27 and N2 supplements, 10 ng/mL human epidermal growth factor, and 10 ng/mL bFGF. For 100 mL: 88 mL DMEM/F12, 1 mL B27, 1 mL N2, and 100 ||L of each human epidermal growth factor (10 |g/mL) and bFGF (10 |g/mL).

7. Media for neural progenitor differentiation: Neural progenitor differentiation media is comprised of Neurobasal media supplemented with B27, 10 ng/mL human neurotrophin 3, and 10 ng/mL human brain-derived neurotrophic factor. For 100 mL: 89 mL Neurobasal, 1 mL B27, and 100 |L of each human neurotrophin 3 (10 |g/mL) and human brain-derived neurotrophic factor (10 |g/mL).

1 hES cells: the hES cell line BG01 was obtained from BresaGen Inc., Athens, GA; cat. no. BG01.

2. MEF feeder cells: MEF feeder cells from strain SVB are obtained from Specialty Media; cat. no. PMEF-N.

3. PA6 cells: PA6 cells are purchased from the Riken Bioresource Center Cell Bank, Koyadai, Japan; cat. no. RCB1127.

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