Table 3 Culture Conditions for Feeder-Free Growth of ES Cells in Synthetic Serum

From: Efficient Germline Transmission of Mouse Embryonic Stem Cells Grown in Synthetic Serum in the Absence of a Fibroblast Feeder Layer

Knockout DMEM (Invitrogen Corporation, Paisley, United Kingdom; 10829-018)

 

500 ml

Serum replacement (Knockout SR; Invitrogen; 10828-028)

 

80 ml

Nucleosides: Adenosine (Sigma, Dorset, United Kingdom; A4036)

80 mg

6.0 ml

 Guanosine (Sigma; G6264)

85 mg

 

 Cytidine (Sigma; C4654)

73 mg

 

 Uridine (Sigma; U3003)

73 mg

 

 Thymidine (Sigma; T1895)

24 mg

 

 Dissolve in 100 ml ddH2O at 37°C and filter sterilize

  

Sodium bicarbonate (7.5% w/v; filter sterilized)

 

9.6 ml

l-Glutamine (Invitrogen; 25030-024)

 

6.0 ml

2-Mercaptoethanol (Invitrogen; 31350-010)

 

0.6 ml

Nonessential amino acids (Invitrogen; 11140-035)

 

6.0 ml

ESGRO (LIF; Chemicon Int., Middlesex, United Kingdom, ESG 1107)

 

1000 units/ml

  1. Cells were incubated at 37°C/5% CO2 in a humidified atmosphere and passaged as described in the text. Media was prepared fresh under sterile conditions and stored at 4°C for a maximum of 2 weeks. Gelatin-treated plates were made by the addition of 5 mls of 0.1% w/v gelatin (in ddH2O) to tissue culture grade flasks/dishes and incubated overnight at 4°C. The excess solution was removed, the plates air dried and stored at 4°C for a maximum of 4 weeks.
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