Medium for ES culturing
View Patent ↗The present invention discloses a medium for a serum-free medium capable of culturing ES cells for a long period while maintaining their undifferentiated state without using feeder cells, and a basal medium for producing the medium thus described. The basal medium of the present invention is characterized by that it has composition shown by Table I. Further, the present invention discloses a medium for ES cells produced with the basal medium.
1. A medium for culturing embryonic stem (ES) cells comprising: (a) insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, oleic acid which has formed a complex with fatty acid-free bovine serum albumin, and LIF (leukemia inhibitory factor; and (b) the basal medium comprising the composition shown by the following Table III.
TABLE III
Components
Concentration (mg/L)
L-alanine
1.78~2.67
L-arginine
40~60
L-arginine HCl
75.8~113.7
L-asparagine H 2 O
13.002~19.503
L-Asparatic acid
6.66~9.99
L-cysteine HCl•H 2 O
7.024~10.536
L-cystine 2HCl
38.058~57.087
L-glutamic acid
6.94~10.41
L-glutamine
439.72~659.58
Glycine
15.5~23.25
L-histidine
3~30
L-hydroxyproline
4~6
L-isoleucine
52.748~79.122
L-leucine
54.58~81.87
L-lysine HCl
73.74~110.61
L-methionine
15.896~23.844
L-phenylalanine
30.392~45.588
L-proline
10.9~16.35
L-serine
24.9~37.35
L-threonine
44.42~66.63
L-tryptophan
7.808~11.712
L-tyrosine
33.888~50.832
L-valine
43.86~65.79
Glutathione
0.2~0.3
Para-aminobenzoic acid
0.2~0.3
Biotin
0.04148~0.06222
Calcium pantothenate
1.746~2.619
Choline chloride
4.992~7.488
Folic acid
2.06~3.09
Inositol
13.48~20.22
Niacinamide
1.8074~2.7111
Pyridoxal HCl
1.6~2.4
Pyridoxine HCl
0.2124~0.3186
Riboflavin
0.2076~0.3114
Thiamine HCl
1.868~2.802
Vitamin B 12
0.273~0.4095
Hypoxanthine
0.816~1.224
Linoleic acid
0.0168~0.0252
Lipoic acid (thioctic acid)
0.042~0.063
Putrecine dihydrochloride
0.0322~0.0483
Thymidine
0.146~0.219
Sodium chloride
5279.8~7919.7
Potassium chloride
284.72~427.08
Calcium chloride
86.644~129.966
(anhydrous)
Calcium nitrate 4H 2 O
20~30
Magnesium chloride
11.444~17.166
(anhydrous)
Magnesium sulfate
48.844~73.266
(anhydrous)
Sodium dihydrogen
43.48~65.22
phosphate (anhydrous)
Disodium monohydrogen
188.408~282.612
phosphate (anhydrous)
Glucose (anhydrous)
1860.4~2790.6
Ferric nitrate 9H 2 O
0.04~0.06
Copper sulfate 5H 2 O
0.0005~0.00075
Ferrous sulfate 7H 2 O
0.1668~0.2502
Zinc sulfate 7H 2 O
0.1728~0.2592
Phenol red
5.248~7.872.
2. The medium according to claim 1 , further comprising 0.001 to 220 mg/L of sodium pyruvate.
3. The medium according to claim 1 , further comprising 0.000692 to 0.00348 of sodium selenite.
4. The medium according to any one of claims 1 to 3 , further comprising 2.5 to 4.5 g/L HEPES, and NaHCO 3 in an amount required for an adjustment to desired pH.
5. A method for culturing ES cells comprising culturing ES cells with the medium of claim 1 .