Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density
A serum-free medium and suspension culture technology, applied in the field of cell engineering, can solve the problems such as the inability to meet the needs of high-density culture of CHO cells, the application status and development trend of difficult animal cell culture, and the limitation of serum-free medium for CHO cells, etc. To achieve the effect of clear ingredients, simple composition and easy preparation
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Embodiment 1
[0065] Example 1: Using the statistical method Plackett-Burman and response surface method to determine the supplementary components of CHO cell serum-free medium and their optimal dosage
[0066] CHO cells (CHO-K1, ATCC Cat.No.CCl-61) were pressed at 3-3.5×10 5 The cells / m were inoculated in a 100ml Erlenmeyer flask with a culture volume of 35ml. Serum-free medium containing 5mM glutamine, 0.1% (v / v) Pluronic F-68 and 25μg / ml dextran sulfate or containing DMEM / F12 (v / v, 1:1) medium with 5% (v / v) serum, the Erlenmeyer flask was placed in a 37°C incubator for culture on a shaker with a shaker speed of 90r / min.
[0067] The design of serum-free medium for CHO cell suspension culture was based on DMEM / F12 (v / v, 1:1), and the Plackett-Burman method was used to investigate the insulin (X 1 ), putrescine (X 2 ), transferrin (X 3 ), trace elements (X 4 , Table 1), ascorbic acid (X 5 ), lecithin (X 6 ), β-mercaptoethanol (X 7 ), glutathione (X 8 ), ethanolamine (X 9 ), β-cyc...
Embodiment 2
[0080] Embodiment 2: the preparation of culture medium and CHO cell culture
[0081] 1. Medium preparation: Add the following substances to the basal medium based on DMEM / F12 (v / v, 1:1):
[0082] Insulin 2mg / L
[0083] Putrescine 0.5mg / L
[0084] Transferrin 5mg / L
[0085] Glutathione 0.5mg / L
[0086] Ethanolamine 1mg / L
[0087] Lecithin 1mg / L
[0088] Hydroxypropyl-β-cyclodextrin 100mg / L
[0089] β-Mercaptoethanol 10μM
[0090] Ascorbic acid 5mg / L
[0091] Said supplement also contains the following trace elements:
[0092] Na 2 SeO 3 5nM
[0093] MnSO 4 ·H 2 O 0.5nM
[0094] Na 2 MoO 4 2H 2 O 5nM
[0095] NaVO 3 5nM
[0096] CuSO 4 ·5H 2 O 5nM
[0097] ZnSO 4 ·7H 2 O 1μM
[0098] FeC 6 h 5 o 7 3μM
[0099] The additive also contains the following substances
[0100] Dextran Sulfate 25mg / L
[0101] Glutamine 5mM
[0102] Pluronic F-68 1000mg / L
[0103] HEPES 15mM
[0104] The above me...
Embodiment 3
[0107] Embodiment 3: Compared with Hyclone's CHO cell serum-free medium SFM-CHO
[0108]0.1% (v / v) Pluronic F-68 and 25 μg / ml dextran sulfate were added to Hyclone's SFM-CHO medium, and the CHO cells (CHO-K1) were cultured in the same culture method as in Example 2. The whole cultivation process lasted 8 days. On the 4th day of culture, the living cell density of CHO cells in the serum-free medium of the present invention and the SFM-CHO medium of Hyclone Company reached 4.2×10 6 cells / ml and 3.3×10 6 cells / ml( image 3 ).
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