Part of Thermo Fisher Scientific Inc.

HyClone
HOME		 Media
Home		 About
Media 		Media
Quality		 Media
Facilities		 Media
Capabilities		 Media
Products		 Media
Support

Technical Brief:
HyQ® SFM4CHO™
HyQ® SFM4CHO™-Utility
HyQ® CDM4CHO™

Adaptation, Cell Maintenance, and Cryopreservation

Direct Adaptation

Medium Preparation: Warm necessary volume of medium for procedure to 37°C for each pass.

1.

Prepare a culture of cells in serum-containing medium. When the culture is at 70% confluence, subculture the cells into the HyClone serum-free medium at a seeding concentration of 2.0 x 105 cells/mL.

NOTE: If the cells continue to attach during this adaptation procedure allow the cells to go to confluence. At this point the cells begin to go into suspension and the procedure can be continued.
2. When cells have reached a density of 1.0-1.5 x 106 cells/mL, sub-culture again at a seeding concentration of 2.0 x 105 cells/mL into fresh HyClone serum-free media.
3.

Continue this schedule for 3 sub-cultures.


Sequential Adaptation

Medium Preparation: Dilute your serum-containing medium with an equal volume of HyClone serum-free medium. This medium will be referred to in this protocol as Sequential Adaptation Medium (SAM). It is necessary to prepare twice the volume of medium needed for the culture vessel that will be used (i.e., for a T75 flask using 25 mL of medium, prepare 50 mL of SAM). Many CHO clones will perform differently, but this procedure can be used with most clones. Prior to each passage, warm necessary volume of medium to 37ºC.

1. Sub-culture the cells into SAM at a seeding concentration of 2.0 x 105 cells/mL. For best results the culture should be approximately 70% confluent.
2. When cells have reached a density of 1.0 –1.5 x 106 cells/ml, subculture again at a seeding concentration of 2.0 x 105 cells/ml into SAM to which an equal volume of serum-free medium has been added.


Cell Maintenance

Maintain the adapted cells by establishing a schedule that allows the cells to be sub-cultured while in mid-logarithmic growth phase. Cultures should be maintained by sub-culturing the cells 2 –3 times per week (seeding concentration:2.0 x 105 cells/ml). Adjust the sub-culture schedule and the seeding density to meet the performance of your cells. The viability should remain higher than 90%.


Cryopreservation

1. The adapted cells should be cryopreserved in a medium consisting of 50% fresh HyClone serum-free medium and 50% conditioned HyClone serum-free medium. To this mixture add DMSO at 7.5%)
2. Prior to relying on frozen cells as a Master Seed Stock, recover the cells from cryopreservation, expand and check for cell growth and recombinant protein production.

Back

 

 

 

Copyright © 2008
Thermo Fisher Scientific Inc.
 
Learn more about Thermo Scientific Life Science Research Products
RNAi
Technologies		 Nucleic Acid
Amplification		 Protein
Research		 High Content
Screening		 For
Europe