CHO Cell Culture

Medium:

EXCELL CD CHO Fusion, cat #14365C, 1x
L-Glutamine, G7513, 4 mM
HT, H0137, 1x
Folinic Acid, F7878, 2 uM

Prepare supplements

–– HT: Add 10 mL of EX-CELL CD CHO Fusion to each vial of HT (2 vials needed for 1L of growth media).
This creates a 50X solution. Discard any unused resuspended HT.

–– Folinic Acid: Resuspend folinic acid in sterile water to create a 1 mM stock solution. Sterile filter. Make 1 mL aliquots and store at -20 °C. Thaw prior to use.

–– Glutamine: Thaw glutamine in 37 °C water bath until completely dissolved. After thawing, glutamine may be stored at 4 °C for up to 2 weeks.
Add media supplements to EX-CELL CD CHO Fusion to achieve the final concentrations listed in the table above.
* Filter sterilize the complete growth media.
* Store growth media at 4 ̊C when not in use. Discard any unused media after 1 month.

Vial thaw for stock culture initiation and maintenance

* Adjust incubator settings to 37 ̊C and 5% CO2.
* Prepare growth media as described above.

Obtain sterile T-75 cell culture flask.

Obtain 15 mL conical tube. Place 8 mL prepared media in conical.

• Obtain frozen vial from LN2 freezer.
  * Immediately thaw vial by gently swirling in 37 °C water bath until just thawed.

Obtain frozen vial from LN2 freezer.

Spray the vial liberally with 70% isopropyl alcohol and place the vial in the biosafety cabinet.
* Transfer cells to the 15 mL conical containing fresh growth media.
* Centrifuge cell suspension for 5 minutes at 220 X g.

Remove supernatant without disturbing the cell pellet.

Add 10 mL fresh growth media to suspension and mix gently by pipetting

Transfer suspension to T-75 cell culture flask.

24 hours post-thaw, remove a cell culture sample and count the cells by trypan blue exclusion.

If culture viability is >80%, then passage into subculture 1 (see below). If culture viability is <80%, then leave in static T-75 culture. Cells may remain in static culture for up to 3 days post-thaw.

Subculture 1

* Set agitation on orbital shaker to 125 rpm
* Remove cell culture sample and count by trypan blue exclusion.
* Transfer the entire culture from the T-75 flask to a 125 mL shake flask.
* Transfer 10 mL fresh pre-warmed growth media to 125 mL shake flask, incubate on the orbital shaker.
* Maintain cultures in 37 ̊C and 5% CO2 incubator.

Stock maintenance (every 3-4 days)
* Remove cell culture sample and count by trypan blue exclusion.
* Determine volume of cells needed to inoculate a new culture at 300,000 cells/mL.
* Transfer the appropriate volume of cells from the existing stock culture to a new flask, and add pre-warmed growth media to bring up to the desired culture volume.

Label cryovials
Prepare freezing media: Growth Media + 7% DMSO.
Remove 600 μL sample from stock cell culture and count by trypan blue exclusion.
* Calculate the volume of cell stock and freezing media needed to obtain a 5x106 to 5x10 6 cells per mL (cryovial)
* Once cell preparation is initiated, work must proceed quickly to get vials into freezer. It is recommended that the total time from removing cells from the stock culture to transferring to the freezer does not exceed 30 minutes.
* Transfer calculated volume of stock culture to appropriately sized centrifuge tube.
* Centrifuge at 220 X g for 5 minutes at room temperature.
* Carefully aspirate supernatant.
* Gently resuspend pelleted cells with calculated volume of freezing media. Mix thoroughly by pipetting.
* Immediately aliquot cell suspension to labeled cryovials. Cap tightly.
* Quickly transfer the vials to prepared freezer buddies and store in -80 oC freezer or transfer vials to a controlled rate freezer for overnight freezing.
* Transfer vials to LN2 freezer within 18-72 hours of freezing.

CHO Transfection with PEI

Seed CHO DG44 cells at a density of 2 X10^6 cells/ml in either a 3-L bioreactor (Applikon) or a 20-L bioreactor (BioEngineering) in RPMI 1640 medium.

Dilute stock solutions of DNA and PEI separately in sterile 150 mM NaCl or 278 mM glucose.  The final volume of each solution is equivalent to 5% of
the volume of the culture to be transfected. Add PEI  solution  to diluted DNA and incubate the mixture at room temperature for 10 min. Add DNA-PEI mix to culture cells and  culture the cells with agitation (200 rpm) for 5 h at 37 oC in an atmosphere with 5% CO2 and 95% humidity.

At 5 h posttransfection, dilute the cultures with one volume of ProCHO5 CDM medium. 

Shake the cultures at 150 rpm during transfection and at 200 rpm after dilution of the culture.

Maintain dissolved oxygen at 20% by sparging air into the culture  pH  at 7.1 using NaOH and CO2.

Feed the culture with glucose in order to keep the level at 4 g/l, and maintain the sodium bicarbonate concentration at 10 mM.