Culture, Transfection and Stable cloning of CHO cells

1. Title : Culture of CHO cells

2. Introduction

2.1 CHO cells are used for the expression of recombinant proteins. This protocol describes procedures for -

2.2 Routine propagation of CHO cells and transfected CHO cells

2.3 A method for transfection of CHO cells and selection of transfected cells in culture

3. Related documentation

3.1 [Trypsinisation of cell cultures]

3.3 QIAGEN Effectene Transfection Reagent Handbook 05/2002

4. Materials

4.1 Hams F12 medium - Prepared by Biological Services (or Purchased from InVitrogen.)

- note : laboratory records should state which source is used.

4.2 Foetal calf serum - Provided by Biological Services - Batch number must be recorded

4.3 Trypsin/Versene - Gibco

4.4 Puromycin (10 mg/ml in Methanol stored -20). (Affiniti Research Products, Exeter, UK)

4.5 General tissue culture plasticware.

4.6 Qiagen Effectene transfection reagent and associated buffers.

5. Procedure (all procedures carried out in Class II biological safety cabinet)

5.a General cell culture

5.1 Thaw cells in Ham's F12 medium with 10% Foetal calf serum.

5.2 Centrifuge at 800�100 rpm (Heraeus Benchtop) x 5-10 mins at 1-8�C

5.3 Resuspend cell pellet in about 8 mls Ham's F12 Medium (with bicarbonate and glutamine) and transfer to a 25cm² (T25) tissue culture flask.

5.4 Incubate at 35-39�C in 5�1% CO₂ humidified incubator.

5.5 Examine at suitable intervals until cells reach near-confluence; then split culture by trypsinisation .

5.b Transfection (using plasmids based on pcIpac vector)

5.6 Plate out trypsinised CHO cells at about 2x10⁶ per 25 cm² (e.g. T25 flask). They should reach 40-80% confluence overnight and are then ready for transfection.

5.7 Centrifuge plasmid in T.E at 14000 rpm in a microfuge for 20 minutes to pellet any contaminating organisms and immediately pipette the a volume containing 3ug from the top of the tube into 450 ul final volume of Qiagen DNA condensation buffer (BC).

5.8 Add 24 ul Qiagen Enhancer and mix thoroughly, incubate at room temperature (15-25^oC) for 2-5 minutes, pulse spin and then add 75ul Qiagen Effectene reagent and stand at room temperature for 15-20 minutes.

5.9 Wash cells with PBSa and add 7 mls fresh growth medium (Ham's F-12 + 10% FCS), then use 1ml of medium mixed with the DNA/Effectene suspension to transfer the reagent into the flask with the CHO cells. Mix gently by tipping and replace the flask in the CO₂ incubator.

5.10 (Optional) After 6-10 hours of culture, decant the medium+Effectene/DNA and replace with fresh culture medium.

5.11 20-30 hours after initial exposure to the Effectene/DNA, decant medium from cells, rinse once with selective medium (Ham's F12 + 10% FCS + 20ug/ml puromycin/HCl) and replace with selective medium (8 mls).

5.12 After 1-3 days of selection, replace the selective medium with fresh selective medium to reduce the number of dead cells in suspension.

5.13 After 7-10 days colonies of transfected cells should be clearly visible to the naked eye. Expect a few hundred to a thousand colonies per T25 flask if the transfection has worked well.

5.14 Before the colonies become confluent, remove cells from the flask by trypsinisation, pellet and resuspend in fresh selective medium and count. It is a good idea to cryopreserve some of the cells at this stage .

5.15 Seed bulk transfected cell cultures, if required, using normal procedures for CHO cell propagation, but with selective medium (Puromycin concentration may be reduced to 15ug/ml).

5.16 For cloning, dilute cells in selective medium to 10 cells/ml and distribute 100 ul per well into 96-well culture plates (1 cell per well). After 3-5 days, observe wells with lowest power of inverted microscope and carefully record which wells have single colonies. (if evaporation is evident, supplement wells with 50-100 ul additional selective medium.

5.17 After 7-10 days, when the wells are 20-100 % confluent, wash wells that had single colonies twice by removing medium and gently pipetting in and out 100 ul PBSa. Remove as much PBSa as possible, but do not allow wells to dry out. Add one drop of trypsin/versene from a pipettor (~20 ul) and observe the wells to see when cells have detached (may be very different times for different clones). Stop trypsinisation by addition of one drop of FCS and transfer cell suspension to a larger culture vessel (24/6 well plate or T25 flask) by rinsing into an appropriate volume of selective medium.

5.18 After cloning, cells are propagated as for untransfected CHO cells, but in selective medium (unless the medium is to be used in cell culture experiments, in which case the puromycin must be omitted.)