Transfection of DG44 Cells & Development of Stable Cell Lines in Defined Medium

• pOptiVEC™-TOPO® TA Cloning Kit: A TOPO®-adapted bicistronic plasmid and reagents for cloning of a PCR product containing a mammalian secretion signal and the gene of interest.
• DG44 cells: DHFR-negative, CHO-derived cells adapted to high density, serum-free suspension culture in CD DG44 Medium that are capable of producing high levels of secreted, recombinant protein.
• CD DG44 Medium: Defined, serum-free medium supplemented with hypoxanthine and thymidine to allow growth of DHFR-negative DG44 cells.
• FreeStyle™ MAX Reagent: A proprietary, animal origin-free formulation for high transfection efficiency of plasmid DNA into DG44 cells.
• CD OptiCHO™ Medium: Defined, serum-free medium formulated for selection and growth of DG44 cells expressing DHFR and the recombinant protein of interest.
• The kit also contains OptiPro™ serum free medium (SFM) for optimal DNA:lipid complex formulation, L-glutamine provided separately for increased media stability, and the surfactant Pluronic® F-68 to control shear forces in suspension culture.

• DHFR-deficient DG44 cells derived from CHO cells (Urlaub et al., 1983), which provide stable and accurate glycosylation (Sheeley et al., 1997; Werner et al., 1998) and are more likely to yield accurate glycoproteins.
• FreeStyle™ MAX Reagent offers high transfection efficiency of suspension CHO cells with low cytotoxicity.
• FreeStyle™ MAX Reagent, CD DG44 Medium and CD Opti CHO™ Medium are animal-origin free.

Shipping/Storage

The components of the OptiCHO™ Express Kit are shipped and should be stored as listed below.
 
TOPO® TA  Cloning Reagents

The pOptiVEC™-TOPO®  TA cloning reagents are shipped on dry ice in two boxes, containing the cloning reagents and the One Shot®  TOP10 Chemically Competent E. coli kit. Refer to the pOptiVEC™-TOPO® TA Cloning Kit manual for detailed description of the kit contents.
 
Store the TOPO® reagents at -20ºC and the One Shot®  TOP10 Chemically Competent E. coli kit at -80°C.
 
DG44 cells
 
DG44 cells are provided as one vial shipped on dry ice containing 1 x 10⁷ cells in 1 ml freezing medium containing 10% DMSO.
 
Important:   Upon receipt, immediately store in liquid nitrogen.
 
OptiCHO™ Express Kit Components

The OptiCHO™ Express Kit components are shipped under various conditions as listed below.
 
Store components as indicated.

• Adapted to serum-free suspension growth in CD DG44 Medium containing hypoxanthine and thymidine.
• Demonstrates high transfection efficiencies with FreeStyle™ MAX Reagent.
• Suspension cultures may be transfected in CD DG44 Medium.

• All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.
• Supplement CD DG44 Medium to a final concentration of 8 mM L-glutamine and 18 ml of 10% Pluronic® F-68 per liter before use.
• Addition of antibiotics is not recommended.
• CD DG44 Medium is light sensitive. For optimal results, store medium at 4ºC, protected from light.

• Frozen DG44 cells (supplied with the kit; store frozen cells in liquid nitrogen until ready to use)
• Complete CD DG44 Medium (prepared as above; pre-warm at 37°C before use).
• 125 ml polycarbonate, disposable, sterile Erlenmeyer flasks with vented caps (available from VWR, West Chester PA, cat. no. 30180-036)
• Orbital shaker set at 130-135 rpm in 37°C incubator with a humidified atmosphere of 8% CO₂

1. Remove the cryovial of cells from the liquid nitrogen and thaw quickly (<1 minute) in a 37°C water bath.
2. Decontaminate the outside of the vial with 70% ethanol. Gently break up any clumps with a sterile pipette tip and aseptically transfer the entire contents of the cryovial into a disposable, sterile polycarbonate 125-ml Erlenmeyer shaker flask containing 30 ml of pre-warmed complete CD DG44 Medium.
3. Incubate cells in a 37°C incubator containing a humidified atmosphere of 8% CO₂ in air on an orbital shaker platform rotating at 130-135 rpm.
4. After 24-48 hours in culture, determine the cell density and viability using the protocol described below.
5. Once the culture has reached >1.2 x 10⁶ viable cells/ml, expand cultures using the subculturing protocol below.

1. Transfer a small aliquot of the cell suspension to a microcentrifuge tube.
2. Determine viability using trypan blue dye exclusion.
3. Determine cell density electronically using a Coulter Counter or manually using a hemacytometer and an inverted microscope.

1. Determine viable and total cell counts.
2. Using the cell density determined in Step 1, calculate the split ratio needed to seed each new shaker flask at 2 x 10⁵ viable cells/ml by dilution.
3. Dilute the cells in 30 ml of pre-warmed complete CD DG44 Medium to give a final cell density of 2 x 10⁵ viable cells/ml.
4. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO₂ in air on an orbital shaker platform rotating at 130-135 rpm.
5. Repeat Steps 1-5 as necessary to maintain or expand cells.

• Complete CD DG44 Medium
• Tissue culture grade DMSO
• Reagents and equipment to determine viable and total cell counts
• Sterile, labeled cryovials
• Sterile, 15 ml-conical tubes
• Automated or manual controlled-rate freezing apparatus

1.   In a sterile, conical centrifuge tube, mix together the following reagents for every 1 ml of freezing medium needed:

2. Filter-sterilize the freezing medium through a 0.22 µm filter and place the tube on ice until use. Discard any remaining freezing medium after use.

1. Grow the desired quantity of DG44 cells in shaker flasks, harvesting when the cell density reaches 1 x 10⁶ viable cells/ml. Transfer cells to a sterile, conical centrifuge tube.
2. Determine the viable and total cell counts and calculate the volume of freezing medium required to yield a final cell density of 1 x 10⁷ viable cells/ml.
3. Centrifuge cells at 1,200 rpm for 5 minutes at room temperature and carefully aspirate the medium.
4. Resuspend the cells in the pre-determined volume of chilled freezing medium.
5. Place cryovials in a microcentrifuge rack and aliquot 1 ml of the cell suspension into each cryovial. 
6. Freeze cells in an automated or manual controlled-rate freezing apparatus following standard procedures. For ideal cryopreservation, the freezing rate should be a decrease of 1°C per minute.
7. Transfer frozen vials to liquid nitrogen for long-term storage.

You will use FreeStyle™ MAX Reagent to transfect suspension DG44 cells with your pOptiVEC™-TOPO® construct. Refer to the pOptiVEC™-TOPO® TA Cloning Kit manual supplied wit the kit for instructions to clone your gene of interest into the pOptiVEC™-TOPO® vector.
 
Plasmid Preparation

The pOptiVEC™-TOPO® plasmid must be clean, sterile, and free from contamination with phenol and sodium chloride for transfection into DG44 cells. Contaminants may kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using the S.N.A.P. Midiprep DNA Isolation Kit.
 
Linearizing the Plasmid

Prior to using the OptiCHO™ Express Kit to transfect DG44 cells with your pOptiVEC™ construct, you may linearize the plasmid. While linearizing your vector may not improve transfection efficiency, it increases the chance that the vector will integrate into the host cell genome without disrupting the gene of interest or other elements required for expression in mammalian cells.
 
We suggest using Pvu I, which cuts once in the ampicillin resistance gene. Other unique restriction sites are possible. A complete restriction map of pOptiVEC™-TOPO® is available at www.invitrogen.com. Be sure that your insert does not contain the restriction enzyme site you use to linearize your vector.
 
After digestion, precipitate the DNA, resuspend pellet in sterile water, and re-quantify using your method of choice.
 
Note:    If an appropriate linearization site is not present, you may transfect the circular plasmid. Transfection efficiency will not be affected.
 
Positive Control

If you performed the control TOPO® cloning reaction to clone the LacZα fragment into the pOptiVEC™-TOPO® plasmid, you may use this plasmid construct as a positive control for transfection. The DHFR gene in the plasmid will allow generation of stably-transfected DG44 cell lines in HT-deficient CD OptiCHO™ medium.
 
Calculate the number of DG44 cells that you will need for your transfection experiment and expand cells accordingly. Make sure that the cells are healthy and greater than 95% viable before proceeding to transfection.
 
Materials Needed

You should have the following reagents and equipment before beginning:

• Suspension DG44 cells cultured in complete CD DG44 Medium at 5 x 10⁵ cells/ml
• Purified, linearized pOptiVEC™-TOPO® plasmid DNA containing your gene of interest
• Optional: purified, linearized pOptiVEC™-TOPO® plasmid DNA containing the LacZα fragment, as a positive control
• FreeStyle™ MAX Reagent (supplied with the kit; store at +4°C until use)
• OptiPro™ SFM (supplied with the kit; pre-warmed to room temperature)
• Disposable, sterile, 125-ml polycarbonate Erlenmeyer flasks
• Orbital shaker in 37°C incubator with a humidified atmosphere of 8% CO₂
• Reagents and equipment to determine viable and total cell counts (e.g., Trypan Blue, hemacytometer, Coulter Counter)
• Vortex mixer

 
Optimal Transfection Conditions

To transfect suspension DG44 cells in a 30 ml volume, we recommend using the following optimized conditions:

• Final transfection volume: 30 ml
• Number of cells to transfect: total of 1.5 x 10⁷ cells (cell density at time of transfection should be 5 x 10⁵ cells/ml)
• Amount of plasmid DNA: 18 µg
• FreeStyle™ MAX Reagent: 15 µl

Note:   Further optimization of culture volume or transfection conditions is not necessary for stable cell line production.
 
Transfection Procedure

Follow the procedure below to transfect DG44 cells in a 30-ml volume. We recommend including a positive control (linearized pOptiVEC™ TOPO® plasmid with LacZa) and negative controls (no DNA, no FreeStyle™ MAX Reagent) in your experiment to help you evaluate your results.

1. At 48 hours before transfection, pass DG44 cells at 3 x 10⁵ cells/ml in complete CD DG44 Medium. Place the flask(s) on an orbital shaker platform rotating at 130-135 rpm at 37°C, 8% CO₂.
2. At 24 hours before transfection, pass DG44 cells at 3 x 10⁵ cells/ml in complete CD DG44 Medium. Place the flask(s) on an orbital shaker platform rotating at 130-135 rpm at 37°C, 8% CO₂.
3. On the day of transfection, perform a viable cell count. To ensure optimal transfection results, viability of cells must be over 95%.
4. For each transfection or control, transfer 1.5 x 10⁷ viable DG44 cells to a new 125-ml flask. Add pre-warmed, complete CD DG44 Medium to a final volume of 30 ml. Place flask in shaker until ready to transfect.

 
Note:   Do not spin cells down prior to transfection, as it will decrease transfection efficiency.
 

5. Gently invert the tube of FreeStyle™ MAX Reagent several times to mix. Do not vortex.
6. Add 18 µg of plasmid DNA and 15 µl of FreeStyle™ MAX Reagent into 1.2 ml of OptiPro™ SFM and mix gently.
7. Incubate the DNA-FreeStyle™ MAX mix for 10 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.
8. Slowly add 1.2 ml of DNA-FreeStyle™ MAX Reagent complex into the 125-ml flask containing cells while slowly swirling the flask.
9. Incubate transfected cell cultures at 37°C, 8% CO₂ on an orbital shaker platform rotating at 130-135 rpm.
10. At 48 hours post transfection, pass cells into HT-deficient, complete CD OptiCHO™ Medium. Proceed to the next section, Selecting for Stable Transformants

1. Determine viable and total cell counts using your preferred method.
2. Dilute the cells in pre-warmed complete CD OptiCHO™ Medium to give a final cell density of 3 x 10⁵ viable cells/ml (see Important, below).
3. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO₂ on an orbital shaker platform rotating at 130-135 rpm.
4. Cells should be passaged in fresh complete CD OptiCHO™ Medium every 2 days for 10-14 days until cell viability increases to >90%.

You will have a population of stably-transfected DG44 cells expressing your protein of interest at various levels. For most bioproduction applications, several clonally-derived cell lines producing your protein of interest are desirable for screening. However, the growth and optimization of each clone is dependent upon the integration locus of the plasmid, the response to amplification using MTX, and the nature of the protein.
 
Depending on your protein production needs, the time and effort required to generate clonal, high-producing cell lines is also variable. Several common pathways from stable pool to clone scale-up are outlined below.



Points to Consider

When choosing a workflow from the options above, you should consider the amount of protein needed for your application, your available resources, and the amount of time it will take to obtain your clonal, high-producing cell lines.
 
Additional media and other products may be purchased separately from Invitrogen.
 
Protocols

• To perform genomic amplification using MTX selection to obtain a population of cells expressing high levels of your protein of interest.
• To perform limiting dilution to obtain single clones expressing high levels of your protein of interest.
• To scale up your clones for protein production, Clone Scale-Up.

Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells by the folate transporter. In the cell, it is converted to a high molecular weight polyglutamate metabolite by folylpolyglutamate synthase, which binds to DHFR and inhibits its activity.
 
If MTX is present in the medium, cells compensate by increasing the DHFR copy number in the genome to overcome inhibition by MTX. Since the gene of interest is integrated into the same genetic locus as DHFR, the gene of interest is amplified as well, leading to increased production of the protein of interest (Kaufman et al., 1985; Tanaka et al., 2002). MTX (as methotrexate hydrate) is available from Sigma (10 mg, catalog number A6770).
 
MTX is toxic to the skin, eyes, and respiratory system. Wear suitable protective clothing, gloves, and eye and face protection when working with MTX. Refer to the product MSDS for complete precautions.
 
Preparing 1 mM MTX

To prepare a 1 mM MTX stock solution:
 

1. Dissolve 10 mg MTX in 22 ml of PBS.
2. Filter sterilize the solution through a 0.22 µm filter.
3. Store in 250 µl aliquots at -20ºC.

• Important: To make complete CD OptiCHO™ Medium with MTX, you will need to use complete CD OptiCHO™ Medium (prepared with L-glutamine) plus the required concentration of MTX.
• Important: Prepare media containing MTX fresh each time. Do not re-use prepared MTX media.

Preparing Media with MTX

Using the sterile, 1 mM MTX stock solution (prepared as described above), prepare media with the appropriate amount of MTX.
 
The recommended selection concentrations per round of selection are 50 nM, 100 nM, 250 nM, 500 nM, 1 µM, 2 µM and 4 µM. Be sure to make enough media containing MTX for your application.
 
Protocol

1. For each clone, spin down cells and aspirate old medium.
2. Seed cells at a density of 2 - 5 x 10⁵ cells/ml in 30 ml of media containing MTX in 125-ml flasks.
3. Incubate flasks at 37ºC/8% CO₂ with shaking at 130-135 rpm for 14-21 days or until viability >90%.
4. Optional: Repeat steps 1-3 with the next highest concentration of MTX.

 
Expected Results

In the experiment below, a pool of stable transformants were created after transfection of DG44 cells with pOptiVEC™-TOPO® vector constructs containing erythropoietin (EPO). The cells were exposed to increasing amounts of MTX, and EPO expression was determined by ELISA after each completed round of amplification (results shown on day 5 after seeding the cells at 2 x 105 cells/well).
 
The amount of EPO produced increases 50-fold after 7 rounds of genomic amplification using MTX




Next Steps

Because MTX selection produces a population of cells producing high amounts of protein, you will have to perform clonal selection by limiting dilution prior to clone scale-up.

• To grow DG44 cells under adherent conditions, use your clone optimization medium of choice, such as Gibco® Custom formulations F17 or CD-CHO-A.
• Because the growth and protein production of each clone are variable, you may need to optimize clonal selection conditions by adjusting the number of cells/well, the cloning medium, and various media supplements.
• Depending on how many clones you wish to screen before scale-up, you may increase the number of limiting dilution plates accordingly. The number of clones obtained from a 96-well plate is variable, depending on the experiment.

1. Determine the number and viability of your cells.
2. Make a 10-fold serial dilution using an appropriate volume of cloning medium such that you will obtain 1 cell per 100 µl.
3. Pipette 200 µl of diluted cells into each well of a 96-well plate to obtain ~2 cells/well.
4. Incubate plates without agitation at 37ºC with a humidified atmosphere of 5% CO₂.
5. Single cell clones will be readily apparent after 14 – 21 days post-plating (see Important below).

• Clones in 96 well plates that are >75% confluent
• Cloning medium of choice
• Complete CD OptiCHO™ Medium
• Sterile tissue culture dishes (96-well, 48-well, 24-well, 12-well, and 6-well) and sterile 125-ml polycarbonate flasks.
• Optional: TrypLE™ Express, if cells do not detach with gentle pipetting.

1. Add 100 µl fresh cloning medium to wells with single colonies and pipette gently until cells in the colony detach.
   
   Optional: If cells do not detach easily with gentle pipetting, aspirate media from wells, add 50 µl of TrypLE™ Express and incubate for 5 minutes at 37ºC. Add 50 µl of fresh cloning medium and pipet up and down until cells detach.
   
   
2. Transfer the entire amount of cells to 48 well plates and add an additional 250 µl of fresh cloning medium to each well.
3. Incubate plates undisturbed at 37ºC, 5% CO₂ until they are 80-90% confluent; usually 2-3 days depending on the clone growth.
4. Proceed to the next volume (i.e. 24-well).
5. Repeat Steps 1-5, transferring cells into a larger format tissue culture plate with each successive round.
6. Once cells are cultured in a 125 ml flask, switch medium to complete CD OptiCHO™ and incubate cells at 37ºC 8% CO₂, with shaking at 130-135 rpm.

Culturing DG44 Cells

The table below lists some potential problems and possible solutions that may help you troubleshoot your cell culture experiment.

• Kaufman, R., Wasley, L., Spiliotes, A., Gossels, S., Latt, S., Larsen, G., and Kay, R. (1985) Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase in Chinese hamster ovary cells. Mol Cell Biol 5, 1750-1759.
   
• Puck, T. (1958). J. Exp. Med. 108, 945.
  
  
• Sheeley, D. M., Merrill, B. M., and Taylor, L. C. (1997) Characterization of monoclonal antibody glycosylation: comparison of expression systems and identification of terminal alpha-linked galactose. Anal Biochem 247, 102-110.
  
  
• Tanaka, H., Tapscott, S., Trask, B., and Yao, M.-C. (2002) Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells. PNAS 99, 8772-8777.
  
  
• Urlaub, G., Kas, E., Carothers, A., and Chasin, L. (1983) Deletion of the Diploid Dihydrofolate reductase Locus from Cultured Mammalain Cells. Cell 33, 405-412.
   
• Werner, R. G., Noe, W., Kopp, K., and Schluter, M. (1998) Appropriate mammalian expression systems for biopharmaceuticals. Arzneimittelforschung 48, 870-880.