FreeStyle™ MAX Reagent i

Introduction

FreeStyle™ MAX Reagent is a proprietary, animal origin-free formulation for the transfection of plasmid DNA into eukaryotic cells that can easily be scaled up to produce large amounts of recombinant proteins. FreeStyle™ MAX

Reagent allows the highest expression levels and transfection rates with lowest cytotoxicity in bio-production applications, and is specifically formulated for use with:

  • FreeStyle™ 293-F Cells (suspension human embryonal kidney cells, Cat. no. R790-07) in serum-free FreeStyle™ 293 Expression Medium (Cat. no. 12338-018)
  • FreeStyle™ CHO-S Cells (suspension Chinese Hamster Ovary cells, Cat. no. R800-07) in serum-free FreeStyle™ CHO Expression Medium (Cat. 12651-014)
  • DG44 Cells (DHFR- suspension CHO cells) in CD DG44 Medium with 8 mM L-glutamine and 18 ml/L 10% Pluronic® F-68 (Cat. no. 12609-012, 12613-014) FreeStyle™ MAX Reagent is intended for use with the FreeStyle™ MAX 293 Expression System (Cat. no. K9000-20), FreeStyle™ MAX CHO Expression System (Cat. no. K9000-10), and OptiCHO™ Express Kit (Cat no. 12745-014).

For more information refer to our Web site (www.invitrogen.com) or contact Technical Support.


Guidelines for Transfection


  • FreeStyle™ 293-F and CHO-S Cells, and DG44 Cells should be cultivated in suspension in a humidified 37oC, 8% CO2 environment on an orbital shaker.
  • CHO-S Cells can be transfected in 0.5X Pen/Strep (Cat no. 15140-122), but many other FreeStyle™ MAX transfections are done without antibiotics. Work under sterile conditions and prevent contamination of your DNA.
  • Clumping of the cells lowers transfection efficiency; prevent this by the suggested frequent passage schedule an  agitation (see below). Do not use anti-clumping agent to the cells during routine culture or prior to transfection. Anti-clumping agent (Cat. no. 0010057AE) may be added post-transfection.
  • We recommend using OptiPRO™ SFM (1X), liquid (100 ml, Cat. no. 12309-050) to dilute the DNA and lipid before complexing.
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Protocol - Culturing of FreeStyle™ Cells

For routine culturing of FreeStyle™ 293-F Cells, shake at 135–155 rpm, and keep cell densities between 0.1 and 3.0 × 106 cells/ml of culture. A cell density above 3.0 × 106 cells/ml will result in a loss of transfection efficiency. For routine culturing of FreeStyle™ CHO-S cells, shake at 120–135 rpm, and keep cell densities between 0.05 and 1.5 × 106 cells/ml of culture. Make sure to supplement FreeStyle™ CHO Expression Medium with L-glutamine (Cat. no. 25030-081) to a final concentration of 8 mM. If large numbers of cells are needed, seed cultures at 0.5 × 106 cells/ml and use cells as soon as they reach a density of 5 × 106 cells/ml (3–4 days).
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Transfecting FreeStyle™ Cells for Protein Expression

Use this procedure to transfect DNA into FreeStyle™ 293-F or CHO-S cells. Use sterile DNA, or filter-sterilize before use (re-quantify your DNA after filtration). All amounts are on a per flask basis for 30 ml cultures in a 125 m  shake flask; for other formats, see Scaling up or Down Transfections (below).

  1. Approximately 24 hrs before transfection, pass FreeStyle™ 293-F cells at 6–7 × 105 cells/ml, shake at 135–155 rpm. Pass FreeStyle™ CHO-S cells at 5–6 × 105 cells/ml, shake at 120–135 rpm. Culture at 37°C, 8% CO2.

  2. On the day of transfection, the cell density should be about 1.2–1.5 × 106/ml. Dilute the cells to 1 × 106 /ml with growth medium. To ensure optimal transfection, viability of cells must be > 95%. Add 30 ml of cells to each flask.

  3. Gently invert the tube of FreeStyle™ MAX Reagent 4 times (do not vortex).

  4. Dilute 37.5 μg of plasmid DNA into OptiPRO™ SFM to a total volume of 0.6 ml and mix. In a separate tube, dilut  37.5 μl of FreeStyle™ MAX Reagent in OptiPRO™ SFM to a total volume of 0.6 ml and mix gently by inverting the tube (do not vortex). Immediately add diluted FreeStyle™ MAX Reagent to diluted DNA solution to obtain a total volume of 1.2 ml and mix gently.

  5. Incubate the DNA-lipid mixture for 10–20 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.

  6. Slowly add 1.2ml of DNA-lipid mixture into the 125ml flask containing cells while slowly swirling the flask.

  7. Incubate transfected cell cultures at 37°C, 8% CO2 on an orbital shaker set to 135 rpm for both FreeStyle™ 293-F cells and FreeStyle™ CHO-S cells. There is no need to change or supplement the medium during the first 6 to 7 days.
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Optimizing Protein Expression in FreeStyle™ Cells

  • Protein expression can be detectable within 4 to 8 hours of transfection, with maximal protein yield usually between 1 and 7 days post-transfection, depending on the protein expressed.
  • When expressing a protein for the first time, perform a time course experiment between days 1 and 9 post-transfection to identify the peak of protein production, and to monitor cell viability.
  • Vary amounts of plasmid DNA and FreeStyle™ MAX Reagent. For 30 ml cultures, try a range between 24–45 μg plasmid DNA and 24–45 μl lipid.
  • For secreted IgG protein production, we have observed peak yields at 5 to 7 days post-transfection.
  • To assess transfection efficiency via a GFP-type fluorescent protein, we recommend monitoring the cultures starting at 24 hours post-transfection.
  • For optimizing protein expression while scaling up culture volumes, see Scaling up or Down Transfections (below).
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Scaling Up or Down Transfections of FreeStyle™ Cells

To transfect cells in different culture volumes, vary the amounts of FreeStyle™ MAX Reagent, DNA, cells and medium in proportion to the culture volume, as indicated in the table below:
 
Cell CultureDilution VolumeDNAFreeStyle™ MAX Reagent
Volume   Flask  Starting Point     Optimization Range Starting Point     Optimization Range
30 ml      125 ml2 × 0.6 ml37.5 μg                 24–45 μg37.5 μl                 24–45 μl
250 ml     1 liter2 × 5 ml312.5 μg               200–375 μg 312.5 μl               200–375 μl
1 liter       3 liter2 × 20 ml1.25 mg                0.8–1.5 mg1.25 ml                0.8–1.5 ml


For culture volumes above 30 ml further adjustments may be necessary:

  • Lower the speed of the orbital shaker if foam is generated. In 1 L cultures, we recommend 70–80 rpm for FreeStyle™ CHO-S Cells, and as close to 135 rpm as possible (without creating foam) for FreeStyle™ 293-F Cells.
  • In 1 L cultures, we recommend incubating the DNA-lipid mixture for 20 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.
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Transfecting DG44 Cells to Generate Stable Cell Lines

Use this procedure to transfect linearized DNA into DG44 cells. Use 30 ml cultures in 125 ml shake flasks; all amounts are given on a per flask basis. See the OptiCHO™ Express Kit manual for culturing and other information (available from our Web site, www.invitrogen.com, or by contacting Technical Support).

  1. At 48 hours before transfection, pass DG44 cells at 3 × 105 cells/ml, shake at 130–135 rpm at 37°C, 8% CO2. Culture in CD DG44 Medium with 8 mM L-glutamine and 18 ml/L of 10% Pluronic® F-68 (Cat. nos. 12610-010, 25030-081, and 24040-032 respectively).

  2. At 24 hours before transfection, again pass DG44 cells at 3 × 105 cells/ml.

  3. On the day of transfection, prewarm the CD DG44 Medium (with 8 mM L-glutamine and 18 ml/L of 10% Pluronic® F-68) to 37°C.

  4. Count cells (viability must be > 95%) and add 1.5 x107 cells in a total volume of 30 ml CD DG44 medium to each flask. Place flask in shaker until transfection.

  5. Gently invert the tube of FreeStyle™ MAX Reagent 4 times (do not vortex).

  6. Add 18 μg of linearized DNA and 15 μl of FreeStyle™ MAX Reagent into 1.2 ml OptiPRO™ SFM (at room temperature) and gently invert to mix.

  7. Incubate the DNA-lipid mixture for 10 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.

  8. Slowly add the 1.2 ml of DNA-lipid mixture 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. Place cells on selective medium (CD OptiCHO™ Medium, Cat no. 12681-011) 48 hours post-transfection. 
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16447.pps     29-Aug-2008