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.

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 × 10 6 cells/ml of culture. A cell density above 3.0 × 10 6 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 × 10 6 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 × 10 6 cells/ml and use cells as soon as they reach a density of 5 × 10 6 cells/ml (3–4 days).

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.

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).

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.

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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