293EF Protein Production

Note

293EF is the same as Invitrogen's Expi293F, features:

• Doubling time in the range of 22–27 hours
• Cell densities of up to 10 × 106–12 × 106 cells/mL in shaker or spinner culture
  
  

Culture of 293EF (Expi293F) Cells

Expi293 Expression Medium is formulated with GlutaMAX-I reagent; for suspension growth and transfection applications, use the Expi293 Expression Medium without any supplementation. 5 mL/L of Antibiotic-Antimycotic containing penicillin, streptomycin, and amphotericin B may be used when required

Store frozen cells in liquid nitrogen until ready to use. To thaw and establish cells:

Thawing

1. Remove the vial of cells from liquid nitrogen and place it in a 37°C water bath for 1 to 2 minutes to thaw the cells rapidly with gentle agitation. Do not submerge the vial in the water.

2. Just before the cells are completely thawed, decontaminate the vial by wiping it with 70% ethanol before opening it in a Class II biological safety cabinet

3. Using a 2-mL or 5-mL pipette, transfer the entire contents of the cryovial into a 125-mL polycarbonate, disposable, sterile, vent-cap Erlenmeyer shaker flask containing 29 mL of pre-warmed Expi293TM Expression Medium.

4. Immediately post-thaw, monitor viable cell density and viability using a hemocytometer with the trypan blue exclusion method or using an automated cell counter. Cell density should be approximately 0.3 × 10^6 cells/mL and cell viability should be above 80%.

5. Incubate the cells in a 37°C incubator with humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 125 rpm.

6. The day after thawing, determine the viable and total cell counts (see protocol on page 6). Cells viability will drop slightly but should remain above 70% and reach over 90% by day 2–4 post-thaw. Thaw out a new vial of Expi293F Cells if the cell viability is below 70%.

7. Once the culture has reached >1 × 10^6 viable cells/mL (typically 2–4 days), subculture the Expi293 F Cells by seeding shaker flasks at 3 × 10^5 viable cells/mL in pre-warmed Expi293 Expression Medium. Use  a 125-mL or 250-mL polycarbonate, disposable, sterile, Erlenmeyer flasks for 30 mL or 60 mL total working volume of cell suspension, respectively.

8. Continue to monitor cell density and viability. Once the culture has reached >3 × 10^6 viable cells/mL (typically 3–4 days), subculture the Expi293F Cells by seeding shaker flasks at 3 × 10^5 viable cells/mL in pre-warmed Expi293 Expression Medium.

Subculture Expi293F Cells

When the cells reach a density of approximately 3 × 10^6– 5 × 10^6 viable cells/mL, typically every 3–4 days, split the Expi293 F culture to 0.3 × 10^6–0.5 × 10^6 cells. When maintaining Expi293F Cells.  Usually a 125-mL or 250-mL polycarbonate, disposable, sterile Erlenmeyer flask is used for 25–40 mL or 50–80 mL total working volume of cell suspension, respectively.

Glass flasks without baffles may be used, but thorough cleaning after each use is essential to avoid potential toxicity, which is more problematic in serum-free cultures.

1. Determine viable and total cell counts

2. Using the cell density determined in Step 1, calculate the split ratio needed to seed the new shaker flask at 0.3 × 10^6–0.5 × 10^6 viable cells/mL.

3. Dilute the cells in fresh, pre-warmed Expi293TM Expression Medium to give a final cell density of 0.3 × 10^6–0.5 × 10^6 viable cells/mL in the desired final volume.

4. Incubate flasks in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 125 rpm.

5. Repeat Steps 1–4 as necessary to maintain or expand cells.

Scale Up

It is possible to scale up the Expi293 F cultures in spinner flasks or bioreactors. The appropriate spinner or impeller speed and seeding density should be determined and optimized for each system. At Life Technologies, we have determined the optimum spinner speed to be 100–130 rpm, and impeller speed in Celligen® stirred tank bioreactors to be 70–100 rpm. We recommend seeding the cells at 0.3 × 10^6–0.5 × 10^6 viable cells/mL.

Transfection

30 mL Transfection

Final transfection volume: 30 mL

• Number of cells to transfect: 7.5 × 107 cells (final cell density of 2.5 × 10^6 cells/mL) with >95% viability
  
• Amount of plasmid DNA: 30 μg
  
• Amount of ExpiFectamine 293 Reagent: 40–100 μL (we typically use 80 μL).
  
• Use 2.7 μL ExpiFectamine 293 Reagent per 1 μg of plasmid DNA transfected
  
  

1. The day before transfection (day 0), determine the number of cells. For each 30-mL transfection, 7.5 × 10^7 cells in 25.5 mL of Expi293 Expression Medium is required.

2. To transfect cells on the following day (day 1), seed the cells at a density of 2.0 × 10^6 viable cells/mL and incubate at 37°C in a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.

3. On the day of transfection (day 1), determine cell viability using an automated cell counter or the trypan blue dye exclusion method.
Important: Cell density should be >3 × 10^6 cells/mL and the viability of cells must be over 95% to proceed with transfection. Cells should not be clumping.

4. Calculate the volume of cell suspension containing the number of cells needed for one transfection. For each 30 mL transfection, 7.5 × 10^7 cells are required.

5. Add the appropriate volume of cell suspension to each sterile, disposable 125-mL Erlenmeyer shaker flask and bring up the volume to 25.5 mL by
adding fresh, pre-warmed Expi293 Expression Medium for each 30 mL transfection.

6. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.

7. For each transfection sample, prepare lipid-DNA complexes by performing the following:

a) Dilute 30 μg of plasmid DNA in Opti-MEM I medium to a total volume of 1.5 mL. Mix gently.
b) Gently mix the ExpiFectamine 293 Reagent by pipetting it up and down before use.
c) Dilute 80 μL of ExpiFectamine 293 Reagent in Opti-MEM I medium to a total volume of 1.5 mL. Mix gently and incubate for 5 minutes at room
temperature.
Note: Longer incubation times may result in decreased activity.
d) After the 5 minute incubation, add the diluted DNA to the diluted ExpiFectamine 293 Reagent to obtain a total volume of 3 mL. Mix gently.
e) Incubate DNA-ExpiFectamine 293 Reagent mixture for 20–30 minutes at room temperature to allow the DNA-ExpiFectamine 293 Reagent complexes to form.

8. After the DNA-ExpiFectamine 293 Reagent complex incubation is complete, add 3 mL of DNA-ExpiFectamine 293 Reagent complex to each shaker flask from Step 6. To the negative control flask, add 3 mL of Opti-MEM I medium instead of the DNA-ExpiFectamine 293 Reagent complex. Each flask should now contain a total volume of 28.5 mL.

9. Incubate the cells in a 37°C incubator with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm.

10. Approximately 16–18 hours post-transfection, add 150 μL of ExpiFectamine 293 Transfection Enhancer 1 and 1.5 mL of ExpiFectamine 293 Transfection Enhancer 2 to each flask. The final volume should be approximately 30 mL in each 125-mL flask.

11. Cells or media (if recombinant protein is secreted) may be harvested beginning at approximately 48 hours post-transfection and assayed for
recombinant protein expression. Time for optimal protein expression depends on the nature of your recombinant protein.

Optimizing Protein Expression

Time course experiment: Harvest cells or media at 24, 48, 72, 96 hours post-transfection.

The Expi293 Expression Medium is designed to support the transiently transfected culture for up to 7 days. Optimal yield of the rabbit IgG
clone expressed from the Antibody Expressing Positive Control Vector occurs at 6–7 days post-transfection

Scale Up Transfection

	Transfection Volume
	1 mL (24-well plate)	2 mL (6-well plate)	30 mL	1 liter	3.8 liter
Total Number of Cells*	2.5 × 10^6	5.0 × 10^6	7.5 × 10^7	2.5 × 10^9	9.5 × 10^9
Amount of DNA	1 μg	2 μg	30 μg	1 mg	3.8 mg
DNA Dilution Volume (in Opti-MEM I)	to 50 μL	to 100 μL	to 1.5 mL	to 50 mL	to 190 mL
Amount of ExpiFectamine 293 Reagent	2.7 μL	5.4 μL	80 μL	2.7 mL	10.3 mL
ExpiFectamine 293 Reagent Dilution Volume (in Opti-MEM I)	to 50 μL	to 100 μL	to 1.5 mL	to 50 mL	to 190 mL
Lipid/DNA Complex Volume	100 μL	200 μL	3 mL	100 mL	380 mL

• Final cell density of 2.5 × 10 cells/mL
  

Control:

Transfect 30 mL of suspension Expi293F cells using 30 μL of the Antibody Expressing Positive Control Vector (i.e., 1 μg of positive control per 1 mL of
Expi293F culture) following the protocol provided on pages 11–12. The rabbit IgG expressed by the control will be secreted into the Expi293
Expression Medium. Expression can be detected by day 3 post-transfection; however maximum yield is obtained 6–7 days post-transfection. The expected
yield of the control IgG is approximately 200–300 mg IgG per liter of culture on day 7

Protein Purification

Components of the Expi293 Expression Medium can strip Ni-NTA resins, interfering with the ability of the affinity resin to bind polyhistidine-tagged
proteins. Therefore His-Assist Nickel Affinity Purification Aid (Cat. no. A14644) should be added to medium if Ni column is used for protein purification.

Use the appropriate amount of resin for the expected yield of protein. Affinity resins have various binding capacities; Protein A Agarose from Invitrogen
(Cat. no. 15918-014) has a binding capacity of >18 mg of human IgG per mL of gel.

1. Prepare resin for binding: Prepare a column containing the appropriate amount of Protein A resin to bind the amount of recombinant protein
produced in Expi293F culture. Pass at least 5 column volumes of 1X PBS over the column to remove the preservative solution.

2. Prepare IgG-containing culture medium sample: Centrifuge the cells out of the transfected culture (10 minutes at 2000–5000 × g). Repeat if necessary.
Filter the culture medium through a 0.2-μm filter (optional). Supernatant (i.e., medium) can be stored at 4°C for several days or –20°C longer term.

3. Bind IgG to Protein A resin: Pass the desired volume of clarified media over the prepared Protein A column.

4. Wash resin: Pass at least 10 resin volumes of 1X PBS over the column. Collect the unbound protein fraction and save at 4°C or –20°C for future analysis.

5. Elute and recover IgG: Elute by adding at least 5 resin volumes of elution buffer and collect the fractions. Neutralize the fractions by adding 1/10th
volume of neutralization buffer.

6. Analyze: Analyze IgG levels in the eluted samples by ELISA or SDS-PAGE.

Vector for Protein Production:

1395 pCMV5-hIgG-Lc-TagGFP-Fc(3c)-WPRE-OriP

Enhancer Plasmid:
1348 pLVX-EF1-VP16-EBNA1c (EF1 promoter): for stable/longterm expression
or 1392 pAAV-VP16-EBNA1c (for transient expression).