Gene Expression in HEK293 Suspensions

In Brief Download the PDF of this protocol

Transfection is inhibited by serum. Use media that is reduced-serum, serum-free ("SFM") or chemically defined ("CDM"). Here are some popular media:

• HEK293 cell culture at viable cell density of 1.5 to 2.0 x 10⁶ cells/mL
• Transfection-grade plasmid DNA (pDNA) with gene of interest, 1 μg per mL of culture to be transfected
• Optional Positive Control: GFP-encoding pDNA

General Guidance

Cell densities used are for typical HEK293 cultures, with maximum viable cell densities of ~4.0 x 10⁶ cells/mL. If using a high-density system, increase the values for cell density linearly. For example, Expi293™ media and Expi293F™ cells can support viable cell densities over 12 x 10⁶ cells/mL instead of ~4.0 x 10⁶ cells/mL, and so should be transfected at 3.15 x 10⁶ cells/mL instead of 1.05 x 10⁶ cells/mL.

The typical pDNA and PEI concentrations (1.0 and 3.0 mg/L, respectively) can achieve high transfection efficiency at viable cell densities from 1.0 x 10⁶ cells/mL up to 5.0 x 10⁶ cells/mL.

If performing the same expression many times, or many similar expressions, we would recommend co-varying on these parameters over corresponding ranges to find the optimal conditions:

Improvements in transfection efficiency are possible with changes outside the scope of this protocol. For further guidance on obtaining better yields please contact us at techsupport@bio-techne.com.

Before Transfection

Subculture and expand cells to obtain culture with viability greater than 95% and viable cell density between 1.5 to 2.0 x 10⁶ cells/mL at time of transfection.

Transfection

1. Immediately prior to transfection, ensure that viability is greater than 95% and viable cell density is 1.5 to 2.0 x 10⁶ cells/mL.
2. Dilute the viable cell density to 1.05 x 10⁶ cells per mL.
3. Invert pDNA and PEI STAR™ 1 mg/mL reagent containers several times to mix well.
4. The final transfection concentration is 1 μg pDNA for each mL of culture to be transfected. First prepare 20 μg/mL of pDNA using 5% final culture volume in a clean vial. For example, 100 mL cell culture requires 100 μg pDNA, prepare 100 μg pDNA in 5 mL of fresh media.
5. To a clean vial, transfer 3 μL PEI STAR™ 1 mg/mL for each mL of culture to be transfected. Use media to dilute to 60 μg/mL (5% final culture volume).
6. Mix together diluted pDNA and PEI STAR™. Invert several times and allow to rest capped at room temperature for 10 minutes. Gently invert the closed container once immediately before use.
7. Use 10 mL of pDNA/PEI mixture for each 90 mL of culture to be transfected. Gradually add mixture to culture while mixing.
8. Incubate cells per typical conditions.

Post Transfection

If using a feed, booster, supplement, or enhancer, these can be added any time six hours post-transfection. Subcultures can also be prepared after six hours.

If using a GFP control, transfection efficiencies over 70% should be observed after 48 hours. For optimized procedures, efficiencies over 80% are reasonable.

Monitor expression levels and harvest upon observing plateaued titers. For typical processes, secreted proteins will be highest at 5 to 7 days post transfection.

References

Boussif et al (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc.Natl.Acad.Sci.U.S.A. 92 7297. PMID: 7638184.

Longo et al (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 529 227. PMID: 24011049.

Support

Support is available by emailing us at techsupport@bio-techne.com.

Gibco, Opti-Mem, FreeStyle, ExpiCHO, and ExpiCHO-S are trademarks or registered trademarks of Thermo Fisher Scientific. BalanCD is a FUJIFILM Irvine Scientific brand. HyClone, HyCell, TransFX-C are brands of Cytiva.