Gene Expression in CHO Suspensions
This is intended as a guide only; for full experimental details please read the references provided.
In Brief Download the PDF of this protocol
Protocol for Transient Gene Expression in CHO Suspensions using PEI STAR™ Transfection Reagent
Materials
- PEI STAR™, 1 mg/mL, pH neutralized, sterile-filtered
- CHO expression medium maintained at 37°C
Transfection is inhibited by serum. Use media that is reduced-serum, serum-free ("SFM") or chemically defined ("CDM"). Here are some popular media:
| Vendor | Suitable Media |
|---|---|
| Thermo Fisher Scientific |
Gibco™ Opti-MEM™ I Reduced Serum Media Gibco™ FreeStyle™ F17 Expression Medium Gibco™ ExpiCHO™ Expression Medium (with ExpiCHO-S™ Cells) |
| Cytiva | HyClone HyCell TransFX-C |
| FUJIFILM Irvine Scientific | BalanCD CHO (Recommended) |
- CHO 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.
- Depending on cell culture media, sodium butyrate or sodium valproate (~500 mM, neutral pH).
- Optional Positive Control: GFP-encoding pDNA.
General Guidance
Cell densities used are for typical CHO 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, ExpiCHO™ media and ExpiCHO-S™ cells can support viable cell densities over ~12 x 106 cells/mL instead of ~4.0 x 106 cells/mL. Accordingly, this system should be transfected at 3.15 x 106 cells/mL instead of 1.05 x 106 cells/mL.
The typical pDNA and PEI STAR™ concentrations (1.0 and 5.0 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 recommend co-varying these parameters over the corresponding ranges to find the optimal conditions:
| Parameter | Range |
|---|---|
| PEI Concentration | 3.50 to 6.50 mg/(L final culture) |
| DNA Concentration | 0.75 to 1.50 mg/(L final culture) |
| PEI/DNA Complex Time | 5.0 to 15.0 minutes |
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
- 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.
- Dilute the viable cell density to 1.05 x 106 cells per mL with media.
- Invert pDNA and PEI STAR™ 1 mg/mL reagent containers several times to mix well.
- 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.
- To a clean vial, transfer 5 μL PEI STAR™ 1 mg/mL for each mL of culture to be transfected. Use media to dilute to 75 μg/mL (5% final culture volume).
- 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.
- Use 10 mL of pDNA/PEI mixture for each 90 mL of culture to be transfected. Gradually add mixture to culture while mixing.
- 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.
Depending on the cell culture media, it may be necessary to add sodium butyrate to the media to obtain gene expression in CHO suspensions. To determine if necessary, prepare 5 post-transfection subcultures and add sodium butyrate to final concentrations of 0, 2.5, 5.0, 7.5, or 10 mM to find appropriate concentration based on the final yields. Sodium valproate can be used instead.
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. Other processes, such as using low temperatures and/or using a batch feed to obtain maximal yields, will change the peak harvest window.
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.