Hello, I am having trouble producing a protein using the Expi293 cell line.
To be clear, I have successfully produced this protein using the mother cell line but after freezing and thawing the daughter cell lines, I keep failing.
When the product first came, I thawed the cells and subcultured them until P3, which is when I froze the cells for later usage. I transfected the mother cell line with the Expifectamin transfection kit and successfully produced the proteins with good yield. The cells were subcultured until P20, which is when I discarded the cells and thawed the cyropreserved cells.
The cells divide in a healthy condition with normal morphology, but they simply fail to produce the desired proteins following transfection. I have tried using a completely freshly new transfection kit and culture media but this didn't help.
The conclusion I have reached is perhaps (by incredibly bad luck) the Expi293 cell line lost its protein producing ability during freezing procedure. Has anyone experienced such problems regarding protein production (even better if you have used the Expi293 cell line)? I would love to hear opinions and tips to make this work.
Dear Youngjae Hong
We are using Expi293 and ExpiCHO routinelly and we never observed a decreasing of expression due to cell frezeeng and storage at -150°C.
Similarly to you we amplified the original stock and at P2 and P3 we freeze several aliquotes. The aliquotes (with 5% of DMSO) were stored at -150°C and we de-froze it every 5-6 months, once the in use aliquote reach the passage 30-35. Our aliquotes are stored at -150°C from at least 5 years but they still work very well after de-freezing?
just some question:
1) how do you freeze and de-freze cells?
2) How did you store it?
3) Are you sure that your transfection reagents are fine?
4) Are you sure that you do not experience some cell contaminations (eg mycoplasma?)
best regards
Manuele
Thanks Manuele Martinelli for your detailed response.
First of all, to answer all your questions,
1-2) I froze the cells using the cell freezing container with isopropanol at -80oC for 2 days and then stored the cells in liquid nitrogen tank (approximately -200oC) with 10% DMSO (according to the Expi293 user guide). When I de-freeze, I use the water bath (37oC) to quickly thaw the cells and transfer them into 30mL of pre-warmed Expi293 media.
3) I'm pretty sure the reagents were fine because we opened a completely new vial just in case the previous one might be contaminated.
4) Looking at the color of the media, smell, and cell morphology under the microscope, contamination doesn't seem to be the case.
Also, I have recently found out that the Expi293 user guide had revised their freezing protocol originally from 10% DMSO to 7.5% DMSO. Could using 10% DMSO be the problem?
We are planning on buying a new vial of Expi293 but I'm afraid the same issue may occur again.
Hi Youngjae Hong,
I've read about instances where 10% DMSO decreases viability of certain cell types. It would be interesting to see what you find if you change DMSO concentration. The change to 7.5% suggests they are aware of an issue.
Did your mother cell line transfect well for 20 passages?
Regards,
Martin
Hello Martin O'Reilly ,
The mother cell line worked fine for 20 passages. I'll try stocking the cells with lower percentage of DMSO once the new cells arrive.
Best regards,
Youngjae
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India