Our lab is facing a difficult problem for almost a year. After thawing the cells are okay, but after 2nd or 3rd passage there are debris present between the cells. We use mycoplasma removed cells but still debris are coming. This is happening with all the cell lines including HEK293T, ShSY5Y, Neuro2A.
Hello Madhuparna
I have a feeling the protocol used for freezing cells is not proper. After thawing do you check the viability of the cells? It has to be greater than 65% to 70%. Usually cell debris disappear after the first passage.
Try replacing the culture medium with fresh medium before subculturing in order to get rid of cell debris. Also revisit your freezing protocol since many of your cell lines are facing the same problem.
Regards.
Hello Madhuparna Chakraborty
We were also facing a similar problem initially. So, I recommend you to be a little more careful with the process of cell dissociation using the trypsin solution, as trypsin would also damage the cells. Also, try to give at least 2 times PBS wash to the cell pallets, in order to get rid of dead cells before seeding them for the next passage.
Hope it will be helpful to you.
Good luck!
I think it is clearly because of an error during the preparation of frozen cells. It may take few passes ( more than 2 or 3 for sure), to get them back to normal. I would recommend you to go for slow passes. I remember having similar problem in the past.
If you have mycoplasma it is a big issue, and we prefer to take new cells rather than cleaning them by different available drugs. If the mycplosma is not the issue, you may check your serum batch, spin carefully the cells when you thaw them and make sure that you dont overgrow the cells.
In general passaging of cells using trypsin or cell scraper causes transient amount of cell damage which is usual. When cells are hardened enough, this issue will be resolved automatically after a few passages.
However contamination, Using media or serum without filteration may also be a reason.
- If cells are in 4ml media wash them with 4ml PBS and reduce trypsin exposure or concentration.
- Perform sterlity test of complete, incomplete media, serum.
-Filter complete media with 0.25 micron filter.
- You can also reduce serum quantity a bit sometimes high serum protein quantities do this.
- If it is after revival, surely it is due to cell damage or contamination.
Hi, I will tray to respond you despite the fact that I am not an expert in cell line culture. I started to do that a few moths ago to establish and maintain Lice cell line based on their nits. I observed this phenomena every time when I do my 2-3 even 4th passage. It was weird but I understand that it's normal like an apoptosis of the extra cell mass. Only embryonal cells remain and start to multiply if the cell maintain is good, you will observe an increasing cell mass in the next passages.
I will take this opportunity to ask a question. In my case, I can't maintain my body lice cell lines. Indeed, I started the project in 2020 summer and until now it's the same results. I loose cells mass in every passage even that I do not perform centrifugation when I change medium. Lice cells are semi-adherant and I do that in plate of 6 wells not in a flask (did not work). I tried to maintain in Leibovitz’s L-15 and Shneider medium, supplemented with 10-20% FBS, 10% tryptose phosphate broth and 1% penicillin-streptomycin (100 units/mL). Cells were incubated at 28 °C without CO2. Same results each time. I'M MISSING SOMETHING !!!!!
I do not want to abandon this project. I want to try again for a last time before the abandon. I will appreciate every suggestion and will be very grateful for that.
Thank you :)
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