Hello all! I am new to cell culture and just recently started to use HEK293T cells (adherent). Sometimes when I passage cells I will need to make sure all cells in the suspension are ideally single (i.e., not clumped or stuck together for whatever reason), for e.g., accurate cell counting or isolation of cell colonies. However, in my case it is quite normal to see cell clumps.
Do you have any suggestions as to alleviating such problem? For example, the time of trypsinization? Or, ...?
Accumax can be used in place of Trypsin. This will not only give you single cell suspensions, but it won't lyse your cells from extended incubations times.
Hello Jian Wang
Some cell lines are loosely attached to the substratum, HEK293T cells being one of them. They have a tendency to detach from the surface. In such a case you may detach the cells by tapping the bottom of the growth medium on the work surface of the sterile hood or by gently pipetting the medium onto the cells.
However, tapping or pipetting nearly never gives a single-cell suspension. So, to detach the cells into single cell suspension, you may either use EDTA alone or use brief trypsinization (less than 1-2 mins). Do not overtreat these cells with trypsin.
Best.
Malcolm Nobre Thanks for the answer! Yes, trypsinisation is what I always do. But still, I come across cell clumps quite often. Like you mentioned in the end - do you think over-trypsinisation could be a reason for seeing those clumps (causes cell death > release of DNA > cells stuck together)?
Yes, you are right. Over-trypsinization is one of the reasons for clump formation. It causes cell death resulting in release of DNA from dead cells. DNA being sticky in nature can cause cells to clump.
In my experience, when HEK293T cells are grown too long and are too crowded, you will tend to get aggregates when trypsinizing.
Hi Jian Wang,
could it be an option to use cell strainers? If you do not need 100% of your cells, filtering your cell suspension will remove of most of the cell clusters and produce a single cells suspension. This is what we do for single clone generation.
Best,
Sandra
Sandra Ackermann Actually it is a good idea. Yes, one important application in my case of making single cell suspension is producing single clones on a dish. However, filtering cell is also a routine step before I do single cell sorting, but I still find big cell clumps in that case.
This was the same when we used standard mesh sizes strainers (40 and 70µm if I remember correctly). We have nowchanged our protocol to 10µm and got better single cell suspensions. However, this might result in a non- representative composistion of the suspension, because you enrich for "non-sticky" cells, but for our application, this was not problematic.
Try poly-lysine coated flasks if possible. Use dilute trypsin, 1ml trypsin+1ml PBS. Do not over-trypsinize. Hope it will help.
You are welcome. No, I used 293 for virus infection purpose, never did any signalling study on the cell line. Based on morphology and growth rate, didn't see any difference between coating and non-coating.
Jialei Sun hello，I would like to ask what concentration of fetal bovine serum is needed to maintain cell monolayers in HEK293 during virus culture. I have used DMEM medium supplemented with 2% FBS when inoculating the virus, and the cells grew very densely for about 24h and then fell out of the flsak 25.
Fuyou Che I used DMEM+5% FBS. Yes, it does grow quickly sometimes. Maybe you can start virus culture from low cell confluency, such as 50%, 75%. Or, you can try infecting the cells at high MOI, such as 10.
Fuyou Che Hey Fuyou, I am super new to cell culture but I have been advised to start with ~30% cell confluency when infecting HEK293T with lentivirus. I use DMEM + 10% FBS, as much as what I use for regular cell culture. Because the gene of interest will be integrated together with a puromycin resistant gene, I apply puromycin at 0.7 ug/mL. And all these combined usually does well in inhibiting cell growth for the first week, and of course eventually cells with integrated genes will dominate. Just something that might help.
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