Hi Syed,

I have used 293T cells for a while now for lentivirus preparations or as regular cell model and unfortunately I never noticed any changes in morphology. The only thing I am can see is that I have never coated my plates with anything. Do you really have to coat the plates? Maybe you could try poly-L-lysine instead of fibronectin?

If that doesn't work, I suggest you get 293 from an other source (even an other lab) to get rid of any eventual contamination by mycoplasm. 

I n any case,  I don't think you should observe any changes.

I hope this will help!

Mathieu

Hi Matheiu,

Many thanks for your kind reply. May be you are right, it may be mycoplasm contamination I will ask if anyone else have the 293 stock to double check.

Many thanks

Syed

Hi Matheiu, 

Do you know a cheap method to detect the presence of mycoplasm in culture, commercially available kits are very expensive.

Many thanks 

Syed 

The changes in cell appearance/morphology can be quite common when changing media. Things to do to avoid this:

1. Equilibrate media to 37C and even incubate at 5% CO2: Try this for DMEM to bring it to the correct pH before changing media. 

2. Avoid leaving cells without media for any prolonged period.

3. Avoid pippeting directly onto the monolayer when changing media. In our experience 293 cells are not that sticky to normal culture plates. Transfected/stable cells are even more sensitive.

4. After media change give your cells sufficient time to recover. 

5. Do not overgrow your cells: change media regularly and passage cells. 

As suggested above check your cells for contamination: mycoplasma is a culture killer but can be easily detected. We conduct routine screening of all new cultures.

good luck

Hi Syed,

You can check for mycoplasma by staining your cells with DAPI. It is a rapid, easy and cheap way to detect if you have any contamination and has the advantage to recognize any kind of mycoplasma when the kits general target only some species.

Here is a protocol I found online to stain the cells with an example of what you would see if there is any contamination: the mycoplasms are situated around the membranes of your cells and so they appear as if you have dots in the cytolpasms.

Hope it helps!

Mathieu 

http://weill.cornell.edu/vascularbiology/protocols_and_downloads/pdf/Mycoplasm-Test_HLA.pdf

The media changes do have initial 1-2 passage changes in little in morphology but after that it adept. Its also depends on if media do not have big changes in ingredient. Contamination is also one of problem but if you observe very early of passage than it should be not the case but mycoplasma is one of the panic stuffs always and better to check it. In my view changes the media do have changes in intial passage and later cells adept to it. 

Daer Mathieu,

many thanks for your kind reply. i will do a check as soon as cells are ready to passage, thans for the link.

Best, syed

Hi Saroj,

Thanks for your input here. Infact my cells are more than at passage 12. But since I have doubt they might have been, infact I am almost sure of mycoplasm contamination, so I have borrowed a 293T cells (which is at passage 5 now) from near by lab to see how they behave in the same condition. Further iwish to continue with this passage 5 cells.

Dear Matheiu & Saroj,

I have another concern too. IN almost all my culture I can see very tiny small black dot like particles (not too much, and also I don't think they are hampering the growth of cells atleast my Neural Stem cells, but they are present in allmost all culture, Infact i have also checked in my colleagues falsk, they are their also. I am not sure what they are but might by myco/bacteria or something, which may be a threat for my 293 cells since they are very fragile cells.

If you people have notice such a think, then please suggest.

And please do suggest, if I use Mycoplasm removel/prophylactic reagent in all my culture with 293T do you think it can negatively impact my lentiviral formation/assembly. Because I am seriously thinking of to use an anti- mycoplam reagent like plasmocin etc. Many thanks

Best

Syed

Dear Ian,

many thanks for your reply too. Please suggest about the above concern I just put up for Matheiu & Saroj. And I firmly believe that i have a serious problem with either with the hood or incubator, bcz when I tried in past to differentiate my NPCs to neuron/astro/oligo, they were a clear visible mould like appearance when grown without P/S.

How about anti-mycoplamic reagent. is it going to negatively effect my lentiviral preparation.

regards, Syed

Hi Syed,

I would advise against using any anti mycoplasma reagent: if you (or someone in your group) has a problem with contamination, you have a problem with poor technique or poor hygiene (Hoods/Culture incubators/lab surfaces/Media) none of which may be your fault!! and using these reagents will not improve that. 

I would suggest routinely checking cell lines for mycoplasma which can be easily done and having a group clean up of cell culture. Discard any infected cell lines immediately Any new cell lines introduced in to the culture room should be tested for mycoplasma. We also have a training scheme for new lab members in cell culture so that they know what to do.

I know this is not always possible but we all have our own media/buffers to avoid cross contamination of stocks. Experiments can be expensive and time consuming so you need piece of mind that your cells are the healthiest they can be.

bw

Ian

You will not see mycoplasma using conventional lab microscopes but bacteria should be clearly visible: Black dots/small rectangular organelles that are constantly moving. it is sometimes difficult to verify 100% that you have contamination because cell debris can also give this impression as well as DNA transfection precipitates. 

One easy way to verify is to take a sample of your media (1ml) and add to LB broth (3ml) and grow at 37C o/n with shaking : if its cloudy the next day then its bacterial contamination. Also check your working stock of media in the same way. You can also streak the media onto an LB plate and grow o/n. 

Dear Ian, 

many thanks for your inputs. i will do the mycoplasm check today as suggested.

Thanks, 

Syed

Its also worth checking that you havnt got your cells contaminated with another line if morphology has changed. it doesnt take much to get cross contamination. If I were you and this is a core cell line I would get it validated to make sure that you havnt got anothe "weed" cell line in there.

Always notice the same with our HEK293 and especially 293FT cells. I believe it`s normal for the line, they are poorly adherent, in fact. Do not think this is something to do with mycoplasma contamination, rather with the plastic quality or else.

Anyway, if you need to avoid this before transection, just use the preconditioned dishes. Plate your HEK293 or 293FT onto the transfection dish the day before transfection, preferably at high density, to have a 100% confluent monolayer the next day. Then, in the morning wash the cells away and discard, and plate the new HEK293 cells onto the preconditioned plates at the density you want to have 1-2 hours later (we plate them ~70% for a calcium phosphate transfection). They are adherent and perfect morphology just 1-2 h thereafter, ready for transfection, no medium change needed. Best,

Sergey is right, I have seen it too but keep in mind use prewarmed media. if you use media straight from refrigerator without prewarming, the cells may even detached.

Seyed dont hesitate to contact me if had any problems.

Bests

Mostafa

Many Thanks All, i think I have overcame with the problem of cell morphology change with high density seeding.

I have noticed the same morphology change on my 293T. When I stain them with Mitotracker, they usually shrinks, lost their morphology. I thought that the PBS could cause it (Mitotracker diluted in PBS), but they shrinks in DMEM-diluted Mitotracker also.

Hello! In our case we have this strange form of HEH293 clústers. Someone saw something similar ?

Rodrigo Hernán Tomas Grau

What are the growth conditions?

I saw something similar when growing HEK293T cells for many days from very low density. Then some clonal islands eventually overcome the restriction to grow as a monolayer and start forming multilayer "bumps". I presume if such clones are reseeded, they will retain this tendency,

Dear Roddigo,

Did you find out what the problem was? I am facing similar issue where the cells are forming clumps instead of spreading as monolayer. Also they detach too often which is abnormal.

Dear Tousif

What a headache when these things happen! we used yo re-sterilized some plates. there we observe that when they have a fissure, line, edge or something off the axis of the surface, the cells tend to grow there and accumulate

Dear Rodrigo,

Thank you for your reply. My plates are completely new. I am confused what's going wrong. Everytime after splitting, the cells attain somewhat circular morphology and starts to detach. The ones that are attached, grow on top of each other and form patches rather than a layer and do not spread across the flask. I changed cells, medium, equipment but no luck.

HEK293T like to grow on collagen-coated plates. Consider coating your plates before growing them. Best luck!

We have used Hek293 cell for a long time for in vitro assays and protein expression.

Yes they are are little less sticky than other cell lines we commonly use i.e. CHO but careful handling especially in the first 24hrs post plating ensures good retention when using normal cell culture plasticware. If using DMEM media then a prior equilibration maybe necessary in CO2 incubator to maintain pH.

Plasticware can have dramatic effects on cell adhesion, maybe worth testing other sources.

Over use of TE i.e. long incubation times, can result in cell clumping, so minimalise exposure.

If you use a different media i.e. DMEM to Optimem then the cells may experience a shock due to changes in conditions leading to changes in morphology.

Dear Syed,

I found the same morphology changes with my HEK293T cells. I use Corning cellbind surface plates. I have noticed that, in some area of my plates, I can find less density cells with shrinkage (skinny) cell morphology. The cells in the rest of areas looks normal and healthy.

It will be great to know why the cells perform like this.

Dear Researcher,

Your query regarding the morphological changes in 293T cells in response to media changes is a pertinent concern in cell culture practices. 293T cells, a human embryonic kidney cell line, are known for their robust growth and are widely used in various research applications. However, like all cell lines, 293T cells can exhibit changes in morphology in response to alterations in their culture environment, including changes in media composition.

In conclusion, it is not uncommon for 293T cells to exhibit morphological changes in response to a change in culture media. These changes can be influenced by factors such as media composition, serum concentration, pH, and osmolarity. Careful monitoring and optimization of culture conditions are recommended to ensure the health and stability of the cell line under the new media conditions.

Best regards,

Check out this protocol list; it might provide additional insights for resolving the issue.