Check your complere medium is with fresh amono acids, L glutamine  this will be one of the first to expire.

Could be the plate treatment is old, you could try coating plate with collagen 

Use dish immediately or air dry for future use. Dissolve collagen 2mg/ml in 0.2% acetic acid. Filter it again through 0.45um filter, then store at 4°C. When coating, dilute the stockedcollagen 1:5 in tissue culture grade water.... 

Make sure co2 is good, and geting air exchange loose cap...

Could try a little extra serum to get them up and doubling from 10 to 15%  fbs

Some freezes are better than others, may take a few passages to get them growing 

From your picture some have nice sudopods and look attached well , but i see the round ball looking ones.  Keep washing them away... if they dont attach, you dont want them any way.

Best,

Thanks Charles. The problem persists in new T25 flasks also, so I guess adherence is not the problem here. 

They actually adhere pretty strongly and washing them away also takes away most cells. I will definitely try increasing the amount of FBS.

Could a very high passage number be a reason for this ? 

I am missing something... do your problem is the filamentous sponge-like aggregate in the central part of the figure, near doubling cells,  and in the lower part of the figure?

Yes. Sorry i missed mentioning that. Also I see that the cells are not spreading well. They are visibly smaller even when they are not confluent. 

Don't worry. Something very similar happened to me once; after trying almost everything (including also what Charles suggested) with the help of a microbiologist I concluded that was fungal contamination.  

If is this the case I recommend you to discard your cells and clean the incubator.

You definitely can remove fungal contamination, but you can't be sure of removing spores.

Thank for the suggestion. But could you also look at the new image and say why they could be forming those micro clusters ? 

The image is from a culture flask seeded approx. 5 hours before the image was taken. The circular blebs near the cell clusters keep growing (number and size) as the cells reach confluency

To me, i think they are dead or unhappy cells.  It does not look like a contamination.  I wouls ri se them away gently

My primary concern is that they are forming these clusters. On top of which these bleb like things are growing. 

My work involves imaging single molecules inside cells and with these clustering it becomes impossible. Could you think of a way to disperse them better ? 

I share the incubator with a person and his HeLa cells have also developed the exact problem. However, he has 3T3 cell lines in the same incubator and they are doing great. 

We do not share any lab consumables/media/PBS/trypsin. Baffling. 

When I said fungal contamination I was referring to such "tangle" (I don't have a better word) that you can observe in your first figure (I have circled it in the image in attachment).

In your second figure I do not see "tangle"; I might be wrong, but such clusters in the second image seems just a group of dead cells or cells having difficulties to attach to the plate. 

Could something in your complete media may be off?  L-glut?

Mycoplasma contamination is a possibility. .. you would have to test for this though.

Are your caps loosly attached...?

Fungi contamination you can see as said above, and bacterium will change pH and medium will turn yellow fast.  

Check the water of your incubator,  hopefully nothing. 

To start fresh with a clean incubator with new water ect, fresh medium, new thaw... maybe best. Fungi are not so fun i  an incubator. 

EtOH everything, assume nothing is steriel... 

Unfortunately the HeLa cell line is very adaptable and hearty, like most cancer cell lines. Keep an eye on them until you get good doubling times, can get a quick hemo count for seeding cells, vs time to total confluent and determine doubling time i hours.  Just from memory 18 to 24 hour doubling time was normal. 

If they just came from a recent thaw, it may be debris of dead cells, and need a bit more time, if too few survived 

May just be time to do a deep clean too... growing blebs dont dound good.

To despense clumps, get a good trypsin digest, pipette up and down gently to despense cell clumps in old flask, look through it to see if clumps are dispensed, sub culture into new plate that has been pre warmed and allow medium to equal ph with co2 before seeding with a gental swirl.

Best of wishes

Did you ever resolve this issue? Has anyone else encountered this? I am seeing the exact same thing in my cultures for 3 weeks now. There is no turbidity in the media, I have tried different suppliers of RPMI-1640, different lots of flasks, and am using frozen aliquots of FBS from a single bottle that has worked well for me in the months prior to this issue arising. I have thawed multiple cell lines from different freeze dates and see this in every culture within 24 hours. I am starting to wonder about impurities possibly coming into the incubator with my CO2 but feel like I am grasping at straws at this point. Any insights would be greatly appreciated!