Recently, we have been grappling with a serious issue whenever we plate out our BHK-21 cells in tissue-culture treated 24 well plates. There appears to be a random pattern of cell death, in which some wells have a characteristic appearance of being shriveled, appearing as though they are almost 'dissolved' in their cell morphology, with ill-defined borders, becoming compact and having a loss of confluency.

Notably, the pattern manifests as follows: in an affected well, only exactly "half" of the well is affected, whereas the other half of the well is fine and healthy. Please note the attached pictures.

Even more baffling, in other wells, sometimes the area of death forms a large, spheroid/oval bubble shape that occupies the centre of the well.

This morphology appears generally after transfection (Transit2020). However, further investigation shows that the onset of death can also appear after simple intervention, and randomly. Please note the following examples:

Case 1:

-23 wells in a 24 well plate is plated at 25,000 cells/well (Falcon)

-24 hours later, transfection with DNA

-24 hours later, appearance of odd/unaccountable cell death in half a well (1/23 wells affected)

-24 hours later, antibiotic selection applied (unrelated protocol)

-24 hours later, number of affected wells rise to 16/24. The no-antibiotic conditions also have this shrivelled appearance

Case 2

-23 wells in a 24 well plate is plated at 25,000 cells/well (Costar)

-24 hours later, transfection with DNA

-24 hours later, all 23 wells are unaffected

-24 hours later, antibiotic intervention as indicated above.

-24 hours later, suddenly, the no-antibiotic control well is affected (media change to all wells), representing 1/23 wells affected

Case 3

-10 wells following NUCLEOFECTION are plated

-1/10 wells are affected.

As you can see, this is not apparently related specifically to any purely defined action, but rather from intervention by the researcher. I have tried this across three different researchers and we all have the same, random issue.

On closer magnification, there does not appear to be bacterial infection, or fungal infection.

Subsequent plating with a different cell line has so far not caused this issue, despite the same media being used. Further results are pending to see what happens when we perform basic tasks on it such as transfection and/or media change.

So far, I believe that we are dealing with some type of cryptic virus that is contaminating our cell culture, and manifests randomly in different wells at low probabilites. What I cannot explain is why the contaminant affects often only half the well or only a small area of a well. It does not correspond to a plaque morphology that I know of. Further to this, I have ruled out the incidence of a bubble in the media causing the area to dry out and dessicate, despite the cells having such an appearance.

Attached are 9 photos. Please note the file named "NORMAL" which is the "typical" cell morphology that I observe when they do not have the shriveled, diffuse appearance for BHK-21 cells. Though, I do notice the "blebs" on the cells, which I think may be due to the temperature change that occurs sometimes when we image the cells.

Any advice would be greatly appreciated. I will sterilize both incubators this week and discard all media.

NOTE, media used to culture the cells is the following: MEM with glutamax with 1% antibiotic-antimycotic. A kit for mycoplasma has also been ordered.

ALSO NOTE: Insofar, this has only been observed in 24 well plates, and not in either the ongoing T75 flask passage (several aliquots plated out), NOR in 6 well plates.