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Fibroblast Cell Culture Issue - Cells grouping together and cell clusters observ

fibroblast cell culture cell clusters abnormal cell growth cell based assay

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#1 Elaine123

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Posted 26 June 2015 - 09:31 AM

Hi All, 

 

I am having a cell culture issue with my fibroblast adherent cells. Below are some of the cell issues that I am having. Hoping somebody can help me out in identifying the cause or the solution to this. 

 

  • Many cell clusters in flask after recovery and all through passaging.
  • Cells grouping together and not spreading out like fibroblast cells should.
  • Low cell counts.
  • Poor growth.
  • Cell clusters in 96 well assay plates.
  • Assays failing because we are getting erratic results.
  • Short cells (not elongated).

 

We have performed many tests to identify the cause of this issue, such as:

  • Contamination (bacterial, fungal and mycoplasma
  • Change in components (FBS, Penn Strep, Trypsin, HBSS, Basal Media)
  • Flask/assay plate/stripette/conical tube material

If it somehow was the basal media - What component/mineral could affect the cells this much by either being at too high/low concentration? The basal media has all the essential materials already added. We only add the FBS and Penn Strep.

 

Below are images of the abnormal cells.

 

Thanks in advance, 

Elaine

 

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#2 bob1

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Posted 27 June 2015 - 02:17 AM

Are these supposed to be immortal cell lines? If not, they could be approaching the Hayflick limit.

#3 Elaine123

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Posted 27 June 2015 - 02:23 AM

Hi Bob, 

 

No these fibroblast cells are not immortal. They are only passaged up to around P15.

 

Elaine



#4 bob1

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Posted 27 June 2015 - 03:55 PM

Sounds like a Hayflick limit problem, which usually strikes between 30 and 50 population doublings - unfortunately irreversible, unless you transform the cells.





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