Fibroblast Cell Culture Issue - Cells grouping together and cell clusters observ - (Jun/26/2015 )

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

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

Hi Bob, 

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

Elaine

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

Hello Elaine

I am facing a similar problem where my cells are grouping up to form a ball like structure in a region of cell culture dish. The images you have attached are similar to the problem I am facing. Can you please inform if you were able to find out the problem and its solution?

Elaine123 on Fri Jun 26 17:31:25 2015 said:

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

 

Hello Elaine

I am facing a similar problem where my cells are grouping up to form a ball like structure in a region of cell culture dish. The images you have attached are similar to the problem I am facing. Can you please inform if you were able to find out the problem and its solution?