I am new to this forum, but it looked like a helpful place to get other's opinions! Please let me know if further information is required.
I am a technician doing some work with Caco-2 cells, and my lab is currently having issues maintaining our cell line. A few notes
- Cells are obtained from ATCC
- Media is M199 with the addition of streptomycin/penicillin (1%), non essential amino acids (1%) and FBS (NZ sourced) (10%)- a media which has been tested for suitability with Caco-2 cells.
- The cells grew well, and were passaged and frozen down successfully over the past 5 or so years, with new vials being brought up frequently for experments (where they are seeded first into flasks, bulked up, then seeded onto inserts)
It was noted that over the last 2 years or so that the cells have not been performing as they had been, with low TER, resistance to passaging (very sticky), and cell clumping following a split (using Trypsin initially, and later we switched to Tryple express).
Within the last few months it has come to our attention that there are smaller cells visable when the Caco-2s have been visualised and counted using trypan blue and a countess machine for cell counting- and also using a haemocytometer. These smaller cells are now visible under 40x magnification inside our flasks also (and they appear to be adhering to the flask, not other cells), and are more prolific. The countess has told us that these cells are less than 20um.
I have attached 2 photos, the first being the cells in the antibiotic media, and the second being cells following a switch to antibiotic free media. If anyone can shed any light on why we might be having these issues it would sure be helpful! The smaller cells are circled in red. These photos were taken from a cell suspension.
Edited to add: Media is holding pH as normal and does not appear turbid. All recommended aeseptic technique is adhered to :-)
Edited by Sprucegoose, 14 August 2014 - 05:26 PM.