The reference cell line (as above) from ATCC turned turbid on D3 upon thawing. I managed to salvage the cells with 2500U/mL of Penicillin/streptomycin. The bacterial culture from supernatant showed presence of pseudomonas fluorescens.
Following 7 days of high dose antibiotic exposure, I have been growing the surviving cells in antibiotic free media for the past 4 weeks. First in 96 well plate then in 6 well plates. Of the 54 wells (6 well plates), 3 well showed residual infection and were discarded. The remaining wells do not show signs of infection; clear media. The cells appeared fine and are dividing (some are forming aggregates). But after three weeks the cells are seen sparcely scattered in a space less than 20% of the plate area, spread only at a single plane. The cells are grown in ATCC recommended media as stated in the product insert: RPMI1640 (Gibco) + FBS10% (Gibco) + 0.05mM BME. The doubling time is suppose to be 3-4 days.
My initial impression was that the surviving cells could be having low clonogenic properties. Until I stumbled upon the patent for H929 as available at http://www.google.com.mx/patents/US4892829
Under Material & Methods > In summary > the following statement was identified:
'It is noted that the use of a serum free defined medium was critical for culture establishment, as the tumor cells initially failed to proliferate in serum containing media.'
Nevertheless, I have found numerous research articles which had used serum containing media for H929 culture.
Is the slow growth:
Expected for cell lines recovering from infection?
Is it because of the media formulation? [The complete media sustained rapid K562 growth indicating that the FBS is not faulty]
Has anyone experience similar situation with NCI-H929?
It appears that the answer is simply that I had not cleared the infection.
At the onset of this problem, the cell suspension under brightfield microscopy at 100x magnification showed presence of dark grainy background speckles that with time increased in numbers. These deposits overwhelm all spaces when the solution turned turbid. I supposed these are the contaminating bacteria. At higher magnification they appear as sphere/rods.
Initially the post-antiobiotic media appeared clear of such infringing material but after approximately 5 weeks they began to reappear. These deposits reappeared earlier in wells with more blunted cell line proliferation. Some of the relatively more active wells also withered away when these deposits reappeared in the background. At the time of this post only 11 of 50 wells still show unadultered media.
Perhaps this is a lost cause.
- Badges
- Science topic