We usually discard a cell line purchased from a commercial source once the given limited passages are over. Has anyone got experience and protocols on evaluating the stability and keeping the cell lines for extended use? We are concerned about BHK-21 and HEp-2 cell lines
Hi Gayan, usually (as I'm sure you do) when we get a cell line or primary cells into the lab we expand them for a few passages and freeze down 3-4 vials for a Master bank. We then continue expanding the remaining cells into a working bank. We use the working bank for experiments (at around the same passage number) and discard these after use. If we run low on working bank cells we return to the Master bank to expand more.
With regards to long term passaging there are a couple o pointers.
1) If the cell line is adherent make sure that during passages you Trypsinise all of the cells in the flask. If you do not do this then you are selecting for a) faster growing cells b) cells that are easier to detach (the cells more resistant to trypsin are left behind in the flask.
2). Use good FBS from a good source - if possible batch test your cells against FBS from various suppliers. If you can grow the cells in media (with various FBS sources) for several weeks and chart their doubling time in culture*1. Also seed the cells at the lowest recommended density - this can highlight small FBS effects (where the cells are under pressure). Once this is done and you have found a good source buy enough stocks to last the duration of the experiment. The reason I mention this is that cells in culture are always under a "selective" pressure from the growth in 2D in vitro. If you can minimise the pressures (with the optimal culture conditions) this will reduce the selective pressure somewhat and minimise the out-growth of "mutant" cells.
Also once you have a good FBS don't split the cells at the lowest density recommended but a little up from this (to maintain a good pool of cells to help reduce "founder effects" etc. (i.e. if ATCC/DSMZ say "split 1-5 to 1-4 use the 1-4 split)
3) Regularly perform a test for the presence of mycoplasma (about once a month or so for long term cultures). Mycoplasma are small bacteria that lack cell walls making them immune to many antibiotics and are often invisible to the eye - they are the "gremlins" of cell culture and can have strange and unwanted effects on cell growth (morphology changes, slow growth etc). The main source of these pests is primary cells and the human performing the cell culture - therefore if you are using primary cells in your lab isolate them from your cell lines if possible (i.e. a different incubator) - and of course only split on ell type at a time in your TC hood - this prevents cross contamination of your cells with another line (i.e. HeLa!).
I hope this helps and I will post more if I can think of any. Phenotypic drift will always occur if cells are grown for a prolonged time - most cell lines have scrambled genomes and the faster growing cells will dominate the culture in time.
Good luck,
Gary.
*1: Population doubling = PD =Log (harvested/seeded)/Log 2
Thus if the PD = 1.5 after 6 days then the population has doubled in 1.5 times in 6 days (= 144 hours) so the d.t. = 144/1.5 = 96 hours.
Hai sir, After purchasing the cell line, after the analysis of the cell count and sterility, the half of the cell you may preserve by the subculturing with Trypsin Versen Glycine, and stored in vapour phase of liquid nitrogen, and every time the liquid nitrogen to be replenished.
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