Although this is a common belief that BY-2 cells are fast growing cell lines, in my own experiment they don't seem to be as fast as I expect. Thus, I decided to change the medium; right now I use Full strength MS with sucrose 3%. I even thought that maybe bigger flasks can help. For now I use little flasks and have 30ml medium in each. Do you have any idea about changing this situation.
Thanks
I think the medium might not be ideal, try something similar to below? (Taken from Kovarik et al. 2012 Bot. J. Linn.)
The BY-2 cell culture was propagated in modified Linsmaier & Skoog (1965) liquid medium supplemented with sucrose (3 g L-1), thiamine (1 mg L-1), KH2PO4 (370 mg L-1), myoinositol (100 mg L-1) and 2,4-dichlorophenoxyacetic acid (0.2 mg L-1). Cells were grown in Erlenmeyer flasks under constant shaking (130 rpm) at 27 °C in the dark. Cell suspensions at a density of approximately 3 million cells per cm3 were passaged at a 1 : 10 dilution into fresh medium usually once a week.
A growth-curve analysis will help. Compare with literature where such curves are very common. 100 ml media in 250 ml flask volumes helped us obtain a very good growth and yield. Shaking speed, temperature, innoculum density/ amount, and subtle changes in media composition are very critical. Change or check one each at a time.
we use 2 mg/L 2,4-D in our culture media. We are using this media (everything is the same as Steven described except 2,4-D) routinely. The growth rate is very fast, while subculture in 1:25 dilution every week.
MS medium is quite OK for BY2 cell culture. It could be that your subculture procedure needs attention. When you take the aliquot for the next subculture, you should swirl the flask, watch where the fine cell fraction is and pick from a dense part. Establish the growth curve (e.g. on a fresh weight basis) and compare how it performs over time. Also, you can check the viability of cells using for example FDA/Evan's blue staining. You need at least 110 rpm on a shaker with wide shaking circle and 500 ml flsks would give faster growth than in smaller flasks.
MS medium including micro & macro elements, 30g sucrose\L, 200mg KH2P04/L, 1mg Thiamine/L, 100mg Myoinositol/L, 0.2mg 2,4-D/L, pH5.8. We keep the cultures in 100ml flasks with 20ml medium. Subculture 1:50 every week. Good Luck!
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