Hi Abu,

regarding your concern the cell lline may not healthy enough:

is there any reason for this (e.g.mutation), or something like this stated in the data sheet of ATCC?

But in general, i would seed the cells initially in a larger culture vessel than a six well (unless it is stated in the data sheet). Because if you seed them initially in a six well, it might be "overcroweded" and the consequnece of this is a bad plating efficiancy or in worst case, none of the cells will attach (i suppose you are using an adherend cell line).

This leads to the main part of your question:

in case you know, that your cell line needs no minimal confluency for growing well you could go for your large culture vessels to obtain a small passage number. BUT keep in mind, that although you have a small passge number, the cell will be able to undergo more population doublings in a large vessel.

Best,

GAD

Dear Guido,

Sorry for having left out some important points. My cell line is in suspension and ATCC recommends 4 x 10^5 viable cells/mL for seeding. (info from product insert)

Guido asked

'regarding your concern the cell lline may not healthy enough:

is there any reason for this (e.g.mutation), or something like this stated in the data sheet of ATCC?'

My answer

'I was informed that the best practice is to immediately put the cell line into culture media upon receiving from ATCC. I was not able to so for technical reason. Hence the alternative is to store in liq nitrogen which I did. I was informed that cell recovery may not be as ideal, that cell recovery may stutter.'

With my primary cells I always first put the cells in a T75 flask, then, when this is confluent, I freeze away 1-2 vials (depending somewhat on cell number) and devide the rest over 2-3 T75 flasks. From each flask, when confluent, I freeze again 3-4 vials and seed 1 new T75 flask and also keep the old T75 flask, wherein always some cells are left that also keep growing. So if you do it this way, you will soon have 20-30 vials with

Hi Abu

You need to defreeze the cells at the confluence recommended by the ATCC. Depending on the cell lines, you would prefer flasks or 100mm dishes or other types but you need to follow the recommendation. You cannot dilute 1/10 every cells. Some cells have a better growth by putting them less diluted. So first, you need to know how much cells you've got at the beginning, thaw them at the recommended concentration and then diluted them as recommended. General conditions of culture may not apply to your particular cell line. If you could tell us which cells you use, we would have the possibility to give you more precise answers.

Best

Dear Abu, in my experience of defreezing not adherent cell lines (suspension cell lines, i.e hematopoietic), it is important that you do not dilute them at first, since they need autocrine/paracrine factors to re-take growth. So, for me it never works scaling up the culture at first. I would advice to keep the conditions the supplier tells in respect of seeding concentration and start with a 5 mL culture, flask standing, not to "gasify" too much the culture. You can also try increasing a bit the FBS concentration. So, when you see the cells are beautiful and happy (round, fat, refringent), you can add some more medium (and still have your first passage) and lay the flask down, to increase contact surface. Hopefully you will have your cells growing steadily now. Then, you can gently think in your scalling-up strategy (1:10 seems a bit harsh, I am afraid I do not know a method to obtain loooots of P2 aliquots! I need at least 4 passages to do so. Unless you have a cocktail of cytokines and growth factors in your protocol). Rememeber to do the passages in the log phase of growth, so check initially the particular kinetics (doubling time) of your cell line.

Cells are living things, you have to understand them and treat them well, otherwise they do not grow and you can loose your seed. For us at least (Brazil) it is very difficult to buy cells abroad by customs restrictions, so the initial care is crucial. Good luck!

I am almost of the same opinion as Rocio. Initially, I also prefer to put the frozen reference stock in a T-25 flask with a 5 ml media. When cells are healthy, we split into somewhere like 1:5 (may vary sometimes) and freeze upto normally P3. It is too expensive and hasselsome process to buy cell lines in India also. Best of luck.

Thank you every one for your input. I am referring to ATCC NCI-H929 cell line.

The general impression I am getting from the thread is I should start slow with T-25 flask as highlighted by Gerald, Rocio and Suparna.

Dear Rocio,

'........So, when you see the cells are beautiful and happy (round, fat, refringent), you can add some more medium (and still have your first passage) and lay the flask down.....'

How much maximum medium will you tolerate in your laid down T-25 flask?

What if I find the T-25 flask filling to its neck, I then transfer the whole content to a T-75 flask and continue adding in media. Will this action increase the passage no?

Dear Abu,

my maximum to laid down a T25 flask is 10 mL. More than that, you have risk of contaminating your culture and spilling-over your incubator. If you decide to go for more, you better keep your flasks standing. In respect of filling the flask up to its neck, I would not advice it if you are using conventional plastic, because you need to maintain a gas interchange surface.

See the basic calculations (Freshney's handbook if I remember well)

0.2 (for minimum volume) to 0.5 (for maximum volume) x T flask cm2

This means that for a T25cm2 flask the volume range is 5 (minimum) -12.5 (maximum) mL for optimal CO2 penetration

T75cm2 flask = 15-37.5 mL

T150cm2 flask = 30-75 mL

BUT, You can use special flasks, like Corning Hyper Flasks or similar that have a gas permeable film and allow to be filled completely with medium and sealed with a solid cap. They are more expensive than traditional, of course, but are worthwile for initial manipulation, I think. Otherwise, you can scale-up your culture in P2 to 75 mL and to 250 in P3 (1:5 split ratio seems ok, as Suparna said). Then you can freeze down your cells.

Best luck with your cultures!

Hi Abu,

Here in Brazil, we are stocking in maximum 8 cryotubes. This is because, depending on were you are stocking your cells (i.e. -80 °C freezer), cells may not resist to long (more than 2 years). So here we grow the cells in a 75 cm2 flask and all the cells from this flask will be freezed. In this flask, depending on cell line, you can have about 10x10^6 cells, than we put around 1x10^6 cells per cryotube with cell freezing media from gibco. But you can also do this freezing media by adding DMSO to a final concentration of 8%. But be carefull, some cell lines suffer differentiation in the presence of DMSO. Though, you need to use glycerin.

I hope my answer could be useful.

Hi Abu,

According to my experience, it is better to use exactly the specefic conditioned media

recomminded by ATCC, use T25 flask in 7-10 ml of the specific conditioned ATCC media.

Please do the following steps:

1- prewarm the suplement media (the specific media but with no serum)

2- thaw the cells quickly then transfere it into the pre warmed media (1:10 dilution)

3- centrifuge at 1000 rpm for 5 min

4-quickly discard the supernatant, resuspend the cells in the last drop by tapping then wash the

cells 1X with supplemented mdia then add the specific culture mdia (7-10 ml) and transfere the

cell into T25 flask incubate at 37 C/5% CO2 for 24 hrs then examin your cells by the inverted phase contrast microscope ,

if you notice dead cells collecte the cells and wash once at low centrifugation (900-1000 rpm)

Keep examing and washing your cells every 24 hr tell you have enough healthy cells for

subculturing and freezing.