Growing RAW 264.7 in suspension culture - (Nov/05/2010 )

I am currently planing to grow RAW 264.7 in a suspension culture by using Techne stirrer bottles. Does anyone has any experience regarding this? Is there any other good alternative method?
Thanks

ouhscr2 on Fri Nov 5 15:51:24 2010 said:

In my opinion it is the only way to grow these cells. We grow both RAW264.7 and J774 (both murine macrophage cell lines) using Techne biological stirrers. My tips are:-

Change the bottle once a month.
Buy Falcon T75 and T175 flasks and use the filtered top on ONE of the side arms of the stirrer. This means that you get adequate gaseous exchange while keeping a barrier to contamination within the incubator.
The stirring speed is very important, 20-25RPM. This will ensure that the cells do not aggregate and settle to the bottom. Too fast and the viability will go down because the cells will be smashing into one another.
Keep the cells at a density between 100,000 and 700,000 cells/ml.
The cells grow extremely fast under these conditions...we try to reduce the growth by using 5-7.5% Foetal Calf Serum.
The Stirrer bottles over time will need to be RESILICONISED. The glass is borosilicate and is used beacause when new the cells do not attach to the glass. However over time, with constant washing and autoclaving, the glass needs to be recoated. This is easy using a siliconising fluid such as REPELCOTE.
Make sure that the silicone seals in the caps are still there and in good condition. This also goes for the rubber that holds the glass stirrer in place.
When preparing an experiment in multiwell plates..it is always best to plate them the day before i.e. give them 24 hours to adhere.

These cells are extremely sensitive to ENDOTOXIN. So be careful with media, FCS/FBS and glassware.

Hope this is useful.

Kindest regards.

Uncle Rhombus.

Thank you very Uncle Rhombus. However, I have a quick question to you. What is the maximum volume of medium I can use in 125mL bottle?
Best Regards,
R2

ouhscr2 on Mon Nov 8 20:19:03 2010 said:

Dear Ouhscr2,

It is best to use the bottles that Techne provide. Generally we use 125,250 and 500ml volume bottles. However if you are short of bottles then a 125ml can hold approximately 200ml. The more media you have in the bottle, the more careful you have to be with handling it i.e. it is easier to contaminate the lid filters(if used).

Kindest regards.

Uncle Rhombus.

Dear Uncle Rhombus,

I have a stupid question to you... do I have to use Techne biological stirrer plate or any stirrer plate can work?

Thank you very much for your sincere assistance.

Regards,
ouhscr2

ouhscr2 on Wed Nov 17 03:43:29 2010 said:

Dear ouhscr2,

We have tried to use a normal stirrer for these bottles. However there are certain problems:

Most stirrers are not equipped to work FOR LONG PERIODS OF TIME AT 37oC and HUMIDIFIED conditions.
The Rate (RPM) at which you need to stir the cells, on most stirrers, is not achievable i.e. 20-25 RPM.
The bottles are very easily knocked off normal stirrers, the Techne ones have a bespoke hardened rubber nipple that keeps the bottles in place.


So in my opinion it is not either possible or ADVISABLE to use any other stirrer.

No question is a stupid question.

Kindest regards.

Uncle Rhombus.

Dear Uncle Rhombus,

In 125 mL volume bottle, we are using 50 mL medium. What is the minimum volume we can use? And what should be the starting cell concentration in 50 or 25 mL medium?

Thanks you very much for your sincere assistance.

Best Regards,

ouhscr2 on Mon Dec 13 16:16:31 2010 said:

Dear ouhscr2,

You should have about 50,000 cells/ml to start with and grow them up to 700,000 cells/ml. In a 125ml bottle the minimum volume should be ideally 40-50mls.

Kindest regards.

Uncle Rhombus.

Dear Uncle Rhombus,

How often do I need to resiliconise the vessels? Do you bake the vessels for how long and at what temperature?

once again thank you very much for your sincere assistance and feedback.

Regards,
ouhscr2

ouhscr2 on Tue Mar 1 15:57:59 2011 said:

Dear ouhscr2,

The bottles should be changed every month/6 weeks. This stops aggregates of cells form building up. Brand new bottles will not have to be re-siliconised for probably a year....this all depends upon your laboratory washing machines and the chemicals used.
However once a year old they should be re-siliconised every other time you use them, the procedure is as follows:-

Get the bottles wash and dried in your lab wash-up facility.
Add 20 ml of siliconising fluid (I use REPELCOTE) and close all caps tight.
Slowly allow the fluid to touch all areas of the glass. Do this for about 1 minute.
If the bottles was perfectly dry then you can re- use this fliud at least 3 times. If however the bottle was wet then the fluid will be contaminated with water and can no longer be used again.
Remove the fluid and let the bottle stand upright for 30 seconds allowing any remaining fluid out.
Autoclave the bottle, making sure that the side arm caps are open i.e. allowing the steam to get inside the bottle. Autoclave for 20 minutes, at 1 Bar pressure and at 121oC.
Once autoclaved and at the right temperature to remove the bottle from the autoclave....IMMEDIATELY tighten all caps.

When using the bottle next make sure that you add 20mls of growth media and wash the inside of the bottle exactly the way you have done siliconising.

I hope you find this useful.

Kindest regards.

Uncle Rhombus.