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Brain tumour tissue enzymatic digestion for culturing cells


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#1 themoon

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Posted 30 August 2009 - 09:14 PM

Hi,
I wanted to know if anyone has a standard protocol for the enzyamtic digestion of brain tumour tissue to develop cell cultures? I know people commonly use collagenase IV however I cannot find a standard protocol.. any ideas?

Thanks very much!

#2 PurpleKnight

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Posted 09 September 2009 - 12:16 PM

Hi,
I wanted to know if anyone has a standard protocol for the enzyamtic digestion of brain tumour tissue to develop cell cultures? I know people commonly use collagenase IV however I cannot find a standard protocol.. any ideas?

Thanks very much!


This is what I've used in the past with great success. You may need to adapt according to your needs of course. Good luck!

Carefully mince the tumor with sterile razor blades. Add RPMI1640 (full media) and wash 2-3 times.
Add appropriate amount of liberase blendzyme IV (8 U in 200 l) in 12 ml of RPMI1640 + 5% FBS + 1% Pen/strep and incubate at 37C for for 3 hours , mixing intermittently (every 20 minutes). Gently disperse the cells by pipeting (trituration) every 20 to 30 minutes. This can be a crucial procedure. It serves to break up the tissue fragments following incubation in the dissociation mix. If done too vigorously, cells will be destroyed lowering viability; too weakly and tissue fragments will be left intact lowering yield. Gentle trituration, using a 10 ml pipette, constitutes filling and emptying the barrel at a rate of about 3.0 ml per sec. Avoid bubbling the cell suspension.
Add neutralizing medium (DMEM full)
Filter the cell suspension through fine mesh (nylon membrane 100 m then 40 m) to get a single cell suspension.
Decant the cells by gently spinning down and remove the supernatant each time. Resuspend the pellet with RPMI1640 full media and spin again. Wash 2 times in total.
Finally seed in T75 flasks and incubate at 37C.

#3 themoon

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Posted 18 November 2009 - 08:41 PM

Thanks heaps for your method! I will try it. What company do you buy your enzyme from?

Thanks




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