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Growing Up Cells


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#1 nbhu

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Posted 15 May 2009 - 03:51 AM

Hi,

This may seem a pretty stupid or basic question but basically im finding it hard growing up my cells to obtain the approx 10million cells per ml required for freezing or for extracting DNA/RNA from.

I have a patient AML3 cell line, and i use alpha MEM media. My current cell count is 1million cells per ml.

This is my first time at cell culture so any help really well explained for dummies like me would be appreciated!!!

#2 rhombus

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Posted 15 May 2009 - 04:53 AM

Hi,

This may seem a pretty stupid or basic question but basically im finding it hard growing up my cells to obtain the approx 10million cells per ml required for freezing or for extracting DNA/RNA from.

I have a patient AML3 cell line, and i use alpha MEM media. My current cell count is 1million cells per ml.

This is my first time at cell culture so any help really well explained for dummies like me would be appreciated!!!



The first thing to check is the serum you are using. A quality serum will definetely affect cell growth rates.
Plastics is the second most important variable: The surface substrate has different properties from all the companies.
Split ratio is the next
The media you use, has this been optimised.

Kindest regards

Rhombus

#3 klinmed

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Posted 16 May 2009 - 01:28 AM

Hi,

This may seem a pretty stupid or basic question but basically im finding it hard growing up my cells to obtain the approx 10million cells per ml required for freezing or for extracting DNA/RNA from.

I have a patient AML3 cell line, and i use alpha MEM media. My current cell count is 1million cells per ml.

This is my first time at cell culture so any help really well explained for dummies like me would be appreciated!!!


Are you trying to grow your cells to 10 million/ml? I cannot think of a cell line that grows to this density.

For freezing you need a healthy, rapidly dividing culture. I would grow to say 6-7 x 10^5 /ml, concentrate the cells by centrifugation, and resuspend the pellet in freezing medium to 10^6ml. Then freeze aliquots of this suspension.

Hope this helps

Edited by klinmed, 16 May 2009 - 01:29 AM.


#4 nbhu

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Posted 16 May 2009 - 03:38 PM

Hi,

This may seem a pretty stupid or basic question but basically im finding it hard growing up my cells to obtain the approx 10million cells per ml required for freezing or for extracting DNA/RNA from.

I have a patient AML3 cell line, and i use alpha MEM media. My current cell count is 1million cells per ml.

This is my first time at cell culture so any help really well explained for dummies like me would be appreciated!!!


Are you trying to grow your cells to 10 million/ml? I cannot think of a cell line that grows to this density.

For freezing you need a healthy, rapidly dividing culture. I would grow to say 6-7 x 10^5 /ml, concentrate the cells by centrifugation, and resuspend the pellet in freezing medium to 10^6ml. Then freeze aliquots of this suspension.

Hope this helps


Thanks for the responses.
I am using Fetal calf serum, 20% as recommended for growing/culturing the cells. Also at the moment i am splitting the cells 1:2 in a total volume of 50ml... if my cell counts are 0.5 x 10^6, do i let them grow another week int he same media and count them, or do i take out 5ml and add 5ml fresh media etc? How do i maintain enough media and allow them to grow to a mazimum density?




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