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Cell Suspension from Frozen Tissue?


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#1 seaboardbc

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Posted 03 February 2010 - 11:16 AM

I am a student researcher at the University of Minnesota. I am looking for a method to produce viable cells from frozen human tonsil tissue (I don't have fresh tonsil or frozen single cell suspensions that I can use). I plan on using the tissue to make a single cell suspension that I will use to examine the different cell populations using flow cytometry based on different cell markers. I was wondering if anyone has a method that works to make single cell suspensions from frozen human tissue and get a good viable cell count?
Thanks
Bren

#2 bob1

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Posted 03 February 2010 - 03:58 PM

How has the tissue been frozen? If it has been frozen without cryoprotectants (e.g. DMSO) then you are very unlikely to get any viable cells out of the tissue, as ice crystals generated during the freezing/thawing will puncture the cell membranes.

#3 rhombus

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Posted 04 February 2010 - 02:32 AM

I am a student researcher at the University of Minnesota. I am looking for a method to produce viable cells from frozen human tonsil tissue (I don't have fresh tonsil or frozen single cell suspensions that I can use). I plan on using the tissue to make a single cell suspension that I will use to examine the different cell populations using flow cytometry based on different cell markers. I was wondering if anyone has a method that works to make single cell suspensions from frozen human tissue and get a good viable cell count?
Thanks
Bren



One word.....Impossible

#4 seaboardbc

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Posted 09 February 2010 - 08:45 AM

How has the tissue been frozen? If it has been frozen without cryoprotectants (e.g. DMSO) then you are very unlikely to get any viable cells out of the tissue, as ice crystals generated during the freezing/thawing will puncture the cell membranes.



Hi~
Sorry it took me so long to get back to you. It was frozen with DMSO and fetal calf serum. I am thawing it next week in an attempt to make single cell suspensions. Any suggestions?
Thanks
Bren

#5 lab rat

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Posted 09 February 2010 - 09:53 AM

Maybe this will help. It's a paper about fetal brain primary cultures, but the techniques (cryopres of whole tissue thru primary culture) should be applicable.

Antonella Peruffo et al. (2004) Primary cultures from fetal bovine brain. Developmental Neuroscience vol. 15, No 11 1719-22.

http://journals.lww....ne_brain.6.aspx
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#6 seaboardbc

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Posted 11 February 2010 - 02:22 PM

Maybe this will help. It's a paper about fetal brain primary cultures, but the techniques (cryopres of whole tissue thru primary culture) should be applicable.

Antonella Peruffo et al. (2004) Primary cultures from fetal bovine brain. Developmental Neuroscience vol. 15, No 11 1719-22.

http://journals.lww....ne_brain.6.aspx


Thank you very much. Next week will be my first attempt to work with the frozen tissue. I'll let you know how things turn out.
Bren

#7 seaboardbc

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Posted 20 February 2010 - 09:28 AM

I thawed the tissue like you would a frozen single cell suspension and then immediately made a single cell suspension. I was able to activate with PMA/Ionomycin, but had a problems with the anti-CD4 and anti-CD3 staining (the intracellular cytokine staining worked) on all samples. I repeated it on an unstained, unstimulated sample and increased the concentration of the anti-CD3 and 4, it was slightly better, but not significantly better. Any suggestions? I will keep you posted.
Bren




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