MDCK Cell :Reply

Excelife excelife at earthlink.net
Fri Sep 4 08:11:24 EST 1998


In article <6sn9at$pds$1 at nnrp1.dejanews.com>, stebby at welchlink.welch.jhu.edu 
says...
>
>
>
>Mi Sun Kang wrote:
>
>> I culture MDCK cell to research toxicity mechaism of heavy metal.
>> MDCK cell is kidney epithelial cell and always had grown very quickly in 
my
>> lab. Because it bind to each other by tight junction, gap junction,
>> desmosome and focal adhesion, it looks like very big aggregates.   But
>> Shape of mdck cell changed into fibroblastic and size of the cell looks 
very
>> small. All the worse, after trypsinizing to subculture, only half the cell
>> survive. Doubling time of the cell is very long.
>> I want to knowf what happen to my cell!!
>> Could character of MDCK cell be changed by continuous subculture?
>
>
>Mi Sun:
>
>Stephen Knight's response to your post is worth reading and taking to heart.
>I'd like to expand on his thoughts a bit.
>
>First of all you should consider who or where you got your MDCK cells from
>and the possibility that you did not receive what you were hoping for.  Many
>a lab has been sent samples with good intentions only to discover that the
>wrong reagent or a contaminated reagent was sent.  If there is any doubt in
>your mind I would suggest getting a good culture from a trusted source.  In
>the United States many of us depend on the American Type Culture Collection
>(ATCC--they have a web site at http://www.atcc.org/).  I do not know a 
source
>in Korea, but I do know that a similar collection of cell lines exists in
>Japan.  E-mail ATCC and they can probably direct you to a contact there.  
The
>catalog number for MDCK cells in the ATCC catalog is ATCC CCL 34.
>
>Second, you should consider what you may have done to the cells in your lab.
>The point is well made by Stephen Knight concerning cross-contamination,
>mycobacterial contamination, and source contamination in your reagents 
and/or
>laboratory ware.  As a rule of thumb you should always freeze ampules of a
>cell line prior to commencing experiments.  It is also a good idea to pick a
>passage number within which you will work.  You ask in your message if the
>character of MDCK cells can change with continuous subculture.  The answer 
is
>that ANY cell line can and probably will change under these conditions. 
>Remember that every day is evolution day in the culture flask.  You are
>continuously selecting for cells that grow best under your conditions. 
>Experiments performed on passage 100 cells may very well not replicate
>experiments performed on passage 10 cells. Its an inherent problem we all
>accept, but it doesn't hurt to remind oneself of this point every so often
>:o)  Remember that the MDCK cell line was generated in 1958.  Today it is
>well known that there are high resistance (type I) and low resistance (type
>II) MDCK cells.  One need look no further than the work performed on the
>sorting of the Na+,K+-ATPase to see how difference in clones of MDCK cells
>can yield contrasting results (For a good read of science policing itself 
see
>the discussion that takes place in "Science" volume 260 pp.550--556.)
>
>In conclusion, problems such as those you have described can be a real
>nightmare.  Go back to your frozen stocks if you have them and double check
>your ability to replicate your data so far.  Do not destroy your sick 
culture
>until you have thoroughly tested for mycoplasma or other contamination.  If
>present, thoroughly decontaminate your culture area before thawing your new
>cells!  In addition, to be safe I would also order some bona fide MDCK cells
>and check the ability to replicate your data in these as well.  To 
safe-guard
>your lab in the future try to quarantine all new cultures you get from
>colleagues and test for contamination before admitting into your stock.
>
>Best of luck,
>
>Steve Dahl
>
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I do appreciate subtlety!

Steve Dahl has correctly pointed out that MDCK is an immortal cell line 
similar in some respects to the HeLa cell line and is not subject to the 
Hayflick limit of reproduction in vivo. 

The protocols and material handling techniques described by both Steve Dahl 
and Stephen Knight are of the utmost importance and most likely include the 
cause of Mi Sun Kangs problems.

However, his description of the cell line still sounds like the results seen 
when a cell line enters senescence.  Since the activation of the enzyme 
telomerase is required for cellular immortalization then it's possible that 
whatever has contaminated his culture has had the effect of inhibiting the 
actions of this enzyme.

Once again, a southern blot or a  fluorescence in situ hybridization,(FISH), 
test to determine telomeric length would put this question to rest.

If, by a long shot, he has found a contaminant that inhibits the actions of 
the enzyme telomerase, and can identify what that contaminant is...  Well it 
wouldn't be the first time cells dying in a petri dish have led to an 
unexpected scientific breakthrough!



Thomas Mahoney, Pres.
Lifeline Laboratories, Inc.
http://home.earthlink.net/~excelife/index.html







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