Vacuoles/Empty spaces in Endothelial cells

P. Friedl di58 at pop.th-darmstadt.de
Fri Mar 31 04:49:28 EST 1995


In article <3l76ni$bsc at mark.ucdavis.edu>, dobates at ucdavis.edu (Dave Bates) says:
>
>In article <3kqav6$of9 at news.acns.nwu.edu>
>djack at merle.acns.nwu.edu (Dowdy Jackson) writes:
>
>> In article <Pine.OSF.3.91.950319022615.20687A-100000 at duke.usask.ca>,
>> Jason at UofSask <jir130 at duke.usask.ca> wrote:
>> >I'm currently culturing bovine brain endothelial cells.  I've noticed 
>> >that in some of the cells there appears to be what looks like vacuoles or 
>> >empty spaces (ranging from small ones to some that are about 1/4 the size 
>> >of the cell) in the cytoplasm of the cells, usually near the nucleus.  
>> >The cells don't seem to be dying as they are able to reach confluence.  
>> >Does anyone have any ideas as to what may be causing the formation of 
>> >these vacuoles/spaces and how can it be eliminated from the culture?  
>> >Could the cause be the serum (calf serum) or the basic fibroblast 
>> >growth factor (human, recombinant) being used?
>> >
>> >TIA for any help/advice or comments given.
>> >
>> >Jason Ram
>> >
>> 
>> 
>> We have seen the same things in our bovine endothelial cells but this usuall
>> happens because of :
>> 
>> a) Cells are close to the max passage number.
>> b) The serum concentration or mitogen concentrations are too low.
>> c) The CO2 in the incubators is incorrect.
>> 
>> We use Endothelial Cell Growth Mitogen from Biomedical Technologies as our
>> mitogen becasue it works well and is cheaper than using fibroblast growth
>> factor.
>> 
>> Dowdy Jackson
>> Department of Biochemistry, Molecular Biology and Cell Biology
>
>I see this from time to time too. I would agree with Dowdy, especially
>on the CO2 and the passage number. Also, be careful about your pH. If
>the media is too alkaline then they get vacuolar (also caused by low
>CO2 or by the tops of the flasks being airtight). Also make sure you
>put fresh L-Glu in your media at least every couple of days. I use
>fetal bovine serum not Newborn Calf. They like this better and I use it
>at 10% (although others say 7% is fine). Keep the passage number below
>20 if you can. For important experiments use 12 or less. A lot of stuff
>changes in these cells as you passage them - from 2 or 3 upwards. There
>was a poster on von Wilebrand Factor expression at ASCB last year which
>said that it is produced at a lower rate after passage 6.
>
>
>------------------------------------------------------------------------
>-
>Dave Bates PhD                                     Tel: 916 752-7081
>Postdoctoral Researcher                            Fax: 916 758-2554 
>Dept of Human Physiology                         email:
>dobates at ucdavis.edu
>University of California at Davis                drink: anything
>Davis, California 95616, USA            No I don't have a sense of
>humour 
>                                                now buy me a beer      
>------------------------------------------------------------------------
>----       
Concerning the suitability of FCS or NCS for endothelial cells: NCS is better.
We tested 9 different lots of NCS and found 3 lots suitable to promote
EC growth at densities of 50-100 cells per cm2, whereas none out of 20 
different lots of FCS was suitable. Ref.: Cytotechnology (1989) 2, 171-179

P.F.



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