Does FBS in dmem inhibit the action of collagenase and elastase? - (Dec/04/2007 )
Hi guys, i'm was trying to extract smooth muscle cells from porcine esophagus for primary cell culture studies and i used a digesting solution consisting of Dmem, FBS, penicillin, streptomycin, amphotericin B, collagenase and elastase. After one hour in the incubator, the minced tissue clumps still looks the same and i did not get any cells after filtering through a 40um cell strainer. Does anyone know if the FBS inhibits the action of collagenase and elastase? Thanks in advance.
Hi,
I would say yes. I use serum free DMEM (plus pen/strep) when extracting the smooth muscle cells from tissue. After 30 minutes incubation I put the supernatant solution through the falcon sieve and add DMEM with 10%FBS gradually to quench the enzymes. (I use Collagenase XI and Coll. 1A).
Hope it will helps you
Cheers,
Anna
Dear Blitzzz,
Yes FBS/FCS inhibits both collagenase and Elastase activity....fact. I have isolated cells from Aorta, Trachea, Cartilage, Skin, Umbilical Vein, Heart muscle etc .....using Pronase, Collagenase (Types I-IV), Elastase, Pronase, Hyluronidase, Trypsin etc.
ALL ARE INHIBITED BY SERUM.
What worries me a little is that you are adding Amphotericin B in your mix. We have found this to be a POTENT inhibitor of many cellular pathways and receptor mediated events. It is therefore a very dirty compound and should not be used.
Our cells experiments, especially with primary isolated PAEC, are well documented.......3 Nature papers in a year and a half in the late 1980's.......Palmer, Ashton and Moncada et al ......release of EDRF/Niric Oxide from Porcine aortic endothelial cells.
Hope this proves useful.
Kindest regards.
Rhombus.
Hi Silver80 and Rhombus, many thanks for your inputs~ I had the amphotericin B in the digesting solution because i used the antibiotic anmycotic solution (from sigma aldrich) which contains all pen, strep and amphotericin. After hearing what you said, i think i'd better change to just pen/strep alone. However, i am concern abt fungi growth. Is it ok not to use amphotericin B at all in the whole isolation process?
Dear Blitzzz,
I know exactly where you are coming from. I isolated primary porcine aortic endothelial cells for over 6 years in the 1980's. Going to abattoirs to get aorta.......not the cleanest of places. I tried at first to use Amphotericin/Pen/Strep in my culture media. I got more clean cells but they were not biologically active i.e. Amphotericin inhibited EDRF release by Bradykinin in these cells (unpublished data).
We adjusted our experimental protocol and aseptic technique as follows:
Collect Aorta DRY, rather than submerged in PBS...this reduced cross contamination.
When back in the lab we washed our aorta x5 in PBS with Pen/strep. Cells collected from each aorta were quarantined from the others......i.e. no POOLING of cells from different aorta's.
Cells were screened for 4-5 days for possible contaminants.
Clean cells finally pooled and seeded onto Cytodex beads in stirrer culture.
BIOLOGICALLY ACTIVE PRIMARY CELLS THAT RELEASED EDRF (NITRIC OXIDE) BY BRADYKININ STIMULATION.
Kindest regards
Rhombus
Dear Blitzzz,
I know exactly where you are coming from. I isolated primary porcine aortic endothelial cells for over 6 years in the 1980's. Going to abattoirs to get aorta.......not the cleanest of places. I tried at first to use Amphotericin/Pen/Strep in my culture media. I got more clean cells but they were not biologically active i.e. Amphotericin inhibited EDRF release by Bradykinin in these cells (unpublished data).
We adjusted our experimental protocol and aseptic technique as follows:
Collect Aorta DRY, rather than submerged in PBS...this reduced cross contamination.
When back in the lab we washed our aorta x5 in PBS with Pen/strep. Cells collected from each aorta were quarantined from the others......i.e. no POOLING of cells from different aorta's.
Cells were screened for 4-5 days for possible contaminants.
Clean cells finally pooled and seeded onto Cytodex beads in stirrer culture.
BIOLOGICALLY ACTIVE PRIMARY CELLS THAT RELEASED EDRF (NITRIC OXIDE) BY BRADYKININ STIMULATION.
Kindest regards
Rhombus
Wow Rhombus, that was exactly what i did: Collected the esophagus, put it in a bottle full of pbs with pen/strep/amphotericin at 4 degree celcius and brought it back to the lab. I'll definitely read up on your articles and see if i can adjust my protocols accordingly. Btw, may i ask if you had put the aorta in a tub of ice or just put the aorta in a bag or sterile flask with nothing inside before bringing it back to the lab? I figured if the aorta is collected dry then it must be at least contained in something clean?
Cheers,
James
Dear Blitzzz,
I know exactly where you are coming from. I isolated primary porcine aortic endothelial cells for over 6 years in the 1980's. Going to abattoirs to get aorta.......not the cleanest of places. I tried at first to use Amphotericin/Pen/Strep in my culture media. I got more clean cells but they were not biologically active i.e. Amphotericin inhibited EDRF release by Bradykinin in these cells (unpublished data).
We adjusted our experimental protocol and aseptic technique as follows:
Collect Aorta DRY, rather than submerged in PBS...this reduced cross contamination.
When back in the lab we washed our aorta x5 in PBS with Pen/strep. Cells collected from each aorta were quarantined from the others......i.e. no POOLING of cells from different aorta's.
Cells were screened for 4-5 days for possible contaminants.
Clean cells finally pooled and seeded onto Cytodex beads in stirrer culture.
BIOLOGICALLY ACTIVE PRIMARY CELLS THAT RELEASED EDRF (NITRIC OXIDE) BY BRADYKININ STIMULATION.
Kindest regards
Rhombus
Wow Rhombus, that was exactly what i did: Collected the esophagus, put it in a bottle full of pbs with pen/strep/amphotericin at 4 degree celcius and brought it back to the lab. I'll definitely read up on your articles and see if i can adjust my protocols accordingly. Btw, may i ask if you had put the aorta in a tub of ice or just put the aorta in a bag or sterile flask with nothing inside before bringing it back to the lab? I figured if the aorta is collected dry then it must be at least contained in something clean?
Cheers,
James
Dear Blitzzz (James),
The aorta were transported dry in an autoclaved 1L glass beaker.....NOT ON ICE. But in a cool box with 2 or 3 freezer packs to cool down the box. The method I descibed earlier works great in the Autumn, Winter and Spring. However rates of infection/contamination do rise in the summer for obvious reasons. In the summer we used to increase the number of washes and length of quarantine time.
In all scientific papers the material and methods are woefully lacking any great detail. You will not find any of these details in our 3 Nature papers in the 1980's (listed below):-
Gryglewski, Moncada and Palmer...... Nature 1986, 320, pages 454-456
Palmer, Ferrige and Moncada.......Nature 1987, 327, pages 524-526
Palmer, Ashton and Moncada.......Nature 1988, 333, pages 664-666
Any other tips please send me a private message as Dominic is sick of me bashing on about excellence in Science. I still think Science and Nature journals are the Gold standard journals. It is these unpublished secrets that are the basis of remarkable papers.
Kindest regards
Rhombus
i heard that 
Dear Dom,
I knew you would.
Kindest regards as ever
Rhombus
Hi guys,
Well, i did a 2nd round of smooth muscle extraction today and results are....not good(sadly). I couldnt get any cells after the disgestion process and all i see in the tissue culture flask are tissue debris. Below are the steps that i used in the extraction process:
1) collected sample from abbatoir ( washed with pbs + strep/pen)
2) Store them in a sterile culture flask and brought back to the lab as quickly as i could (around 20 mins)
3) washed again x3
4) remove all visible connective tissues and cut the muscle layer into small pieces (all less than 2mm at most)
5) wash the pieces again
6) put them in 10 ml of hanks buffered solution + pen/strep + collagenase (concentration of collagenase is 0.6mg/ml)
7) incubate them at 37 degree celcius for 3 hours and shaking them once in a while
8) take out, centrifudge away the solution, use complete medium to resuspend and put the suspension through a 40um cell strainer to seperate the cells from the undigested clumps. Finally, i pour them into a tissue culture flask
hmm.. i'm not sure what went wrong. i can only think of a few possibilities:
a) concentration of collagenase may be too low
b ) the cells may be stuck at the 40um cell strainer or,
c) i may be cutting the wrong part of the tissue (which i really don't think so)
Any ideas guys? Meanwhile i will leave the flask overnight in the incubator and see what happens the next morning. Will let you guys know.
Cheers ,
James