Pituitary Primary culture- need help - Need protocol/ troubleshooting (Feb/12/2008 )

Hi All,

I am attempting to perform primary culture of rat pituitary cells in order to study the effect of a neuropeptide on TSH and Prl release

Anyways, I have done several test runs using one or two rat brains and the best yield I have gotten from whole pituitaries is at least 320,000 cells. The published yield is around 1-2 million cells per anterior pituitary (and Im using whole pituitary- makes my yield that much more sad)

If anyone has any suggestions or a protocol that they use to get better results it would be greatly appreciated. Currently my protocol is as follows:

Extract pituitary and wash in pbs
Dice finely with sterile scalpel
Incubate in serum free DMEM/ F12K + 0.5 % collagenase type I at 37C for 30 - 40 minutes (Ive tried shorter incubation with even worse results) mixing by hand periodically and removing clumps by pasteur pipetting every 10 minutes
Add 8ml PBS & spin 1250 rpm (don't have x g conversion handy)
wash & spin again
remove supernatent
add .03% EDTA/ PBS
spin again & remove sup
incubate with .05% trypin/ pbs 20 min
add media with serum & strain through 70-uM nylon filter
spin again in 50ml connicle& resuspend in media
Count cells
Bang head on lab bench in frustration

I've noticed that I get a lot of clumping once the diced pituitary is put in collagenase solution- I tried removing these by running the large clumps through a pasteur pipette which seems to increase yield a little- I know it's not great for viability, but I'm trying to just really improve total yield at this point by breaking up the clumps.

If anyone has much experience with this and can offer some tips or has a good protocol I would greatly appreciate it. Thanks!

May be its clumping because of all the DNA released after different enzymatic digestions. Try adding some DNAse to your cells after collagenase or after trypsin. It can definitely make things less clumpy.

Thanks for the advice. Dnase definitely made it less clumpy and improved yield some. The yield is still not very good though- could I somehow be losing cells during the wash step centrifugations- I am only doing one brain at a time and it might be enough tissue to stay down after a spin?

Any ideas?

Thanks

We often did multiple animals for pituitary cells. The yield is not a lot but you definitely need lots of animals even for a single experiment. One loses a percentage with every wash so its better to start with a higher number of cells.

Do you happen to remember approximately how many mice or rats it took for each data point- I have read about 500,000 cells is necessary per well and am not sure what kind of yield people get. In the literature I have seen 1-2 million cells/ rat pituitary but that seems really high ( I'm pulling in about 300,000 per pituitary lately). Thanks again

Thank you much- I'll try it out with a load of rats and see if I can get results.

Hi all,

My prolem is slightly different. I have a couple of questions regarding use of collagenase in isolating primary endothelial cells.

Q.1. I have been using collagenae Type 1 (Worthington) for both the heart and the lung tissues. But I have read in the company product info page that Collagenase Type 4 has leesser tryptic activity and is better at maintaining the integrity of cell surface protiens. does anyone have any experience in using different types of Collagenases?

Q. 2 Also what is the use of EDTA in the isolation procedure. I know it is a Calcium chelator but how does the presence of calcium affect the isolation of primary cells?

Q.3 Also I have never used Trypsin in my isolation procedure, is using trypsin necessary?

thanks a lot for all the information you all are sharing with the others.

It has been a lot of help.

P.maj