Smooth muscle cell culture - (Nov/13/2007 )

Hi ,

Has anyone cultured human smooth muscle tissue in completely serum free medium? I want to use DMEM plus ITS and EGF, FGF but I dont know if it will work. I did some search in the literature and I know there are serum free media design for smooth muscle cell, but the nutritient suplements for those media unfortunately have FCS as a compound:(

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Nope But I found out the hard way (whilst doing my honours year) that they needed high glucose medium!
Good luck
Clare

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I used to culture hSMC from clonetics and if i remember well, they are selling a kit (bullet kit) to make media (serum, EGF, insulin and other thing i can't remember). The important thing with this primary cells is that you can't use them for more than 8 passages.
In order to synchronize the cells i withdrawned the serum for 2 days but the shape of the cells changed which is never a good sign.
Good luck
bob

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Dear Silver80,

Just a few pointers with regards to the experience we have had over the years:

Isolation of SMC: We have over the years isolated primary cells from saphenous vein/thoracic aorta. There are 2 methods, Explants or Dissociated tissue. From animals, cells grow well from young/old rats. When it comes to human primary cells, patients have to be younger than 30 for the cells to grow well. We always grow the cells in high glucose DMEM + 10/15% FCS ( normally NZ sourced FCS which is the best serum). WE HAVE NOT ATTEMPTED TO GROW IN SERUM FREE CONDITIONS. The commercial Primary SMC lines from Clonetics and Biowhittaker grow in "Defined Media" BUT HAVE 2% FCS, Bovine brain extract, hydrocortisone etc ( bullets kits)......SO THEY ARE NOT SERUM FREE. Also if you intend to use these commercial lines, there are other "secret ingredients" they do not tell you about. They will not devulge their media constituents, but will if pressed tell you if " X " is in the media.

REMEMBER that cells are only a model for easy experimentation and results have to be carefully interpreted. In culture the SMC cells instantly revert from their normal CONTRACTILE PHENOTYPE to what is descibed as a SYNTHETIC PHENOTYPE.....again not a good sign.

It is very important to check that the marker you want to observe is their in your cells. If the cells are isolated from primary tissue, check for:
a) Alpha actin staining: a specific marker for SMC.
MYCOPLASMA TEST ALL PRIMARY CELLS.

As ROM1 has correctly stated, all primary smooth muscle cells SHOULD BE USED IN LOW PASSAGE, as they change dramatically over time in culture. As he says the cells change morphology over time AND IS DEFINITELY NOT A GOOD SIGN, as SMC should have a characteristic "hill and valley morphology".

I do hope this does not put you off completely, but experimentation is an art and easily corrupted by poor design. In my opinion it is useful to use A7R5/A10 SMC cell lines ( Rat Aortic smooth muscle cells) to do preliminary experiments.....the advantages are:
i) Cell lines are more stable over time/passage, and can be more easily manipulated into serum free conditions.
ii) Mycoplasma testing and characterisations have been done already.
iii) It saves alot of time and money on enzymes/antibodies/mycoplasma testing.......AND YOUR PRECIOUS TIME by using cell lines.
iv) They are more suspectable to transfection than primary cell lines.
v). It is alot easier to produce large cell numbers and therefore do larger experiments and do they more frequentely.


Disadvantages:
i) They have been virally/chemically transformed and are very different in alot of ways to primary cells.


I hope this is useful, please send me a private message if you need more information.

Kindest regards.

Rhombus.

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Dear all,

Thank you for information I appreciate your help .
I forgot to write that I am focusing on primary myometrium smooth cell cultures :/, but if it won't work we would switch to cell lines then. And I am going to try serum free stuff next week, but I am not convinced that it will work.


Silver:)

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