fibroblast primary culture - (Apr/01/2009 )

I've been trying to isolate skin fibroblasts from rat's skin and used different methods and protocols
which wasnt successful, the skin has trouble attaching to the plastic surface (now i'm trying to isolate them from rabbit's skin )
also i\m not sure what serum concentration i have to add 10% or 20% its different depending on the protocol.
Also i have trouble with culture contaminants fungi mostly and yeast.
i'm using DMEM 20% FBS and antiboitic solution with amphotericinB 1ml/100ml
are there any substitutions for amphotericin cuz its very expensive.
Also should a culture dish hood stay slightly open in incubator or be closed with parafilm

thank u

Hi Lyeshmanioz,

I regularly make primary cell cultures. While I normally prepare follicular dendritic cells (of fibroblastic origin) from lymph nodes, I will be happy to give advice. I also have access to others who prepare cell cultures from endothelial cells.

An excellent book on cell culture is Freshney's Culture of Mammalian Cells.
Hardcover: 672 pages
Publisher: Wiley-Liss; 5 edition (July 29, 2005)
Language: English
ISBN-10: 0471453293
ISBN-13: 978-0471453291

Thanks,
lab rat

Yeast and fungi are really common on skin from any mammal. You will need to sterilise the outer surface of the skin before you take it for culture. To do this shave the skin to remove any hair, then wipe with surgical prep style solution (usually iodine in isopropanol) and then cut inside the area of the skin that has been sterilised. Rinse excised skin in PBS a few times to remove the iodine and IPA and then generate your fibroblasts from there.

thank you guys
the problem is i work in the area full of flying yeast they are everywhere

leyshmanioz on Apr 5 2009, 06:21 AM said:

You need to work in a laminar flow hood or a bio-safety hood, these should stop yeast and bacteria from getting in your cultures during preparation, so long as they are maintained well.

As far as your problems with attachment, have you tried any adhesion factors? In my cultures (DRG neurons), I consider fibroblasts a contaminant, but I can tell you that with Poly-L-Lysine and/or Laminin, they attach just fine. Also, look for culture plates that are treated for adherent cells. They sometimes cost a little more, but you get much better attachment.

gfischer on Apr 9 2009, 08:36 AM said:

they're adhere very well, but now i have problems with contamination with fungi , even though i use antimicotic, fungi appear in second passage.
Does Trypsin affects cells when dethaching them from plastic, and whats the difference between trypsin and versen solution.

how to use this plastic thing to detach cells

whats the best culture dish, petri or flask, contaminationwise

thanks

Your best bet to prevent contamination is to use a flask, especially the ones with a filter in the lid so you don't have to leave the it partly unscrewed for gas exchange. Your attachment problems with fibroblasts may not be due to the surface at all, but rather the amphotericin B, it is quite cytotoxic to all eukaryotic cells, just more so to fungi/yeast.

As you are having such problems with contamination, I suggest a complete review of your technique and where you are working. If you have never done cell culture before, I suggest reading R.I. Freshney's Culture of Mammalian Cells (the same book Lab Rat suggested) and getting someone experienced to show you sterile technique. If you work in a yeast/fungus/bacteria lab - find another area to work in, I can't stress this enough, unless you are working in a bio-safety hood and have excellent technique, you will end up with contamination frequently. Be aware that a lot of contamination is cryptic, as in you can't see it, but it is still there - a few dozen bacteria per ml is not visible, but it will have an effect on your cultures. Have a look for posts by Rhombus if you want more info - he is the expert.

Regarding amphotericinB:

Sigma offers it relatively cheap while the same antimycotic is offered at expensive levels by Invitrogen/Gibco as "fungizone".