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Primary mouse lung fibroblast cultures

Primary mouse lung fibroblast cultures - Cell Biology and Cell Culture

Primary mouse lung fibroblast cultures - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


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Old 02-10-2011, 04:11 PM
Pipette Filler
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Default Primary mouse lung fibroblast cultures



I am trying to establish a primary culture of fibroblasts from mice. I was following a protocol previously used in our lab that isolates them from lungs, and I recently tried another protocol that isolates fibroblasts from ear clips. No matter what I do, no matter which reagents I use, I cannot get fibroblasts to grow (and these are protocols that should just work with minimal effort!).

I have been removing the tissue (lung or ear clip), rinsing in PBS, mincing with a razor, treating with trypsin (I've tried 0.05% for 1 hr and 0.25% for 10 min), rinsing with DMEM (+15% FBS +1% antibiotic) and then plating. I also tried an additional 1hr, 3hr, and 5hr 0.3% collagenase digestion on the lung tissue. With the lungs, I have a goopy mess with bits of tissue and over time just dead cellular debris at the bottom, and with the ear clips I am getting nothing but floating bits of tissue in media. Bacterial contamination is not a concern.

Anyone have any ideas why I cannot get fibroblasts to grow? I do not think I am over-digesting the tissue, and another individual followed the same protocol and it did not work for her either. Does anyone else get the goop (reminds me of cellular membrane debris, but I still have lots of bits of tissue)? Do I need to supplement my media with anything extra? Sing the cells a song, read them a book?

This is supposed to be very easy, and it's holding up several months of planned experiments until I can get these cells to grow.

Sincerely, Fibroblast Hater (Caitlin)
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Old 02-21-2011, 05:52 PM
Pipette Filler
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Default Re: Primary mouse lung fibroblast cultures

Have you tried a smaller volume of media? Shoot me an email because our membranes are Silicon based which we have had feedback and publications showing our adhesion and growth rates are better than glass or PS platforms.


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Old 07-29-2011, 10:34 PM
Pipette Filler
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Default Re: Primary mouse lung fibroblast cultures

Quote:
Originally Posted by cemacnair View Post
I am trying to establish a primary culture of fibroblasts from mice. I was following a protocol previously used in our lab that isolates them from lungs, and I recently tried another protocol that isolates fibroblasts from ear clips. No matter what I do, no matter which reagents I use, I cannot get fibroblasts to grow (and these are protocols that should just work with minimal effort!).

I have been removing the tissue (lung or ear clip), rinsing in PBS, mincing with a razor, treating with trypsin (I've tried 0.05% for 1 hr and 0.25% for 10 min), rinsing with DMEM (+15% FBS +1% antibiotic) and then plating. I also tried an additional 1hr, 3hr, and 5hr 0.3% collagenase digestion on the lung tissue. With the lungs, I have a goopy mess with bits of tissue and over time just dead cellular debris at the bottom, and with the ear clips I am getting nothing but floating bits of tissue in media. Bacterial contamination is not a concern.

Anyone have any ideas why I cannot get fibroblasts to grow? I do not think I am over-digesting the tissue, and another individual followed the same protocol and it did not work for her either. Does anyone else get the goop (reminds me of cellular membrane debris, but I still have lots of bits of tissue)? Do I need to supplement my media with anything extra? Sing the cells a song, read them a book?

This is supposed to be very easy, and it's holding up several months of planned experiments until I can get these cells to grow.

Sincerely, Fibroblast Hater (Caitlin)
Hi Caitlin,

Here's my lab protocol. I use this often and it always works well for me. The snot you keep getting is why you need the DNAse in the enzyme solution.

0.2% trypsin
0.1% collagenase IV (Gibco 17104-019)
400ug/ml DNAseI assuming 2000 U/Mg
Made in HBSS-CMF

Digest one minced lung in 20ml shaking at 300rpm at 37 deg 30 min
Strain to a fresh tube through a 70um cell strainer
Add 5ml FBS and hold sup on ice

Use another 20ml of enzyme solution to rinse the minced tissue from the strainer into a fresh tube and digest again.

Again strain sup and spin both for 5 min at 600xg

You may do a RBC lysis step as well or just resuspend pellet in DMEM/F12 w/10% FBS additional L-Glut and antibiotics. If you don't do the RBC lysis, change the media the next day and rinse with media as well to remove the RBCs. You should be able to put the digest from 2 lungs into a T225 and have a confluent flask in a week.

Good Luck.
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