I am trying to isolate fibroblasts from normal murine mammary tissues extracted from laboratory mice in July 2016 and stored in RNAlater at -20 C. I would greatly appreciated any help with troubleshooting or suggestions as I have been unable to isolate any fibroblasts.
I have been attempting the protocol found in the publication "Establishing Primary Adult Fibroblast Cultures From Rodents" (V. Gorbunova, 2010). I have attempted the protocol a few times with no success in fibroblast isolation. A few details of the protocol and changes I have made:
- Initially I remove the tissue from RNAlater and washing with PBS, then cut the tissue with sterile scalpels into about 5 mm pieces.
- Next, I have digested the tissue with Liberase TM (contains collagenase I and II) two different ways: on hotplate stirrer at 37 C stirring on low setting for 75 minute or by incubating tissue for 18 hours at 37 C. Tissue fragments do appear separated and media cloudy. I pipette the liberase and tissue solution vigorously up and down post digestion.
- I follow the protocol exactly in regards to deactivating the Liberase with total 30 mL DMEM w/ 15% FBS and 1X antibiotic, centrifuge at 524 x g for 5 min, remove supernatant from pellet, and repeat 2 more times.
- I resuspend the final pellet in 10 mL DMEM w/ 15% FBS and 1X antibiotic and plate on 10 cm tissue culture dish
- Initially, I did not have access to a 3% oxygen tank, so I incubated the tissue culture dish containing the resuspension at 37 C, 5% CO2 and 1% O2 with no fibroblast isolation. Last week I gained access to a 3% oxygen tank and attempted the same protocol but incubated at 37 C, 5% CO2 and 3% O2. I checked the cells after 7 days but did not see any fibroblasts isolated. I add new media and placed the tissue in the incubator for another 7 days (currently at day 4).
Please let me know if you have any suggestions on changes or ways to troubleshoot this protocol for better fibroblast isolation. Thank you in advance.
Why did you choose RNAlater as the storage solution? I don't believe you will get live tissue after storing it in RNAlater at -20C, but the RNA should still be ok. Have you verified that the cells are still living?
As for soft tissue digestion, 75 minutes seems like overkill. 10-30 minutes should be adequate.
Instead of enzymatic digestion, can you try crushing the tissue through a ~70um cell strainer and just culturing that? Or even just chop up the tissues samples and culture. If the cells are still alive, you should see fibroblasts start crawling out of the tissues.
I agree with Charles. Along the way, you can stain the cells with Trypan blue to estimate cell viability.
Thank you Charles and Michael for your answers.
How can I verify the tissue is still viable without being able to extract cells? Is it better to stain a tissue fragment with trypan blue or digest the tissue and stain with propidium iodide then analyze with cell cytometry? Other suggestions on how to verify tissue viability are appreciated.
Secondly, if I opt to use fresh gland tissue, I will have to travel 25 minutes from the animal facility to the research lab facility. What is the best way to store the fresh tissue during this commute (saline, media with FBS, or RNALater, etc.)?
Thank you again for your help!
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