I have tried to get some lung fibroblast from mice by using the following method: perfusing the lung with PBS and cut the lung into small pieces, about 1mm. I used the digestion buffer: 1mg/ml collagenase IV and 50U/L Dnase in 5%FBS DMEM. The digestion tube was kept shaking in a 37℃ incubator for 30 minutes. After digestion, I centrifuged the tube at 1000rpm for 5 minutes and discard the supernant. Resuspended the pellet in RBC lysis buffer and kept on ice for 4 minutes, neutralized and centrifuged it and discard the supernatant, resuspended the cells in 10% DMEM and culture. After 24 hours, I change the medium.
However, the cells I extracted seemed attached and growed very slowly. Maybe after a week, I can see the shape of attached fibroblast. There still need several days to let them grow into confluence. As I don't have much experience in growing the lung fibroblast, could someone tell me that whether it is right or not for taking such a long time for these fibroblast to attach and grow?
Another question is, the cells I extracted showed a myofibroblast phenotype and seems like to differenciate in a very early passage(maybe passage 2-3). Whether this question is related to the intense or insufficient of the digestion?
Hello Chen,
I have tried the following method to isolate lung fibroblasts from mice. This method works very well.
"After cervical dislocation or decapitation, excise the lungs and collect it in sterile PBS. Mince the tissue into bits of approximately 1 mm size and subject it to enzymatic digestion using dissociation medium(DM) (116.4 mM NaCl, 23.4 mM HEPES, 0.94 mM NaH2PO4, 5.4 mM KCl, 5.5 mM dextrose, 0.4 mM MgSO4, 1 mM CaCl2, 1mg/ml BSA, 0.5 mg/ml collagenase type IA, 1 mg/ml trypsin, and 0.020 mg/ml pancreatin, pH7.4). Digestion should be done in a shaker incubator for 10 minutes at 37oC. Collect the digest and centrifuge it at 1500 rpm for 5 minutes. Discard the supernatant and resuspend the pellet in 1ml of culture medium (with 10% FBS). Meantime repeat the digestion (Total 10 times, 10ml DM/digestion). Pool the cell pellets and then centrifuge it at 1500rpm for 7 minutes. Discard the supernatant. Resuspend the pellet in 4ml of culture medium and seed it into two 35 mm cell culture dishes. Incubate them in a humidified CO2 incubator at 37oC in 95% air-5% CO2 for 150 minutes. At the end of this period, discard the supernatant (contains unattached cells and debris) and rice the dishes (contains adherent fibroblasts) for 3-4 times with the complete culture medium. Keep the cells in a humidified CO2 incubator at 370C in 95% air-5% CO2. At 20 hours after the isolation, wash the cells and incubate it with culture medium maintain in a CO2 incubator at 370C."
Regarding the myofibroblast phenotypes:
The use of standard polystyrene culture plates will activate fibroblasts to myofibroblast phenotype. To reduce the activation/differentiation of fibroblasts, plate and maintain the cells in collagen-coated soft hydrogel-bound polystyrene plates (hydrogel stiffness 8 kPa or less).
Best
Hello Dr. Pramod Sahadevan , thank you so much for answer my question! I have other questions that what kind of medium are you using for treating these fibroblast? Are you using the high glucose (4.5g/L) or low glucose (1g/L) DMEM? Are you culturing these cells in 10%FBS ? And how many passages could they be passed ?(fibroblast are showing differentiation and there are many don't attach after several passage, I want to see how many passage they could reach).
I use M199 (M4530) low glucose (1g/L) with 10%FBS. I notmally complete my experiments with maximum passage 3. I will not continue after p3.
Dr. Pramod Sahadevan , thank you! I'm now trying the matrigel perisoft dishes. Are you using these dishes? It seems different in morphology of these fibroblast in the hydrogel dishes. Do you have some experience about that?
For getting the fibroblasts ( to reduce fibroblast activation), we are using collagen-coated soft hydrogel-bound polystyrene plates from "Matrigen". Yes, the cell's morphology is slightly different compared to the hard culture plates but they behave well.
Thank you Dr. Pramod Sahadevan ！We are now trying some Perisoft dishes. What are the stiffness you are using? In my experience, it's hard for me to use trypsin to digest the cells from the collagen-coated soft hydrogel-bound plate even I have changed the no serum medium for more than 30 minutes. Have you followed their recommondation and bought TrypLE and CYTOONE® CELL SCRAPER for your cells?
Can you tell me how to extract cell protein from the dishes? That's also a problem for me.
The stiffness that we used is 8 kPa. No, i haven't used their products. You can use Trypsin-EDTA (2.5 mg/ml trypsin, 0.38 mg/ml EDTA solution). After washing the cells with warm PBS, Incubate the cells for around 3-5 minutes with Trypsin-EDTA (warm, 37oC). Stop the trypsinization reaction with complete medium. Collect the cells and centrifuge to get the pellet. Add cell Lysis buffer to the pellet and process it further for the protein extraction.