I am isolating smooth muscle cells form abdominal aortic aneurysm tissue. In brief I cut obtained tissue into small pieces and digest it overnight. The cells grow very well in M199 medium with 10% FSC and antibiotics, however, after three passages I get more and more fibroblasts. Can anyone advice me how to diminish their population? As I know that fibroblast are easier detached I tried to trypsinize cells for one minute, then get rid of the medium and again use trypsine to detached the rest of the cells. This brought some benefits but maybe you have some other ideas. Many thanks for your help!
As fibroblasts attach generally quickly back to the culture dish after trypsinisation, you can try a "platting". It is based ont the difference of speed of adhesion of fibroblast compared to muscle cells. To do a platting, you need to trypsinise everything, put the cells with medium on culture dishes for 15-20 mn in incubator. If you are lucky, fibroblasts will start to attach but not your muscle cells. So you take only S/N (containing mainly non fibroblasts cells) and put it back in a new plate. If you get not a pure enough population, you may want to do a second plating.
And you may also combine your way of "quick trypsinisation" with a plating.
Hope it works, I never try it with muscle cells.
As fibroblasts attach generally quickly back to the culture dish after trypsinisation, you can try a "platting". It is based ont the difference of speed of adhesion of fibroblast compared to muscle cells. To do a platting, you need to trypsinise everything, put the cells with medium on culture dishes for 15-20 mn in incubator. If you are lucky, fibroblasts will start to attach but not your muscle cells. So you take only S/N (containing mainly non fibroblasts cells) and put it back in a new plate. If you get not a pure enough population, you may want to do a second plating.
And you may also combine your way of "quick trypsinisation" with a plating.
Hope it works, I never try it with muscle cells.
What are you using to digest? We isolate SMC from the aortic root by cutting into small pieces, adding 600U/mL collagenase, incubating overnight, then spinning down and plating. This typically gives a small population of higher purity SMCs.
Try negative FACS sorting for CD90+ population (fibroblasts), and then western blot for vimentin (mesenhymal marker) and desmin (muscle cell marker) for input negative Scott and positive sort population. Once you optimize this system (might take month), the protocol will give you ability to work with purest cell population.
Two things . Digest with collagenase to release more muscle cells,( as emily suggested). do pre plating for 1 hour , fibroblast will attach rapidly , passage supenatant to fresh plate , repeat 1 to 2 times more.
It worked great for embryonic cardiac myocytes.
Miltenyi biotec sell anti-fibroblast microbeads, they are generally for the removal of ES cell feeder layers but they might be useful for you application.
Under culture conditions, primary smooth muscle cells would retain morphological features no more than 2-3 passages of . I had similar problem and I was advised so. Most importantly when differentiating them as the number of passages increases they wont differentiate properly.
Fibroblasts are first to attach and last to detach.Moreover they are susceptible to high glucose .Therefore you can also try DMEM with 4.5 Gms glucose to selective arrest growth of fibroblasts.In keratinocyte culture from skin,the fibroblasts are killed by use of cholera toxin (10 to the power -11M).
Manya and Emily thank you for your suggestions. For digestion I use collagenase/elastase/trypsin mix. Now I will try plating after 1h and see what the results are.
You have to use medium with D-valine instead L- valine cosidering fibroblasts do not grow in this kind of medium.
Hi Dear
Below article may be useful to troubleshoot your problem.
http://www.cellbiolint.org/cbi/024/0001/cbi0240001.htm
All the best
We have two protocols for primary smooth muscle culture. we used to isolate it from bladder. one is Collagenase digest : Collagenase Type IA (Sigma C9891): Na-Tyrode, pH 7.41, Collagenase Type IA, 0.3 mM CaCl2 , BSA (0.1%),Dissolve, filter-sterilize, warm to 37 C, DMEM w/o serum, DMEM + 10% FCS, 1% PS, Na-Tyrode no addds, sterile, pre-warmed
Protocol:
we collect tissue in DMEM w/o serum and Isolate SM bundles, free of other epithelium tissues and adventitia/connective tissue. then we Cut into small pieces , transfer into a 50 ml facon tube with the rest of Collagenase sln, incubate at 37oC for 15-20 min shaking occasionally. then we Triturate cells through fire-polished cold Pasteur pipette to disperse gently and spin down 2 min, 500 rpm, wash with Na-Tyrode, spin again. afterward we resuspend the cells in DMEM+FCS, ca 5 ml per large petri . we plate out cells and tissue clumps on TC petri dishes (Cell+, Starstedt), 5 ml per dish. After clumps attach (overnight), add 5 ml medium per dish, incubate 37oC 5% CO2 at least one week w/o moving. Examine the cells, feed survivors and split when confluent
* optional: After trituration pass the suspension through 100 um Nylon cell strainer (Falcon) to get rid of tissue pieces/cell clumps
the other methos is Papain+Collagenase digest
for this method you need:
1. Papain (Sigma P4762)
Na-Tyrode, pH 7.4 w/o additives 10 ml
1 mg/ml papain 10 mg
1 mg/ml 1,4 dithioerythritol (D8255)10 mg
1 mg/ml BSA 10 mg
Dissolve, filter-sterilize, warm to 37oC
2. Collagenase Type IA (Sigma C9891):
Na-Tyrode, pH 7.4 10 ml
1 mg/ml Collagenase Type IA 10 mg
0.3 mM CaCl2 2 ul 1.4 M sln
1 mg/ml BSA (0.1%) 10 mg
Dissolve, filter-sterilize, warm to 37oC
3. DMEM + 10% FCS + 1% PS
Protocol:
Collect tissue in serum-free DMEM, dissect SM bundles using sterile scissors
Mince into small pieces; incubate in Papain sln (50 ml Falcon) 20 min, 37oC
Transfer pieces into another Falcon with Collagenase solution, or spin down 500 rpm, 3 min and then change the solution. Incubate in Collagenase 10 min 37oC.
Triturate though a Pasteur pipette, then pass through 100 m Falcon cell strainer (Cupboard 139, Antoinette, 4th floor): let drop through, collect into 50 ml Falcon
Spin down 500 rpm, 3-5 min, resuspend in DMEM+10% FCS,PS, wash and spin again. Resuspend cells in DMEM+FCS, plate on Cell+ dishes and coverspips/multiwell plates.
Why do you think it's fibroblasts you are getting? are you using some kind of marker? As someone pointed out earlier, SMCs dediferentiate in culture and they look like fibroblasts. Good luck on that!
You can try using magnetic sorting of your smooth muscle cells by tagging them with alpha smooth muscle actin beads,but be careful,some fibroblasts also show alpha SMA.Try other SMC specifc marker based MACS.
Hi aleksandra
Based on attachement and detachment properties of both smooth muscle cells and fibroblasts, you can try cold PPS on the culture, muscle cells will detach but not with very high density. Finally, centrifuge the suspended cells and replate with M199.
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