Our lab is attempting to study trans-infection of HIV-1 from primary Alveolar macrophages. The Alveolar macs are provided from HIV+ BAL samples. Issues are that the cultures are obviously contaminated with fungi and bacteria from the patients nose, mouth, trachea, etc.
Are there any means to prevent contaminants from exploding in the culture media as trans-infection assays take several days to complete??? These cultures are heavily contaminated with what seems to be fungi by light microscopy.
Also, any advice on general culturing of Alveolar macrophages is most welcome. Much obliged and thanks.
Hi,
I regular culture AMs from lung organ transplant patients. Try sieving the BAL through a 100micrometer cell strainer (will hopefully remove some hyphae/mucus). I plate in RPMI with Pen/Strep and after an hour of adherance wash with warmed RPMI x2 and replace with fresh RPMI + Pen/Strep.
We study fungal infection so I don't add an antifungal and you occasionally get contamination but this is unavoidable for us as I don't want to add Amphotericin to the media.. that could be an option if you want though
Hope that helps
Anand
Thank you very much. That is quite helpful.
One additional note...I frequently produce human MDMs from enriched monocytes with great success. The AMs I've seen from the BAL samples do not appear viable. The AMs retain a round morphology and do not seem to adhere and spread upon the culture surface. Any basic culturing conditions, ie coatings, factors and such that could promote survival and viability would be beneficial.
Again, thank you for sharing your expertise. It is much appreciated as I have never worked with AMs and the lab which the samples are coming from have no long-term culture experience. They keep the AMs in culture for only a single day. We need them viable for about a week or more if possible. Seeding densities for viable cultures would be a tremendous help as well.
Macrophages from BAL adhere to plastic relatively fast, specially if you cover your plates with collagen (see reference on how to grow primary bronchial epithelial cells for protocol for coating your plates) . Once they adhere (approximately 20-30 min incubation at 37C and 5% CO2, delicately rinse the wells and change to a media containing gentamicine at the appropriate concentration. If you would like to keep the cells for long term (more than 3 days), you may try adding 5% human AB serum to the media, hard to say if it would work. Each culture behaves a bit different, sometimes mycoplasma or other organisms (perhaps viruses) damage the cells in less than 3-4 days.
As Silvio mentioned, AMs generally adhere very fast. The amount they spread out is dependent on the activation state when they are isolated. When active they spread similar to MDMs but not always. I usually seed at 5x105 for standard tissue coated plates. You can use collagen/fibronectin coating but its not essential. We usually keep the cells for 3-4 days. Longer than this I'm not sure given the contamination as Silvio mentioned but you could try. Probably add 10% human serum and change media at day 3. Good luck!
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