I am trying to differentiation human derived monocytes from blood into macrophages. I am culturing them in collagen coated plates (at 0.3 mg/mL) for 7 days. First I culture them for 6 days with 100 ng/mL M-CSF at 37 degrees +5%CO2 at a density of 1 million per mL per well. On day 6, I remove the media without disturbing the attached macrophages, spin my cells in the media and remove the supernatant. Then I resuspend them in 25 ng/mL of IFN-gamma (1O^6 IU/mL) and 1ug/mL of LPS. The media I used for my macrophages include RPMI +10% Human serum + 1% Penicilin/ streptomycin. By day 7, I remove the media, then wash with PBS and then add 500uL of warm 0.25% trypsin and try to remove the attached cells by pipetting vigorously as and incubate at 37 degrees for 5 minutes. Then I neutralize the trypsin using R10 and add everything to my media that was previously removed and spin everything. Usually my recovery barely gets as high as 20% and even by day 6 I already see a difference when comparing how the cells look on the microscope before and after changing the media. I do not know how to increase the recovery and have tried the same experiment at least 10 times where the recovery is usually around the same percentage of initial plated cells.
Trypsinization and vigorous pipetting can seriously damage the macrophages. There are some other culture and detachment protocols that have better survival: https://www.nature.com/articles/srep42495
You can also use non-tissue culture plastic plates so the macrophages attach more weakly, then use Ca++/Mg++ -free PBS incubation for 20-30 minutes at 37C, followed with your protease + 0.5mM EDTA for 20 min at 37C.
hi Juan Diego Sanchez ,
I agree with Ellen A G Chernoff ,
but you can try reducing the trypsinization timing and also can try using serum for trypsin neutralizaion.
hi Juan,
simply use accutase instead of trypsin.
Also, what do you mean by saying that recovery is below 20%? Cell detachment or cell viability?or percent of macrophages in cell suspension?
After trypsinising the cells, are there still many cells left on the plates? Cause then, it could be the problem with the detachment protocol. It could be the trypsin concentration or duration. Also, is there any particular reason you are using collagen coated plates?
On the other hand, if not many cells left on the plates upon trypsinising, you might want to look into the problem with centrifuge or even pipetting.
This is a different response: these cells are dying because they are living beyond their means without the normal growth factors required to keep them alive, even the best protocol will still get significant cell death - enough to activate and alter the functions of the surrounding cells. M-CSF is not enough for monocyte-derived macrophage survival indefinitely. These are not like bone-marrow precursors (who have a slightly longer lease of life). A question you may want to ask is: is this physiologically relevant? What I mean by this is that monocytes floating in the blood do not get recruited to an inflamed tissue then wait 7 days to differentiate into a bigger cell before they work. They are recruited from the bone marrow and work instantly, differentiation is a by-product of their function unless a bigger cell is needed (i.e. IL-4+M-CSF stimulation in parasite infections). With this in mind, experiments with monocytes in the short-term are preferable, you wont see cell death, will have more cells to work with and you can justify their use compared to macrophages. Morphology is not the primary outcome of your experiments right? Also think of atherosclerosis - monocytes enter the plaque site, get full of lipid then die, so death is a natural endpoint for these cells without the right growth factors. Gut macrophages are one of the few non-inflammatory macrophages that are known to be definitely monocyte-derived (but they still have a relatively short life span), the rest are a mix of foetal tissue-derived which are very slowly replaced by monocytes (but tissue specific growth factors govern this). If you are wanting to investigate gut macrophages then these likely require different (gut specific) growth factors. Otherwise for monocyte work/ inflammatory macrophage, stick to the effective cell: the monocyte! (recommended max = 3 days of culture, but experiments start day 0).