The overall goal of this procedure is to improve the availability of monocytes and to minimize the variation in obtained cell densities during MCSF induced differentiation of human blood monocytes into macrophages. This method can help answer key questions concerning human macrophage biology. The main advantage of this tactic is that human monocyte derived macrophage cultures can be readily obtained with reduced dependency on donor availability.
In addition desired macrophage cell density and the homogeneous cultures can be consistently achieved. To begin this procedure, obtain the mononuclear cells by leukapheresis from human donors and then enrich the monocytes by continuous counterflow centrifugal elutriation of mononuclear cells. After that, count the cells using a hemocytometer.
Next, centrifuge the monocytes in a 15 milliliter polypropylene tube for five minutes at room temperature. Then, remove the supernatant and gently re-suspend the cells in FBS followed by Dimethyl Sulfoxide to achieve a final concentration of 90%FBS, 10%Dimethyl Sulfoxide, and 50 times 10 to the six cells per milliliter. Afterward add one milliliter of cell suspension to an individual cryofile.
Place the cryofiles in a cell freezing container, and transfer it to the minus 80 degrees Celsius freezer for 24 hours. After 24 hours, transfer the container to a liquid nitrogen cyrofile tank for long term storage. In this step, thaw a cyrofile of cells by briefly keeping it in a 37 degrees Celsius water bath.
When the cell suspension has thawed about 70%remove it from the water bath immediately and transfer it to room temperature. After the cell suspension has been completely thawed, transfer one milliliter of the cyrofile contents to 50 milliliters of RPMI 1640 medium with two millimolar L-glutamine, 50 nanograms per milliliter M-CSF, 25 nanograms per milliliter IL-10, and 10%FBS at 37 degrees Celsius. Then, transfer 25 milliliters of monocyte suspension into two 75 square-centimeter plastic cell culture flasks.
Incubate the cultures at 37 degrees Celsius for 48 hours. Remember do not change the medium until 48 hours;otherwise, the cells won't be well attached and will be rinsed away. After 48 hours, rinse the cultures three times with 10 milliliters of pre-warmed RPMI 1640 medium.
Gently remove the culture medium to avoid dislodging any loosely attached cells. Subsequently, add complete medium and culture the cells for one week. Change the medium every two days until the monocytes differentiate and proliferates efficiently to become confluent.
Now, rinse the differentiated macrophages in the flask three times with 10 milliliters of 37 degrees Celsius DPBS without calcium and magnesium. Subsequently, add 10 milliliters of 37 degrees Celsius 0.25%Trypson EDTA solution. Next, place the flask in a cell culture incubator for 10 to 15 minutes.
Following this, approximately 90%of the macrophages should have rounded and detached. Then, add 10 milliliters of RPMI 1640 medium containing 10%FBS to stop trypsinization. If necessary, use a cell lifter to completely remove the cells.
Sometimes in this procedure many macrophages remain attached. These macrophages can be removed following trypsinization by passing a cell lifter across the flask surface. Afterward, transfer the macrophage cell suspension from the flask into a 15 milliliter polypropylene tube.
Centrifuge it for five minutes at room temperature and discard the supernatant. Then gently simply re-suspend the macrophages in one milliliter of complete medium. Based on the cell count, add additional complete medium to achieve the desired seeding cell density.
Then, plate the macrophages in 1.5 milliliters of medium per well of a 12 well plate. One times 10 to the fifth cells in 1.5 milliliters of medium per well, usually produces a near confluent culture. Following seeding, incubate the macrophages overnight at 37 degrees Celsius to allow cell attachment before using them in experiments.
This figure shows the face contrast microscopic images of cryopreserved fresh monocytes during their MCSF induced differentiation into macrophages at low magnification. The images of monocytes were taken on day three and seven of culture in flask, and on day eight, one day after their harvesting and re-plating in the 12 well culture plate. Shown here is the comparison of fresh versus cryopreserved monocytes used to produced macrophages at high magnification.
The phase loosened vacuoles are macropinosomes. After watching this video you should have a good understanding of how to cryopreserve and later differentiate human monocytes with MCSF into macrophages. Subsequently, the macrophages can be cultured at desired cell densities for experiments.
