Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

The overall goal of this procedure is to generate human macrophages in high quantity and quality with standard lab equipment. This is achieved by first centrifuging blood from Buffy coats on a FI call gradient to collect the P BMCs cs. Next on a per call gradient, the monocytes are separated from lymphocytes.

Then the monocytes are seeded in Teflon coated cell culture bags and differentiated. After six or seven days, macrophages are harvested and seeded for subsequent studies. The obtained cells should express the typical markers of mature macrophages and respond to various stimuli such as co cultivation with tumor cells or stimulation with LPS, The main advantage of this technique over existing methods such as counterflow, centrifugation, or magnetic activated cell sorting, is that human macrophages can be generated with standard lip equipment without the need for expensive reagents or instruments.

While this method is easy to perform, individuals new to this method might struggle with visualizing and separating the different cell populations in the density gradients. Therefore, visual demonstration of these steps will help to overcome these difficulties. This protocol is performed in duplicate to make balancing the rotors easier.

Begin by disinfecting two bags of the buffy coat fraction from layered blood. Then transfer the Buffy coats to two 50 milliliter tubes for each buffy coat. Prepare three 50 milliliter tubes with 15 milliliters of room temperature FI call solution at 1.077 grams per milliliter.

Now slowly and carefully, layer 30 to 35 milliliters of puffy coat on each tube of FI call. The layers should not mix centrifuge these tubes without breaking at 400 G for half an hour. At room temperature between the two phases, a white ring of peripheral blood mononuclear cells will form.

Remove these layers with a plastic pipette. Combine the layers of the three tubes from each donor into two 50 milliliter tubes per donor. Wash the collected cells by adding one millimolar P-B-S-E-D-T-A up to the 40 milliliter line, and then centrifuge them at 300 G for 10 minutes without breaking at room temperature.

Now aspirate and discard the supernatants. Then repeat the wash and P-B-S-C-D-T-A resuspend and pool the pellets from each donor and 20 milliliters of RPMI plus FCS without phenol red. There should now be one tube of cells per donor.

Now prepare the second density gradient mix 23.13 milliliters of room temperature per call solution with 1.87 milliliters of 10 XPBS in a 50 milliliter tube. Prepare one tube of the mix for every two samples. Next, transfer 23 milliliters of the mix to a new 50 milliliter tube.

Then add 27 milliliters of RPMI plus FCS with phenol red. The result is a 46%ISO osmotic per call solution. Now for each donor transfer, 25 milliliters of the 46%solution to a 50 milliliter tube and carefully layer on the cell mixture.

The two phases should be distinguishable by their colors without a break. Centrifuge the cells for half an hour at room temperature and at 550 G.A white ring of monocytes will pool between the two faces. Collect these cells into a 50 milliliter tube with a plastic pipette.

Wash the monocytes with one millimolar P-B-S-E-D-T-A, filled to the 50 milliliter mark. Then centrifuge them at 400 G without breaking for 10 minutes at room temperature, discard the supernatant and resuspend the pelleted monocytes in 20 milliliters of medium. Then proceed with the next section.

This protocol uses FEP Teflon coated culture bags as a culturing chamber. Begin by estimating the number of collected monocytes. Monocytes are large and often irregularly shaped.

Do not count the smaller lymphocytes. If 100 to 150 million cells of one donor are available, prepare 180 milliliters of supplemented RPMI. If there are fewer cells, prepare 30 milliliter volumes of medium for every 30 to 50 million cells.

In a 20 milliliter aliquot, carefully mix the cells into the 180 milliliters of prepared medium. Then load the culture bag with the cells with a 50 milliliter perfu syringe attached to the plug on the culture bag. Remove the syringe, push out the remaining air and cap the plug with a cone.

Now incubate the bags for six to seven days with 5%carbon dioxide. After six to seven days of incubation, transfer the culture bags to ice to harvest the cells completely immerse the bags in ice and allow them to cool for an hour to three hours to detach the cells. Next, gently pull the bags over an edge about 10 times.

Then thoroughly disinfect the outside of the bags. Replace the plug cone with a 50 milliliter syringe. Then draw out the cell suspension and transfer it to a 50 milliliter tube.

When all the tubes are collected, spin them down at 400 G for 10 minutes At room temperature, now aspirate the supernatant and pool all the cells into 10 milliliters of RPMI plus FCS. Then count the cells as before. Only count the macrophages as residual lymphocytes may be present.

Then use the cells for the application of interest to reuse the culture bags. Wash them twice with 70%ethanol. Then fill them with 50 milliliters of 70%ethanol and incubate them overnight at room temperature the next day, rinse the bags three times with sterile PBS.

Then wrap them in sterilization paper and autoclave them. A bag can easily be reused 10 times the density gradient. Centrifugations yield a white interface containing lymphocytes and monocytes.

Here we examine the first density gradient. May Grunwald. Staining of these cells shows both a high nucleus cytoplasm ratio, typical of lymphocytes and bean or ring shaped nuclei, typical of monocytes.

These stains show the results of the second gradient. Each buffy coat yields about 150 million monocytes, which can be differentiated into about 70 million macrophages. In 20 preparations, 47%of the monocytes became macrophages.

The purified macrophages can be further enriched by adherence to plastic surfaces, a feature that is not shared by the contaminating cells. Once plated, most macrophages show a fried egg morphology while others have a stretched spindle like phenotype. The cells are characterized by the expression of several markers.

Typical for mature macrophages. Expression of CD 11 B argues against a dendritic differentiation, as does the lack of CD 2 0 9 expression. After the differentiation, the cells remain functionally and metabolically active for five to seven days.

Calcium staining of viable cells shows their ability to take up red labeled extracellular vesicles shed from tumor cells. Additionally, the macrophages can be activated, for example, stimulation with lipopolysaccharide results in the expression of several pro-inflammatory genes. Following this procedure, the isolated macrophages can be subjected to a huge spectrum of functional assays in order to answer basic questions about macrophage biology in health and disease.

After watching this video, you should have a good understanding of how to generate high numbers of human macrophages. However, it's important to remember that you're working with human blood samples, which might be potentially infectious.