The overall goal of this study is to compare two different methods of human monocyte isolation for dendritic cell generation in vitro and to characterize the CT11c, CT14 cell surface expression of the resulting monocyte derived populations by imaging flow cytometry. These monocytes isolation protocols can contribute to the development of reliable methods for the purification of human dendritic cells for research and clinical applications. The main advantage of these protocols is that they facilitate the isolation of human monocytes and their differentiation into viable and functional monocyte derived cells.
In addition, this is the first study to characterize the specific population of monocyte derived dendritic cells by single cell imaging flow cytometry. This method can also be applied as an alternative for the isolation of other rare cells at an adequate yield for downstream research applications. Visual demonstration of these methods is critical as it can be challenging to handle the blood samples safely without the appropriate instruction and experience.
Begin by diluting the blood sample to a one to one ratio with PBS in a T75 flask. Then, add 15 milliliters of density gradient solution into a 50 milliliter centrifuge tube and carefully layer 25 to 30 milliliters of the diluted blood over the gradient. To achieve a proper separation of the peripheral blood mononuclear cells, load the blood onto the density gradient solution quickly, but carefully and without mixing the layers.
Separate the cells by centrifugation, followed by the transfer of the white blood cell interface layer into a new 50 milliliter conical tube. Wash the cells two times in PBS, re-suspending the final pellet in 10 milliliters of ammonium chloride potassium lysing buffer for 15 minutes at 4 degrees celsius. Then wash the cells two more times in PBS.
To isolate monocytes by the adherence method, culture five times 10 to the seven cells of the resulting PBMC pellet per 10 milliliters of complete medium in a new T75 flask for two hours at 37 degrees celsius and five percent carbon dioxide in a humidified incubator. At the end of the incubation, discard the non-adherent floating cells and gently wash the adherent cells two times with PBS. Then, feed the adherent cells with complete cell culture medium supplemented with human GMCSF and IL4 and return the culture to the incubator for five to seven days.
To isolate the monocytes by magnetic separation, after collecting the PBMC by density gradient separation transfer the white blood cell layer to a five milliliter polystyrene tube and re-suspend the cells in PBS at a five times 10 to seven cells per milliliter concentration. Next, add 50 microliters of a human monocyte enrichment cocktail per milliliter to the cells with mixing and incubate the cells with the cocktail at four degrees celsius for 10 minutes. Then add 50 microliters of magnetic particles per milliliter of cells with careful mixing and incubate the cells at 4 degrees celsius for five minutes.
At the end of the second incubation, add PBS to the cells to bring the total volume up to two point five milliliters and mix by pipetting two to three times. Then place the polystyrene tube in an appropriate magnetic device at room temperature. After two and a half minutes, with the tube still in the magnet, decant the purified monocyte fraction into a new 15 milliliter conical tube.
Then culture the purified monocytes and complete cell culture medium supplemented with GMCSF and IL4 for five to seven days as just demonstrated. After seven days of differentiation, dispense one times 10 to the six cell aliquats of the monocyte derived dendritic cells into the appropriate number of one point five milliliter tubes, and add 50 microliters of heat inactivated human serum to each sample to block any non-specific finding. After 10 minutes, centrifuge the cells and re-suspend the pellets in the appropriate fluorescence conjugated primary antibodies for 20 minutes at four degrees celsius protected from light.
Then wash the cells two times in one milliliter of PBS, resuspending the pellets in 100 microliters of PBS per one times ten to the six cells. Just before the analysis, add one microliter of DAPI to each tube. Then, read the samples on a single cell imaging flow cytometer according to the manufacturer's instructions.
On average, monocytes isolated by the adherence method account for approximately six point two percent of the total peripheral blood monocyte cell, or PBMC, population, while the monocytes isolated by magnetic separation account for up to 25 percent of the total PBMC. Monocytes isolated by either method are equally viable. MDDCs differentiated from monocytes purified by both methods were first gated based on DAPI negative or viable cells from total single cells, followed by gating of each cell population.
Both isolation methods yielded a low single CD14 positive population and both methods yielded a high total CD11c positive population. Further phenotypic analysis was performed on all the CD11c positive cells and even though magnetic isolation gives a higher percentage of double positive CD11c positive CD14 positive cells, this effect was non-significant when compared with the adherence method. These double positive CD11c positive, CD14 positive cells may be early stage differentiated MDDCs that have not yet dropped the CD14 monocytic marker.
When analyzing the single CD11c positive cells, the adherence method gives the highest yield of CD11c positive, CD14 negative cells. These single positive cells may be late stage differentiated MDDCs. Once mastered, the adherence and magnetic isolation techniques can be completed in three to four and one to two hours respectively if they are performed properly.
While attempting this procedure, it's important to remember to have fresh dendritic cell differentiation cytokines every 48 hours when the medium is replenished. Working with biohazard fluids such as blood, can be extremely harmful, and the use of appropriate personal protective equipment and disposal methods should always be taken when performing these procedures. This study paves the way for researchers in the field of immunology to explore the use of human monocytes and dendritic cells for therapeutic treatment.
After watching this video, you should have a good understanding of how to optimize human monocyte isolation for generating different populations of monocyte derived cells in vitro.
