These individual human audio model uses relevant immune cells as a powerful tool to predict acute immune response of substances including nanomaterials and knowledge drugs. These medical combines human epithelial cells and human primary immune cells to assess those specific reactions. The use of fresh and frozen monocytes provides flexibility for the experiment plan.
Numerous studies have shown that this model can provide an insight into the interstellar communication between a behavior sense and immune sense. As this demonstration includes all the important details for studing the model a personal trait in cell culture, visual instructions to replicate experiments. Several steps in the bladder's protocol are complex and require special handling.
Therefore they're demonstrated with all the required details that they can be easily followed. For the isolation of peripheral blood monocytes from human Buffy coats, split the harvest Buffy coat bag contents, evenly between 250 milliliter conical tubes. Carefully bring the total volume in each tube 250 milliliters with PBS and mix the tubes with three gentle inversions.
Then split the Buffy coat PBS solution equally between four new 50 milliliter tubes, then add 10 microliters of CD 14 positive magnetic beads the seventh total cells to the tube and mix well by pipetting. Upon filling, detach the pipette from the pipetter and immediately plugged the upper opening of the pipette with a thumb then placed the filled pipette at the bottom of one tube and unplug the pipette opening to allow density gradient medium to slowly flow beneath the buffy coat PBS mixture, until one milliliter of the density gradient medium is left inside the pipette. When density gradient medium has been added to each tube in the same manner separate the cells by density gradient centrifugation and use a serological pipette for collecting the white two to three millimeter thick peripheral blood mononuclear cell layer from between the plasma and the density gradient medium layers.
The best way to connect the layer is to remove the plasma first and then you suppress our logical pipettes to collect the cells. Pull the peripheral blood mononuclear cells from each set of two tubes into one new 50 milliliter tube and wash the cells two times with a total volume of 50 milliliters of fresh PBS per wash. Then discard supernatant and resuspend the pipette in each in five milliliters of fresh PBS and pool the cell suspensions into a single tube for counting.
After counting, centrifuge the cells again and resuspend the pelette to 80 milliliters of magnetic separation buffer per attend to the seventh cells. Then add 10 microliters of CD 14 positive magnetic beads per attend the seventh total cells to the tube and mix well by pipetting. After a 15 minute incubation at four degrees Celsius rinse a magnetic column with three milliliters of magnetic separation buffer and add the cells to the column.
Wash the column three times with three milliliter volumes of buffer per wash and transfer the column into a 15 milliliter tube containing one milliliter of fresh magnetic separation buffer. Then use the plunger and five milliliters of buffer to flush the CD 14 positive cells into the tube. Do induce differentiation of the magnetic bead isolated CD 14 positive cells.
First, resuspend the cells at a one times attend to the six cells per milliliter concentration in cell culture medium. Or MDDC differentiation, add 10 nanograms per milliliter of and of granula site macrophage colony stimulating factor to the tube and add three milliliters of cells to each well of a six well cell culture plate, or MDM differentiation add 10 nanograms per milliliter of macrophage colony stimulating factor to the cells and plate the cells as just demonstrated. To set up a human alveolar epithelial cell tissue triple cell co-culture model, first transfer 1.5 milliliters of pre-warm cell culture medium into the appropriate number of Wells of a 12 well plate and place a cell culture insert into each well.
Next add 500 microliters of epithelial cell suspension at a density of five times 10 to the fifth cells per milliliter into the apical side of each insert and cover the plate for a four day incubation in the cell culture incubator. Or MDDC seeding replaces supernatant from the Wells of six well plates used for MDDC differentiation, with one milliliter of fresh warm cell culture medium and use a cell scraper to detach the adherence cells from each well. Wash each well three times with the supernate and in each well and combine all of the cell solutions into a single centrifuge tube.
The harvested MDDC by centrifugation and sterile tweezers to place the inserts from the 12 well plates upside down in a sterile Petri dish. Scrape any epithelial cells from the Basal side of the insert and resuspend the MDDCs in fresh cell culture medium do a concentration of 4.2 times 10 of this cells per milliliter. Then add 150 microliters of cells onto each insert so that the entire basal surface of the insert is equally covered with the liquid without bubbles.
For MDDM seeding after harvesting the differentiated MDDMs as demonstrated for the MDDCs, dilute the cells to a 2.5 times 10 to the four cells per milliliter of fresh cell culture medium concentration and add 500 microliters of cells down the wall of each epithelial cell and MDDC containing cell culture insert, then place the lid onto the plate and place the plate in a cell culture incubator for 24 hours. The next day aspirate the supernatant from both the apical and basal areas of each cell culture insert and Wells. Then you sterilize tweezers to lift the inserts from the Wells and add 600 microliters of fresh prewarm to cell culture medium to each well.
After six days of differentiation, the MDMs appear round in shape while MDDCs form clusters made up of cells with a more elongated shape and observable protrusions. After three days of growth on membrane inserts, the epithelial cells form a dense A significant increase in lactate dehydrogenase release is observed in the cell culture medium of the basal compartment upon exposure to a positive control for membrane rupture, proving the responsiveness of the model to a cytotoxic substance. No increase in lactate dehydrogenase release as measured upon apical stimulation with TNF alpha or LPS.
Statistically significant increases in the release of IL6 and IL8 are observed in both LPS and TNF alpha treated samples compared to control untreated cells. No differences in cell morphology are observed between the co-culture models using MDDMs and MDDCs from fresh peripheral blood monocytes compared to those using thawed cells in LPS and TNF alpha exposed co-culture, a disrupted epithelial layer was observed. It is possible to use other types of human cells such as airway or bronchiolar cells, other end points can also be analyzed.
Mainly researchers have been inspired by this technique using a similar to set up but with different cell types.
