Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

This co-culture method will help answer key questions surrounding the cell contact mechanisms behind mesenchymal stem cell regulation of macrophage phagocytosis, and therefore illuminate the role of MSC in regulating innate immunity. This technique can be applied in high-throughput analyses. The bioparticles conjugated to pH-sensitive dyes fluoresce only in acidic phagolysosome environments.

Hence, washing and quenching are necessary. This method can also be applied to a variety of co-culture models that include neutrophils and other phagocytes. Demonstrating the procedure will be Ethan Chetkof, an undergraduate currently in my lab.

To begin with, observe the confluency of cultured mesenchymal stem cells. When 70-80%confluency is achieved, aspirate the growth medium from the cultured cells in a 100 millimeter dish and add five milliliters of PBS. After aspirating the PBS, add two milliliters of 0.05%trypsin EDTA solution and incubate the cells for three minutes.

After three minutes, check the cell detachment using an inverted microscope. And if the cells are not detached, incubate for one to two minutes again. Once all the cells are detached, add five milliliters of fresh growth medium to the plate.

After rinsing the plate using the trypsin medium mixture, use a sterile serological pipette to collect cells into a clean, sterile 50 milliliter conical tube. Next, pellet down the cells by centrifugation. After aspirating the supernatant, resuspend the cell pellet in 10 milliliters of fresh growth medium by gently pipetting up and down.

For cell counting, add 10 microliters of cell suspension into a 1.5 milliliter microcentrifuge tube containing 30 microliters of 0.4%Trypan Blue solution, then add 10 microliters of the mixture under the coverslip of a hemocytometer counting chamber. Under the inverted or Brightfield microscope, count the unstained viable cells using a 10X objective lens and four one-square millimeter chambers and calculate the cell number as described in the text manuscript. Use the equation C1V1 equals C2V2 to determine the desired volume of the fresh medium and cell suspension required to prepare the cell suspension at a final concentration of one times 10 to the fifth cells per milliliter.

Use a multi-channel micropipetter to add 100 microliters of the cell suspension in the wells of a 96-well plate according to the plate design. Use a micropipette to add 530 microliters of the cell suspension to each one of the chamber slide according to the plate design and incubate overnight. Prepare 10 milliliters of activation medium containing interferon gamma at a concentration of 250 nanograms per milliliter for activating the macrophage cells in one 100-millimeter dish.

Aspirate the growth medium from the macrophage cell cultures and rinse the cells with five milliliters of the activation medium devoid of interferon gamma. After aspirating the rinsing medium in the experimental dish, add 10 milliliters of activation medium supplemented with interferon gamma. And in the control dish, add 10 milliliters of activation medium without interferon gamma, then incubate the cells for 16 to 24 hours.

At the end of the incubation, for preparing the co-cultures, remove the activation medium from the macrophage cells and add five milliliters of fresh growth medium. Use a cell lifter to gently scrape the cells and collect the cells into a 50 milliliter conical tube. Next, count the cells using Trypan Blue exclusion and hemocytometry, followed by preparation of control and treated macrophage cell suspensions at the concentration of two times 10 to the fifth cells per milliliter as demonstrated previously for the mesenchymal stem cells.

Gently aspirate the medium from the experimental wells containing mesenchymal stem cells. Use a multi-channel micropipette to add 100 microliters of the treated and the control macrophage cell suspensions in the appropriate experimental wells according to the plate design and incubate overnight as demonstrated. Gently aspirate the medium from the four-walled chamber slide containing mesenchymal stem cells.

Use a micropipette to add 530 microliters of the macrophage cell suspension to the appropriate wells according to the design and incubate overnight. Add one milliliter of live cell imaging medium to the vial containing one milligram of Zymosan particles and collect the medium particle mixture into a glass culture tube. After rinsing the vial with an additional one milliliter of imaging solution, transfer the rinsed imaging medium in the glass tube, then add three milliliters of additional imaging solution to achieve 0.2 milligrams per milliliter Zymosan particle suspension.

Vortex the Zymosan suspension using quick pulses for 30 to 60 seconds, then use a probe sonicator to sonicate the suspension with 60 quick pulses. After aspirating the medium, rinse the experimental wells and reagent blank wells with 100 microliters of live cell imaging solution. Next, aspirate the live cell imaging solution from the wells and add 100 microliters of the Zymosan suspension.

Open the plate tray of the fluorescent reader using the touch pad interface. Set the plate without the lid in the tray with the A1 well in the upper left corner. Close the tray using the touch pad and click on the green read button in the top menu.

To conduct the phagocytosis assay, after rinsing the first experimental well with 750 microliters of the imaging medium, add 400 microliters of the Zymosan suspension leaving the remaining wells in the growth medium. Next, place the slide on the incubated stage of the imaging system. Use the imaging software.

Select the Brightfield option under the locate tab, and then click on the eyepiece icon. With the 10X objective in place, use the eyepiece and focusing knob to focus on the cells. Select the acquisition tab.

Open the experiment dropdown menu and select a set of wavelengths that includes EGFP filter set to accommodate 509 nanometers excitation and 533 nanometers emission wavelengths of the pH-sensitive dye. When about two minutes are left on the timer, click on the 20X icon in the objective menu to change the objective to 20X, then click on the channels menu, select EGFP filters, and in the exposure menu, set the exposure time to 400 milliseconds to detect the pH-sensitive green fluorophore, and then click on live. After adjusting the focus, click on stop to turn off the light and check the box next to the top time-lapse experimental setting.

In the focusing strategy menu, select software auto-focus and from the dropdown menu, select smart and core settings to reduce the time of exposure to light while the auto-focus is running. In the time-lapse menu, set the desired number of acquisitions to 30 to 60, the interval time to one minute, and then click on the start experiment button to begin the acquisition. The effect of interferon gamma on the co-culture was studied.

The representative results demonstrate that the co-culture of macrophages with mesenchymal stem cells enhances the phagocytic activity of the macrophage. Interferon gamma treatment reduces the activity of the macrophage. However, in the presence of mesenchymal stem cells, the macrophage phagocytic activity was partially rescued.

The optimal cell densities are critical in these studies. And when the macrophage cells were plated at too low of a density, changes in fluorescence intensity were not detected. When the macrophage cells were plated at a high density, the fluorescence intensity was elevated rapidly in all groups and differences were not discerned.

One of the most important things during this procedure is to ensure that the cell densities are optimized and kept consistent between experiments.