The overall goal of this procedure is to separate the two main cell populations obtained from human muscle biopsy samples with a high efficiency and yield to allow the evaluation of specific phenotypic and transcription factor markers. This is achieved by first dissociating, a human muscle biopsy sample into a single cell suspension In the second step. After a seven day culture, the cells are separated by immuno magnetic beads sorting into CD 56 positive, which are myogenic cells and CD 56 negative fractions, which are the fibroblasts.
The cells are then stained and imaged for the desired phenotypic and protein markers of interest. Ultimately, immunofluorescence microscopy and quantitative image analysis can be used to measure the intensity of selected nuclear localized transcription factors within the sorted cell populations. The main advantages of this technique, which uses immuno magnetic cell sorting over existing methods like fluorescence activated cell sorting, are that it is gentle, allows excellent sorting of human primary muscle cells with the high yield and viability and can be performed quickly.
The implications of this technique extend towards increasing our understanding of the cellular basis of the fibro fatty muscle degeneration observed in aging, as well as in a number of muscle pathologies To isolate the muscle derived precursor cells as soon as possible after obtaining the biopsy. First, determine the weight of the tissue sample and then swell the muscle in basal medium several times to wash the sample clear of excess blood. Let the muscle sediment at room temperature for 30 to 60 seconds and then aspirate all but the final two to three milliliters of medium from the tube.
Next, invert the tube onto a sterile petri dish to allow the remaining fluid to carry the muscle onto the dish. Now clean the sample of any visible pieces of any obvious fat or connective tissue. Then rotate the dish to mobilize the remaining media, and then aspirate all of media and add three milliliters of one collagenase and disbe enzyme solution per 100 to 400 milligrams of tissue, and cut the muscle sample into very small one to two millimeter cubed pieces.
When the sample is sufficiently minced. Use a wide boar 25 milliliter pipette to transfer the tissue fragments and enzyme solution into a sterile 10 to 20 milliliter conical tube. Wash the plate with a further three milliliters of enzyme solution and then use a 10 milliliter pipette to transfer any remaining muscle fragments to the tube.
Place the tube at 37 degrees Celsius for 60 minutes. Reating the tissue suspension every 15 minutes with a 10 milliliter pipette. Then terminate the enzymatic dissociation with at least an equivalent amount of fresh rewarmed growth medium.
Next, pass the cell suspension through a 100 micrometer filter to remove any large pieces of debris and then centrifuge the cells resus. Suspend the pellet in seven to eight milliliters of growth medium, and then incubate the cells in an uncoated T 25 tissue culture flask. A 37 degrees Celsius with 5%carbon dioxide for seven days.
At the end of the week, trypsin eyes the cell monolayer for three minutes. When the cells have detached, add five to 10 milliliters of skeletal muscle growth, medium to prevent over digestion and app pellet the cells by centrifugation. Then after counting, reserve some of the cells for cell lineage marker characterization and wash the remaining cells in 15 milliliters of PBS centrifuge gain.
This time resuspend the pellet in 170 microliters of room temperature max sorting buffer and pipette gently to resus. Suspend the cells at 35 microliters of well mixed anti CD 56 antibody conjugated magnetic micro beads. Mix the cell solution a few times by pipette and incubate the mixture for 15 minutes of four degrees Celsius with gentle vegetation.
At the halfway point after the incubation, wash the cell and bead solution with 10 milliliters of sorting buffer centrifuge and reus. Suspend the pellet in one milliliter of fresh sorting buffer. Next, lubricate the pres separation filter and column with 500 microliters of sourcing buffer.
And then immediately mix and transfer the entire one milliliter of cell suspension through the pres separation filter and into the column, rinse the column three times with one milliliter of sourcing buffer power wash, collecting the non retain fibroblast fraction, passing through the column into a sterile 50 milliliter conical tube containing a small amount of growth medium. Then remove the column from the magnet and depress the plunger into the top of the column to collect the CD 56 positive cell fraction. In a separate 50 milliliter conical tube containing warm growth, medium growth medium is emitted from this step.
If double sorting is to be performed, then contemplate the positive and negative collected cell fractions. If double sourcing is required for extra purity, do not place media in the CD 56 positive collection tube on the first sort, but repeat the steps starting from the filter and column lubrication at the appropriate experimental endpoint. Fix the CD 56 positive cell cultures in their culture medium, using an equivalent volume of ice cold, 8%para formaldehyde.
The 10 minutes with gentle vegetation. Then aspirate the fixative and wash the cells twice with PBS to immunostain the cell surface antigens. Block the cells for at least one hour in 1%BSA in PBS, and then label the cells with the relevant primary and then secondary antibodies.
Optimize the detector gain laser power and exposure settings relevant to your microscope and stain. Before imaging, the cells take the images of the cells in the desired number of detection channels and in more than six different fields of view. For analysis, open the appropriate TIFF image files and drag the images into one another to overlay the appropriate corresponding channels.
Each channel will appear as a separate layer in the layers panel. Next in the analysis window, choose select data points and then custom and select the desired measurements. To analyze the nuclear fluorescence intensity, open the color range dialogue box and select sampled colors from the dropdown menu.
Then holding the shift key, select the tones specific to the labeled nuclei. Click save to store this color range selection mask for use with other images selected in the same session. Once the nuclei have been selected, write click and select fill.
Then choose black from the dropdown menu and confirm that the opacity is set to 100%Next, click select and inverse. Then right click and choose Fill again and choose a white fill from the dropdown menu. Perform a watershed algorithm to separate any partially overlapping nuclei on the now binary nuclear layer.
Go to select then color range, and then shadows to select the individual separated nuclei. Then transfer the selection to the layer containing a 16 bit gray scale image of the desired marker and click record measurements. Finally, export the selected measurements as text files to the appropriate analysis software for further processing oil.
Red staining of the purified cell populations in combination with immuno staining for adipogenic and myogenic lineage markers reveals that only the fibroblast fraction is capable of a epigenic differentiation with the massive accumulation of fat by the fibroblasts being visible to the naked eye and with further demonstration of their complete transformation by the strong expression of nuclear PPAR gamma by these cells by day 15 of treatment, these cells have released any remaining TE seven A connective tissue antigen onto their substrate. By contrast, myogenic cells maintain their normal phenotype, including their expression of desmin and myosin heavy chain without upregulation of nuclear PPAR gamma expression. In this figure, an example of the quantitative analysis of a field of Desmond positive myogenic cells and the field from which these data were obtained are shown illustrating the variation of myogenic expression in individual nuclei at this specific seeding density and time point.
In this graph, the utility of the method to directly compare transcription factor levels in different cell types on a cell by cell level is demonstrated. For example, these CD 56 negative muscle fibroblasts express a high level of the nuclear adipogenic transcription factor, PPAR gamma, whereas assorted CD 56 positive myogenic cells maintain only very low levels of nuclear receptor transcription factor after exposure to adipocyte inducing medium. This technique allows researchers in the field of muscle biology to explore the mechanisms of cell fate decisions in healthy or pathological human muscle samples.
After watching this video, you should have firstly, a good understanding of how to obtain high yields of purified and well characterized human muscle derived cells, and secondly, how to perform objective quantitative analysis of cellular constituents identified by immunofluorescent staining.
