The overall goal of this procedure is to deliver a novel cytoplasm replacement therapy to the gastrocnemius muscle of mice using plasma membrane vesicles, or PMVs derived from mouse bone marrow mesenchymal stem cells or BMSCs. This method can help answer questions with supplementation of cells with young cytoplasm from other BMSC via PMV fusion can ameliorate or even reverse the age associative phenotypes. The main advantage of this technique is that PMV encapsulation of BMSC cytosol and can be instantly induced by a mechanical extrusions.
When you are demonstrating this method it is critical as you're extruding and constructing steps are difficult to learn by testing to actually owning. To generate the PMVs, first culture mouse BMSCs in complete DMEM culture medium in one well of a six well plate in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. When the cells have reached 90%confluency, rinse the well with 0.5 milliliters of calcium free PBS and detach the cells with 0.5 milliliters of 0.25%trypsin-EDTA.
After three minutes at 37 degrees Celsius, tap the plate with the palm of the hand a few times to facilitate cell dissociation and stop the digestion with one milliliter of culture medium. Transfer the cells into a five milliliter conical tube for centrifugation and resuspend the pellet in four milliliters of fresh culture medium. Then divide the cells between two wells of a new six well plate and return the plate to the cell culture incubator for 24 hours.
The next day, harvest the cells from one well by trypsin digestion as just demonstrated. Resuspending the pellet in 0.5 milliliters of fresh culture medium after collection by centrifugation. Load the cells into a one milliliter insulin syringe and attach the syringe to a filter unit.
Quickly depress the plunger to squeeze the cells through the filter, and discard the extruded medium. Then load and dispense 0.5 milliliters of extra culture medium through the filter as just demonstrated, collecting the medium from the second extrusion. Seed 150 microliters of the collected medium into a 35 milliliter glass bottom dish and allow the vesicles to settle for about 10 minutes.
Then confirm the presence of PMVs under an inverted phase contrast microscope equipped with a CCD camera using a 20 times objective lens. To trace the mitochondrial enclosure and membrane potential in the PMVs, stain a 90%confluent BMSC culture with an appropriate mitochondrial dye or a mitochondrial membrane potential probe respectively in fresh culture medium for 30 minutes at 37 degrees Celsius. Then rinse the cells three times with 0.5 milliliters of PBS per wash and harvest the cells by trypsin digestion for PMV generation as just demonstrated for their visualization by confocal microscopy.
To trace the cytoplasmic localization of the PMV proteins, first dilute two micrograms of supercoiled plasmid DNA for an EGFP expression cassette and six microliters of cationic transfection reagent in individual 0.5 milliliter tubes containing 50 microliters of serum free DMEM. Vortex the tubes, followed by a brief centrifugation. Then add the transfection reagent drop wise into the tube containing the diluted DNA solution.
Vortex the DNA transfection mixture robustly for one minute, followed by a brief spin in a 10 minute incubation at room temperature. Next, feed a 30%confluent BMSC culture in a six well plate with two milliliters of fresh culture medium and add the DNA mixture drop wise to the cells. 48 hours after their transfection, harvest the BMSC by trypsin digestion and prepare PMVs as just demonstrated.
The PMVs can then be analyzed by confocal microscopy using a 100 times oil objective. Before injecting the PMVs, label a BMSC culture with a membrane staining fluorescent cell tracker dye in one milliliter of fresh culture medium at 37 degrees Celsius for 30 minutes and harvest the cells by trypsin digestion. Resuspend the labeled cells in 0.5 milliliters of fresh culture medium supplemented with one microgram of polyethylene amine and prepare PMVs as demonstrated.
Then concentrate the PMVs by centrifugation and resuspend the pellet in 100 microliters of fresh culture medium with gentle swirling. Next, load the cells into a one milliliter insulin syringe equipped with a 30 gauge needle and disinfect the outside of the gastrocnemius muscle of an anesthetized BALB/c mouse with 75%ethanol. Then slowly inject the entire volume of PMVs into the muscle and allow the animal to recover under a heat lamp.
After 12 hours, sprinkle 75%ethanol over the muscle, open the skin with scissors, and harvest the entire gastrocnemius muscle at both ends. Rinse the muscle with PBS and place it under a fluorescence microscope. Using the 20 times objective lens, remove the non-fluorescing muscle tissue pieces with a sharp razor blade and mince the remaining strongly red fluorescing pieces into about nine cubic millimeter cubes.
Embed each cube in an optimal cutting temperature medium. Then submerge the cubes in liquid nitrogen for five minutes. And store the tissue samples at minus 20 degrees Celsius until their analysis.
PMVs generated from BMSCs as demonstrated predominantly exhibit a size in the range of about three micrometers in diameter as examined under phase contrast microscopy using the 20 times objective. BMSC transfection with a fluorescent expression cassette for 48 hours illustrates the cytoplasmic encapsulation of the EGFP protein by the PMVs. Further, standing with a fluorescent mitochondrial dye reveals that a large fraction of the PMVs contain mitochondria.
The majority of which stain positive for a mitochondrial membrane potential dye, indicating the functionality of the mitochondria. Polyethylene amine treatment prior to their en vivo administration prevents PMV aggregation during their centrifugal concentration for cellular component removal. Membrane dye labeling of the BMSCs before PMV generation facilitates the generation of fluorescence trackable PMVs.
Indeed, visualization of frozen sections of the gastrocnemius muscle after intramuscular delivery of labeled PMVs indicates PMV delivery to the muscle cell cytoplasm probably via endocytosis. When attempting the PMV generation procedure it's important to remember to load the syringe with the appropriate number of cells to depress the plunger quickly when flushing the cells through the filter. After its development, this technique paved the way for researchers in the field of stem cell therapy to explore the treatment of age related diseases in mice.
After watching this video, you should have a good understanding of how to prepare PMV from BMSC for the delivery of cytoplasm into the gastrocnemius muscle of mice.
