The overall goal of this procedure is to transfer previously isolated mitochondria from donor cells such as mesenchymal stem cells to target cells such as glioblastoma stem cells. This procedure can help answer key questions in the metabolism field such as the metabolic and biological effects of the mitochondria that are transferred spontaneously between cells by processes such as tunneling nanotubes or microvesicles. The main advantage of this technique is that the effects of the mitochondria can be assessed independently of other factors present in the micro environment such as cytokines or metabolites and also of other compounds that can also be trafficking in the tunneling nanotubes.
For imaging purposes, MSC mitochondria and GSC can be labeled with vital dyes before the mitochondria transfer by MitoCeption. To begin, after pre-labeling GSCs with vital dyes according to the text protocol, collect GSC neurospheres in a 50 milliliter tube by centrifugation at 270 times g at 20 degrees Celsius for five minutes. Use five milliliters of HBSS to wash the cells and spin the tube again.
Aspirate the supernatant and with 100 microliters of 0.25%Trypsin-EDTA gently re-suspend the GSC pellet. Then incubate the cells at 37 degrees Celsius for three minutes. Next, add 10 microliters of 20 millimolar calcium chloride in two microliters at 10 milligram per milliliter of DNase I to the tube.
Dissociate the neurospheres by using a P200 pipette to gently pipette up and down 30 to 50 times avoiding the formation of bubbles. Add 10 microliters of 5%Trypsin inhibitor in 10 milliliters of HBSS to the cells and centrifuge the GSCs for seven minutes. Then discard the supernatant and 10 milliliters of GSC Basal Medium to the sample.
Now, using a Thoma counting chamber, count the cells before spinning again. Add the appropriate volume of GSC proliferation medium to reach a cellular concentrate of 10 to the sixth GSCs per milliliter. Then seed 10 to the fifth GSCs per well of a 96 well plate.
Centrifuge the plate so that the GSCs settle at the bottom of the wells. Check the cells under the microscope. Then incubate the plate at 37 degrees Celsius.
To carry out MSC mitochondria isolation, begin by adjusting the microcentrifuge temperature to four degrees Celsius. Prepare two 1.5 milliliter tubes labeled A and C containing reagents for the mitochondria extraction. Prepare two additional tubes labeled MSC and MiTo.
Keep the tubes on ice. Also prepare tubes for the mitochondria serial dilutions that will be set up with the mitochondria preparation. Use 10 milliliters of PBS pre-warmed to 37 degrees Celsius to wash the MSCs.
Then with two milliliters of EDTA-free Trypsin, wash the cells for 10 seconds and add an additional one milliliter of Trypsin before incubating the cells at 37 degrees Celsius for five to 10 minutes. The use of EDTA should be avoided as per protocol, in particular for MSC Trypsinization and for protease inhibitor used in the mitochondria preparation. Recover the MSCs by adding 10 milliliters of Alpha MEM 10%FBS and transfer the suspension to a 50 milliliter tube.
Then centrifuge the cells. Discard the supernatant and add 10 milliliters of Alpha MEM 10%FBS to the cell pellet. Then use a Malassez counting chamber to count the MSCs before spinning down four to five times 10 to the fifth cells.
Discard the supernatant, add one milliliter of ice cold Alpha MEM 10%FBS to the cell pellet and transfer the cells to the previously prepared MSC-labeled tube while continuing to keep it on ice. Then centrifuge the MSC tube at 900 times g at four degrees Celsius for five minutes and remove all residual medium from the tube. Next, add 200 microliters of mitochondria isolation reagent A with EDTA-free protease inhibitors to the MSCs and vortex at medium speed for five seconds.
Then leave the tubes on ice for exactly two minutes. Add 2.5 microliters of mitochondria isolation reagent B and vortex the cells at maximum speed for 10 seconds before placing the tubes back on ice. Repeat the vortex every 30 seconds for five minutes.
Now, add 200 microliters of mitochondria isolation reagent C with EDTA-free protease inhibitors to the tube and mix the contents by tilting the tube approximately 30 times. Centrifuge the tubes at 700 times g at four degrees Celsius for 10 minutes. Transfer the supernatant containing the MSC mitochondria to the previously prepared MiTo tube.
Centrifuge the sample at 3, 000 times g at four degrees Celsius for 15 minutes. After discarding the supernatant, use 200 microliters of reagent C to rinse the mitochondrial pellet. Then spin the suspension at 12, 000 times g at four degrees Celsius for five minutes to pellet the organelles.
To transfer isolated MSC mitochondria to GSCs, add 200 microliters of pre-cooled GSC proliferation medium to the mitochondrial pellet. Dilute the mitochondrial preparation in GSC proliferation medium to consistently allow the addition of 20 microliters of mitochondrial suspension to the GSCs. Next, slowly add the mitochondria close to the bottom of the well.
To control MSC mitochondria vital dye leakage, add the same amount of mitochondria preparation to the wells of a 96 well plate containing GSC proliferation medium only. Centrifuge the plates at 1, 500 times g at four degrees Celsius for 15 minutes. Then immediately place the cultures at 37 degrees Celsius.
The following day, to analyze the control wells for vital dye leakage, seed GSC cells as described earlier, centrifuge the plate, and then incubate the cultures at 37 degrees Celsius for one hour. Centrifuge the 96 well plate containing only mitochondria. Then aspirate the medium from the GSC 96 well plate and replace it with the mitochondria supernatant.
Incubate the GSCs with the mitochondria supernatant at 37 degrees Celsius for two hours. Finally, carry out analysis of mitochondria transfer, FACS analysis and confocal imaging according to the text protocol. GSCs are cancer stem cells grown as neurospheres to preserve their stem cell properties.
Just before mitochondrial transfer, they are seeded as single cells to allow a high mitochondria transfer efficiency and the following day as shown here, the cells are observed again forming neurospheres. In this figure, fluorescently-labeled MSC mitochondria can be seen localized inside Gb4 GSCs. The MSC mitochondria appear throughout the GSCs similar to the GSC mitochondria.
The transfer of MSC mitochondria could be observed for most GSCs in 3D reconstruction from GSC confocal images confirm the intercellular localization of the transferred MSC mitochondria. Pre-labeling GSCs with both blue cellular and red mitochondrial vital dyes allowed visualizing the localization of the transferred MSC mitochondria seen in green relative to the endogenous GSC mitochondria. As shown here, MitoCeption with increasing amounts of fluorescent MSC mitochondria led to increased FACS signals and with several orders of magnitude higher than the control signal.
The mitochondria transfer protocol presented here performed on single cell GSCs showed a dose-dependent response and relative mean fluorescence intensity. Finally, although MSC mitochondria transfer was also achieved on GSC neurospheres, the efficacy of the transfer to GSCs under these conditions was lower as compared to mitochondrial transferred to GSCs first seeded as single cells. Once mastered, this technique can be done in three hours if it's performed properly.
While attempting this procedure, it is important to remember to keep the mitochondria preparation on ice at all times. Following these procedures, other methods like FACS, confocal microscopy, QPCR on mitochondrial DNA can be performed in order to visualize and quantify the MSC mitochondrial transfer to the target cells. Functional studies can also be performed to determine the biological effects of the transferred mitochondria.
Keep in mind that these studies will generally be performed with lower amounts of mitochondria than those used to visualize the mitochondria transfer. This technique will allow to explore the outcome and biological effects of mitochondria that are spontaneously transferred between cells either in normal physiological or pathological conditions and will thus contribute to develop novel therapeutic treatments.
