Protocol for Mitochondria Transfer between Mesenchymal Stem Cells and Glioblastoma Stem Cells

As essential components of cellular metabolism and energy generation, mitochondria are known to be involved in a number of illnesses. Recent studies have revealed that mitochondria may move across cells and affect how cells operate. The technique for transferring mitochondria from mesenchymal stem cells (MSCs) to glioma stem cells (GSCs) is described in this procedure. Through the process of separating mitochondria from MSCs and introducing them to GSCs, it is possible to examine the impact of mitochondria on GSC metabolism, proliferation, and responsiveness to therapy.

Experimental Materials and Recommended Services

Name	Comments	Related Products and Services
Mesenchymal Stem Cell (MSC)	Human mesenchymal stem cells, or MSCs, are multipurpose stem cells that are employed in studies on regenerative medicine and tissue repair. They have the ability to develop into numerous cell types, such as fat, cartilage, and bone cells.	GFP Labelled Human Mesenchymal Stem Cell Genome Editing Gene Knockdown Stable Cell Lines Gene Knockout Stable Cell Lines Gene Overexpression Stable Cell Lines miRNA Overexpression Stable Cell Lines
Glioblastoma Stem Cells (CSC)	Tumor stem cells called glioblastoma stem cells (GSCs) are essential to the growth and recurrence of glioblastoma. Researching GSCs aids in the development of new cancer therapies as well as better understanding of the properties of tumor stem cells.

Procedure

Glioblastoma stem cells (GSCs) can benefit greatly from the effective transfer of mitochondria from mesenchymal stem cells (MSCs) in a variety of biological applications. However, carefully planned experimental protocols and approaches are needed to achieve effective mitochondrial translocation. Using fluorescence-activated cell sorting (FACS) and confocal imaging, this approach offers a comprehensive scheme covering the methods for identifying MSC mitochondria and GSCs, as well as the processes for separating and moving mitochondria from MSCs to GSCs.

Fig. 1 illustrates the workflow for transferring isolated MSC mitochondria to GSCs via MitoCeption, detailing the 7 steps involved in the process and subsequent GSC analyses. (doi: 10.3791/55245) Figure 1. Workflow for transferring isolated MSC mitochondria to GSCs via MitoCeption. Step numbers in the graphic relate to the protocol section and represent the seven steps involved in MitoCeption and subsequent GSC analysis. Steps 1 and 2 can be bypassed, beginning the process on day 2, if MSC and GSC labeling is not used for functional analysis. Images using phase-contrast technology depict Gb4 GSCs as neurospheres on day 1, in a unicellular culture prepared for the transfer of MSC mitochondria on day 2, and 24 hours after the transfer on day 3. 100 µm is the scale bar. Step 4 presents a typical 5-micrometer-scale photomicrograph of isolated MSC mitochondria that has been pre-labeled with MitoTracker Red CMXRos before to extraction from MSCs. (Nzigou Mombo, B., et al., 2017)

Day 1

1. Labeling of Mesenchymal Stem Cell (MSC) Mitochondria

Seed human MSCs at a density of 4 x 105 MSCs in a 100 mm culture dish using 10 ml αMEM/FBS 10% two days prior to mitochondria production.
Rinse MSCs with 4 ml PBS, then add 4 ml of αMEM/FBS 1% that has been prewarmed to 37°C.
For 30 minutes at 37°C, incubate cells with the necessary mitochondrial vital dye.
Eliminate dye solution, give cells two rinses using 4 ml of heated αMEM/FBS 1%, and then replace them with 4 ml of αMEM/FBS 10%. Cells are incubated at 37°C.
Adjust the culture medium (10 ml αMEM/FBS 10%) after two hours and thirty minutes.

2. Labeling of Glioblastoma Stem Cells (GSC)

Dissociate globular stem cells (Gb4 cell line; 10 x 106 cells) cultivated as neurospheres on cell culture flasks covered with poly-HEMA.
Seed 105 cells/well of GSCs on a 48-well plate, on GSC proliferation media.
For five minutes at 20°C, centrifuge the plate at 270 x g.
For thirty minutes at 37°C, incubate cells with the necessary cell vital dye.
To each well, add 500 µL of GSC basal media, then centrifuge again.
Add the medium again, then centrifuge.
Fill each well with 500 µL of GSC proliferation media, then incubate for 30 minutes at 37°C.

Day 2

3. Seeding of Glioblastoma Stem Cells

Gather 10 × 106 GSC neurospheres using centrifugation.
Centrifuge after HBSS cell washing.
After resuspending the GSC pellet in trypsin-EDTA and letting it sit at 37°C, add DNase I and CaCl2.
Pipette gently to dissociate the neurospheres, then add the HBSS and trypsin inhibitor.
Re-suspend GSCs in GSC basal media after centrifuging them.
In the GSC proliferation medium, count the GSCs and modify the cellular concentration to 106 GSCs/ml.
In a 96-well plate, seed 105 GSCs per well, then centrifuge.
Plate should be kept at 37°C until used again.

4. MSC Mitochondria Isolation

Use warm PBS to wash MSCs, then trypsinize and harvest cells.
Centrifuge and count MSCs.
Centrifuge the reconstituted MSC pellet in ice-cold αMEM/FBS 10%.
Centrifuge after adding Mitochondria Isolation Reagents A, B, and C in that order.
Repeat centrifugation after transferring the supernatant containing MSC mitochondria to a fresh tube.
Centrifuge the mitochondrial pellet after rinsing it with Reagent C.

5. Transfer of Isolated MSC Mitochondria to GSCs (MitoCeption)

To the MSC mitochondria pellet, add the GSC proliferation media that has been pre-cooled.
Add the diluted mitochondria preparation to the GSCs in a 96-well plate after diluting it in GSC proliferation media.
After centrifuging a plate containing MSC mitochondria and GSCs, incubate it.

Day 3

6. Analysis of Mitochondria Transfer by FACS and Confocal Imaging

GSC samples should be trypsinized, resuspended, and then transferred to FACS tubes in order to be ready for FACS analysis.
To limit mitochondrial vital dye leakage, seed GSC cells and then incubate them.
Place the plate in a centrifuge and add the mitochondria-only plate's supernatant to the medium.
Incubate and perform FACS analysis.
Prepare GSC samples for confocal imaging by resuspending in culture medium and seeding in glass bottom culture dishes.
Perform confocal imaging on GSCs with transferred MSC mitochondria 24 hours later.

This protocol provides a detailed explanation of several important steps related to the labeling of MSC mitochondria and GSCs, the separation of MSC mitochondria, and the transfer of MSC mitochondria to GSCs (MitoCeption). These processes include the extraction of MSC mitochondria, the production of GSCs, the optional tagging of MSC mitochondria, and the transfer of mitochondria to GSCs. The technique also describes how to use confocal imaging and FACS to analyze mitochondrial transfer efficiency.

* For research use only. Not intended for any clinical use.

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