Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro

Summary

Here, we present a protocol including mitochondrial tracing, direct co-culture procedures of mesenchymal stem cells (MSCs) and retinal pigment epithelial cells (ARPE19), as well as the methods for observing and statistically analyzing tunneling nanotubes (TNT) formation and mitochondrial transfer to characterize mitochondrial exchange via TNTs between MSCs and ARPE19 cells.

Abstract

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods.

We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

Introduction

Mitochondria serve as the primary energy source for most cell types, with mitochondrial dysfunction particularly impacting high-energy-demanding tissues like the retina¹. Metabolic alterations in the retina can trigger a bioenergetic crisis, ultimately resulting in the death of photoreceptors and/or RPE cells². Mesenchymal stem cell (MSC)-based therapies have demonstrated efficacy in treating ocular degeneration, and one of the precise mechanisms underlying the beneficial effects of MSCs on retinal tissues may be attributed to functional mitochondrial transfer^3,4,5,6. In 2004, Rustom et al. first reported the phenomenon of mitochondrial transfer through a novel cell-to-cell interaction facilitated by tunneling nanotubes (TNTs)⁷.

In 2D culture, tunneling nanotubes (TNTs) are identified by their thin (20-700 nm) membrane protrusions ranging from tens to hundreds of nanometers in length, which are suspended over the substrate and can directly establish connections between two or more homotypic and heterotypic cells. These structures are notably enriched in F-actin and facilitate the transport of cargo, such as mitochondria, between cells. Additionally, TNTs possess openings at both ends, enabling the continuity of cytoplasmic content between interconnected cells⁸.

It is difficult to detect TNT-mediated mitochondrial transfer in vivo due to the dense cellular arrangement and challenges in tracking mitochondria. In vitro experimentation, utilizing cell co-culture and mitochondrial tracing techniques, allows for the observation of TNT formation and mitochondrial transfer^8,9. We also observed the phenomenon of TNT-mediated mitochondrial transfer by co-culturing MSCs and retinal pigment epithelial cells in vitro¹⁰.

Many previous studies have only observed unidirectional mitochondrial transfer from MSCs to other cells^3,4,5,6. Previously, we also tried to analyze the bidirectional mitochondrial transfer using two kinds of cells labeled with mito-tracker green and mito-tracker red, respectively, but the crosstalk of the dyes interfered with the experimental results. To study mitochondrial bidirectional transfer more precisely, here, we constructed two cell lines with different mitochondrial fluorescence using the lentiviral transfection technique, and subsequently, observed and analyzed the phenomena of TNT formation and mitochondrial bidirectional transfer by direct co-culture in vitro.

In brief, a step-by-step and actionable protocol is described here as to how to trace mitochondria, co-culture MSCs with ARPE19 cells, and analyze TNT formation and mitochondrial transfer. The results of this experiment demonstrated TNT-mediated bidirectional mitochondrial transfer, which not only proved that mitochondrial transport is a common physiological phenomenon but also showed the potential therapeutic ability of MSCs on retinal cells.

Protocol

1. Generation of MSC-mito-GFP and ARPE19-mito-RFP cell lines

1. Cell culture
   NOTE: Only MSCs are used here as an example.
   1. Culture human MSCs in a 6-well plate in hMSC medium with 1% penicillin and streptomycin (see Table of Materials) until the cell density reaches 80%-90%. Depending on the density of cell growth, change the medium once every 2-3 days.
      1. Remove the original medium and wash the cells once with phosphate-buffered saline (DPBS) (see Table of Materials). Add 1 mL of the trypsin-EDTA-DPBS mixture (0.05% Trypsin-EDTA: 0.25% Trypsin-EDTA diluted with DPBS at a ratio of 1:5; see Table of Materials), and incubate for 3-4 min (depending on the cells) in a 37 °C incubator.
         NOTE: Observe cell digestion under a microscope and stop digestion if most of the cells are rounded and detached.
      2. Gently tap the 6-well plate to detach the cells adhered to the surface, and then add 1 mL of fresh complete medium to terminate the digestion.
      3. Gently resuspend all the cells with a pipette gun and subsequently transfer all collected cells into a 15 mL centrifuge tube.
      4. Centrifuge the cells at 200 × g for 5 min.
      5. Aspirate the supernatant and resuspend the cells by adding 1 mL of fresh medium. Take 10 µL of cell suspension for cell counting.
      6. Eighteen to 24 h before lentiviral transfection, seed MSC cells at a density of 1 × 10⁵ cells per well in 6-well plates, ensuring that the cell density at the time of transfection is approximately 50%.
2. Transfection of lentivirus
   NOTE: All operations related to lentiviral transfection need to be performed in a biosafety cabinet.
   1. Replace the initial medium with 2 mL of fresh medium the next day, and add 25 µL of lentivirus suspension (5.00E+08 TU/mL) with adjuvants, followed by incubation at 37 °C.
      NOTE: Lentivirus packaging was performed using the Mito-GFP (pCT-Mito-copGFP) plasmid (see Table of Materials and Supplemental Figure S1) by a company.
   2. After 6 h of incubation, replace the virus-containing medium with 2 mL of fresh medium. Continue to incubate and change the medium once a day.
      NOTE: Significant mitochondrial fluorescence expression is observed in MSCs 48 h post transfection, with further enhancement evident at the 72 h time point.
   3. Screen successfully transfected cells by supplementing the culture medium with puromycin (1 µg/mL) 2 days post transfection. Concurrently, add an equivalent concentration of puromycin to mesenchymal stem cells lacking viral transfection as a control.
      NOTE: The mito-GFP viral vector is engineered to include the puromycin resistance gene.
   4. Change the medium daily with the introduction of puromycin (1 µg/mL). Stop puromycin addition upon near-complete cell death in the control group.
      NOTE: The medium does not contain this concentration of puromycin. The addition of puromycin is required only when the medium is added to a 6-well plate.
   5. Continue to culture without puromycin until cells are passaged and frozen. Name this type of cell as MSC-mito-GFP.
      NOTE: ARPE19 cells were derived from ATCC Cell Bank and cultured in DMEM/F12 containing 10% FBS and 1% penicillin and streptomycin (see Table of Materials), and the methods of their digestion and passage are the same as that of MSCs. Plasmid Mito-RFP was modified from Mito-GFP by a company. Finally, we named the successfully transfected cells as ARPE19-mito-RFP.

2. Direct co-culture of MSC and ARPE19 cells

NOTE: In this co-culture system, MSC-mito-GFP cells will serve as the donor cells while ARPE19-mito-RFP cells will function as the recipient cells. To distinguish between donor and recipient cells, we traced recipient cells.

1. Label ARPE19-mito-RFP cells with CellTrace Violet (see Table of Materials) in the cytoplasmic membrane.
   NOTE: The excitation and emission of dye CellTrace violet are 405 nm and 450 nm, respectively.
   1. Prepare a CellTrace Violet (see Table of Materials) stock solution by adding 20 µL of dimethyl sulfoxide (DMSO) to a vial of CellTrace Violet Reagent and mixing well immediately prior to use.
   2. Grow the ARPE19-mito-RFP cells to the desired density of 80%-90%.
   3. Dilute the CellTrace Violet stock solution in prewarmed (37 °C) DPBS to the desired working concentration (1:1,000). This is the loading solution.
      NOTE: The dilution of CellTrace Violet must be performed with DPBS and cannot be replaced with culture medium as this will lead to very poor staining results.
   4. Remove the culture medium and replace it with 1 mL of loading solution. Incubate the cells for 10 min at 37 °C.
      NOTE: If conditions permit, the staining can be observed under a fluorescence microscope after 5 min. Staining can be terminated as soon as the desired results are achieved, as some cells may not be able to tolerate prolonged exposure to DPBS.
   5. Remove the loading solution, wash the cells 2x with DPBS, and replace with 2 mL of fresh complete medium. Incubate the cells for at least 10 min after staining to allow the CellTrace Violet to undergo acetate hydrolysis.
      NOTE: This step is necessary for the cells to return to their optimal state and to avoid interfering with subsequent experiments.
2. Prepare a 24-well plate by adding 50 µL of DPBS into each well. Subsequently, insert the cover glass (with a specified diameter of 15 mm) (see Table of Materials) into the plate and allow it to settle for 1 min. Remove the DPBS from the wells, utilizing negative pressure to ensure the cover slide is close to the bottom of the dish.
3. Trypsinize donor MSCs and recipient ARPE19 cells as described in step 1.1.1.
4. Cell counting
   1. Place 10 µL of cell suspension on a counting plate for enumeration.
      NOTE: If the density of cells is too high, they can be diluted 10-fold with medium and then proceed as above.
   2. Seed 10,000 MSCs and 10,000 ARPE19 cells in 500 µL of medium per well of a 24-well plate. Mix the cells well before adding them to the 24-well plate. See the calculations shown below given an MSC count of A/mL and an ARPE19 cell count of B/mL.
      NOTE: For 4 wells, 40,000 MSCs and 40,000 ARPE19 cells are required in 2 mL of medium.
      Volume of MSC suspension (C) containing 40,000 MSCs = 40,000/A µL
      Volume of ARPE19 suspension (D) containing 40,000 MSCs = 40,000/B µL
      Make up the volume to 2 mL with 2 - (C + D) mL of medium.
5. Observe the cells under a microscope, shake the plate until the cells are evenly distributed, and then incubate them in the culture for 24 h.

3. Indirect co-culture of MSC and ARPE19 cells in a transwell system

1. Perform cell culture, labeling, digestion, and counting as described in steps 2.1 to 2.4.1.
2. Seed 20,000 ARPE19 cells in 1 mL of medium per well of a 24-well plate.
   NOTE: In this experiment, we used ARPE19 cells without labeled mitochondria.
3. Place the insert into the well where the cell has been seeded.
4. Seed 10,000 MSCs in 100 µL of medium in the upper cell chamber per well.
5. Repeat step 2.5.

4. Cytoskeleton staining

NOTE: Protect from light throughout the experiment.

1. Remove the medium from the 24-well plates. Wash all cells once with 500 µL/well of 4% polyformaldehyde (PFA), add 500 µL of fresh 4% PFA, and fix the samples for 20 min.
   NOTE: The insets of the transwell system are discarded.
   NOTE: Because nanotubes are very fragile, we washed the cells directly with 4% PFA instead of DPBS to maximize the retention of intact nanotubes.
2. Remove the fixative and wash the samples for 3 x 10 min with DPBS.
3. Add 500 µL/well of blocking solution composed of 0.5% Triton X-100 (see Table of Materials) and 4% bovine serum albumin (BSA) (see Table of Materials) and incubate for 1 h at room temperature.
4. Remove the blocking solution and wash the samples for 3 x 10 min with DPBS.
5. Preparation of phalloidin staining solution (see Table of Materials)
   NOTE: The excitation and emission of phalloidin are 650 nm and 668 nm, respectively.
   1. Prepare stock solutions by dissolving the vial contents in 150 µL of anhydrous DMSO to yield a 400x stock solution.
   2. Prepare staining solution by diluting the 400x storage solution to 1x with DPBS. This is the phalloidin staining solution.
6. Add 200 µL of phalloidin staining solution to each well and incubate for 1 h at room temperature.
7. Recover the staining solution and wash the sample 3 x 10 min with DPBS.
   NOTE: When washing the cells with DPBS, it will be better to leave the plate on a shaker.
8. Pick out the cover glass with a syringe needle and allow it to air dry.
   NOTE: The optimal state is one in which no visible liquid can be seen on the cover glass but it is also not dry.
9. Apply a small amount of mounting medium (see Table of Materials) onto the slide and carefully flip the cover glass on the slide to ensure that the medium evenly coats the entire surface. Seal the edges with nail polish.
   NOTE: Wait for a few minutes to allow the nail polish to dry out.
10. Capture a confocal image promptly or transfer it to a slice box and store it at 4 °C for a brief duration.
    NOTE: Ideal storage time is 2 weeks. If this time is exceeded, the imaging effect may deteriorate.

5. Confocal imaging

NOTE: Confocal imaging is performed according to the operation manual and may vary between microscopes. Here we give only some of the key steps.

1. Start the confocal microscope and open four laser channels (405, 488, 561, 640) according to the operation manual.
2. Access the software on the computer and perform preliminary parameterizations of the software.
   1. Add the fluorescent channels CF-405, CF-488, CF-561 and CF-640 within the Process Management panel.
   2. Adjust the exposure time of each fluorescence channel to 200 ms and set the gain intensity to 2 in the Camera Control panel.
   3. Adjust the laser/LED combiner to a value of 80.
3. Manipulate the external control panel of the microscope to adjust the objective lens to a magnification of 20x and ensure that the lens is positioned at its lowest setting.
4. Position the slide in an inverted orientation on the carrier table.
5. Activate the 405 nm laser and manipulate the focus using the coarse and fine focusing spirals. Observe the sample under the eyepiece until blue fluorescent cells are visible.
6. Select Live Imaging in the software interface on the computer, switch to the CF-405 channel, and readjust the fine focus helix until a distinct image of the cells exhibiting blue fluorescence is visible on the computer screen.
7. Adjust the objective lens to 40x magnification and transition to the CF-640 channel, then carefully adjust the focus until a distinct cellular microfilament structure is visible.
8. Maintaining a constant focal length, transition to the CF-405, CF-488, and CF-561 channels sequentially, and modify the parameters of optimal exposure time, gain intensity, and laser/LED combiner for imaging within each channel, as necessary.
9. Activate the Z Image Stack function and define the beginning and ending points of the Z-axis by manipulating the focal length to capture an image with depth using the Z-axis.
10. Click the Start button in the Process Management panel to take a photo.
11. Select 10 fields of view randomly for each slice.
    NOTE: There should be no overlap between any two fields of view.
12. Activate the time-lapse function, specifying a total imaging duration of 24 h and a photo interval of 5 min following identification of the desired field of view.
    NOTE: To capture a time-lapse sequence, it is necessary to inoculate cells in a glass-bottomed dish and position them within a ventilated box containing 5% CO₂ gas.
13. Save and export all images.
    NOTE: To enhance the scrutiny of TNT and mitochondrial transfer, super-resolution imaging was employed. Super-resolution imaging was performed using commercialized HIS-SIM, termed HIS-SIM (High Intelligent and Sensitive SIM) provided by a company. Images were acquired using a 100x/1.5 NA oil immersion objective.

6. Data analysis

1. For the statistical analysis of each image, quantify the total number of cells, the number of nanotubes, the number of Violet-positive ARPE19-mito-RFE cells, the number of Violet-positive cells exhibiting green fluorescence (representing the number of ARPE19 cells with mitochondrial transfer from MSCs), and the number of Violet-negative cells exhibiting red fluorescence (representing the number of MSC cells with mitochondrial transfer from ARPE19 cells).
   1. To quantify TNT formation, calculate the ratio of the number of intercellular nanotubes to the total number of cells.
   2. Calculate the mitochondrial transfer rate as the ratio of the number of Violet-positive cells exhibiting green fluorescence to the total number of Violet-positive cells (representing the mitochondrial transfer from MSCs to ARPE19 cells) or the ratio of the number of Violet-negative cells exhibiting red fluorescence to the total number of Violet-negative cells (representing the mitochondrial transfer from ARPE19 cells to MSCs).

Results

The schematic diagram illustrating the direct co-culture of mesenchymal stem cells (MSC) and ARPE19 cells is depicted in Figure 1. MSCs, engineered to express mito-GFP, as the donor cells and ARPE19-mito-RFP cells with violet-labeled cytoplasmic membranes as recipient cells were co-cultured at a ratio of 1:1. Following a 24 h co-culture period, the cells were stained for phalloidin and examined using confocal microscopy. The resulting cell populations included MSC-mito-GFP cells, ARPE19-mito...

Discussion

Numerous studies have demonstrated that the phenomenon of TNT-mediated mitochondrial transfer is a prevalent physiological process in various types of tissue cells^10,11,12,13. Functional mitochondrial donation from MSCs to cells with mitochondrial dysfunction exhibits strong therapeutic potential^3,14,15

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco	25200-056
4% paraformaldehyde	Solarbio	P1110
6-well plate	NEST	703001
15 mL centrifuge tube	BD Falcon	352097
24-well plate	NEST	702001
ARPE19 cells	ATCC	CRL-2302	Cell lines
Bovine serum albumin (BSA)	Beyotime	ST025
CellTrace violet	Invitrogen	C34557
Cover slide	NEST	801007
DMSO	sigma	D2650
DPBS	Gibco	C141905005BT
DMEM/F-12-GlutaMAX	Gibco	10565-042
Fetal Bovine Serum (FBS)	VivaCell	C04002-500
FluorSave Reagent	Millipore	345789
MSCs	Nuwacell	RC02003	Cell lines
ncMission	Shownin	RP02010
Pen Strep	Gibco	15140-122
pCT-Mito-GFP	SBI	CYTO102-PA-1	Plasmid; From  https://www.systembio.com/mitochondria-cyto-tracer-pct-mito-gfp-cmv
Puromycin	MCE	HY-B1743A
Pipette	Axygen	TF-1000-R-S
Phalloidin	Invitrogen	A22287
Triton X-100	Solarbio	T8200
Transwell plate	Corning	3470