Method Article

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

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DOI:

10.3791/66917

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October 4th, 2024

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1Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University

In This Article

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 transfer3,4,5,6. 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 transfer8,9. 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 cells3,4,5,6. 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-RFP cells, ARPE19-mito-RFP cells containing mito-GFP, and MSC-mito-GFP cells containing mito-RFP. Notably, the presence of mito-GFP in ARPE19 cells and mito-RFP in MSCs suggested mitochondrial bidirectional transfer.

Specific details of TNT formation are presented in Figure 2. These structures can establish intercellular connections between cells of the same type (Figure 2A-C), as well as between cells of different types (Figure 2D). Tunneling nanotubes (TNTs) serve as conduits connecting adjacent cells, exhibiting a distinctive F-actin-rich membrane structure with open ends facilitating cytoplasmic continuity and the transport of mitochondria. Utilizing Z-axis imaging, it is evident that TNTs are not firmly anchored to the extracellular matrix but rather suspended within the culture medium, thereby distinguishing them from conventional cellular protrusions. Mitochondrial transfer is evident in Figure 3, where violet-positive ARPE19 cells exhibit dispersed, green-dotted fluorescence indicative of mitochondria originating from MSCs (Figure 3A). Violet-negative MSCs containing red-dotted fluorescence represent the mitochondrial transfer from ARPE19 cells (Figure 3B). Figure 4 shows TNT formation and mitochondrial transfer under super-resolution imaging, from which we can see more details of mitochondrial transfer via TNT. Video 1 shows the dynamics of mitochondrial transport through TNT.

To further demonstrate that the prerequisite for mitochondrial transfer is physical contact, such as TNT, rather than mitochondrial secretion and uptake, we used a previously reported co-culture method10. As shown in Figure 5A, MSCs and ARPE19 cells were seeded in the upper and lower chambers of a transwell plate, respectively. They were separated by a filter with 0.4 µm pores, which blocked the direct contact between MSCs and ARPE19 cells but allowed the passage of mitochondria. After 24 h of co-culture, ARPE19 cells in the lower chamber were assayed and little mito-GFP was observed from MSCs in the upper chamber (Figure 5B). These results suggest that mitochondrial transfer requires direct contact between MSCs and ARPE19 cells.

Ultimately, the quantification of TNT formation and mitochondrial transfer was conducted, with the corresponding results depicted in Figure 6. MSCs were significantly more capable of donating mitochondria than ARPE19 cells (p = 0.0286). These findings indicate that MSCs possess the capability to transfer mitochondria to retinal pigment epithelial cells.

figure-results-1
Figure 1: Schematic view of co-culture of MSCs and ARPE19 cells. MSCs expressing mito-GFP and ARPE19-mito-RFP cells labeled with CellTrace Violet are co-cultured for 24 h at a ratio of 1:1. Mito-GFP in violet+ cells marks the transfer of mitochondria from MSCs to ARPE19 cells, while Mito-RFP in violet- cells marks the transfer of mitochondria from ARPE19 cells to MSCs. Abbreviations: GFP = green fluorescent protein; RFP = red fluorescent protein; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.

figure-results-2
Figure 2: TNT formation between MSCs and ARPE19 cells. White arrowheads indicate TNT formation, green arrowheads indicate mitochondria with mito-GFP and red arrowheads indicate mitochondria with mito-RFP. (A,B) TNT formation between ARPE19 cells. (C) TNT formation between MSCs. (D) TNT formation between MSCs and ARPE19 cells. Z-axis imaging showing TNTs suspended in culture medium. Scale bars = 25 µm. Abbreviations: MSCs = mesenchymal stem cells; TNTs = tunneling nanotubes. Please click here to view a larger version of this figure.

figure-results-3
Figure 3: Mitochondrial transfer from MSCs to ARPE19 cells. (A) The white dashed circle indicates mitochondrial (mito-GFP) transfer from MSCs to ARPE19 cells, and (B) the yellow dashed circle indicates mitochondrial (mito-RFP) transfer from ARPE19 cells to MSCs. Scale bars = 20 µm. Abbreviation: GFP = green fluorescent protein; RFP = red fluorescent protein; MSCs = mesenchymal stem cells. Please click here to view a larger version of this figure.

figure-results-4
Figure 4: TNT formation and mitochondrial transfer between MSCs and ARPE19 cells by super-resolution microscopy. (A and B) Mitochondrial transport from MSCs to ARPE19 cells. (C) Detail of TNTs under super-resolution imaging. (D) Green mitochondria from MSCs present in ARPE19 cells under super-resolution imaging. Scale bar = 5 µm. Abbreviations: MSCs = mesenchymal stem cells; TNTs = tunneling nanotubes. Please click here to view a larger version of this figure.

figure-results-5
Figure 5: Almost undetectable mitochondrial transfer in indirect co-culture in transwell system of MSCs and ARPE19 cells. (A) Schematic view of co-culture of MSCs and ARPE19 cells in transwell system. (B) Representative images of ARPE19 cells in the bottom of the transwell plate after indirect co-culture with MSCs for 24 h. Please click here to view a larger version of this figure.

figure-results-6
Figure 6: Quantification of TNT formation and mitochondrial transfer. (A) TNT formation, (B) mitochondrial transfer. Abbreviations: M = MSCs = mesenchymal stem cells; A = ARPE19 cells; TNTs = tunneling nanotubes; M-A = TNT formation between MSCs and ARPE19 cells; M-M = TNT formation between MSCs; A-A = TNT formation between ARPE19 cells. n = 4, mean ± SD, Mann-Whitney U-test, *p < 0.05. Please click here to view a larger version of this figure.

Supplemental Figure S1: Mapping of plasmid pCT-Mito-copGFP. Mito-GFP (CYTO102-PA-1) was purchased. The Mitochondria Cyto-Tracer, pCT-Mito-GFP (CMV), fuses with copGFP via a Cox8 tag, resulting in the labeling of mitochondria with copGFP. This copGFP fusion is driven by the CMV promoter, enabling robust expression in cell lines such as HeLa, HEK293, and HT1080. Mito-RFP was modified from mito-GFP by replacing GFP with RFP; it carries a Puromycin resistance gene. Lentiviral packaging of this plasmid is done by specialized companies. Please click here to download this File.

Video 1: TNT-mediated mitochondrial transport between MSCs and ARPE19 cells. TNT is generated through the bidirectional movement of MSC and ARPE19 cells, facilitating the transfer of mitochondria from MSC cells to ARPE19 cells. Abbreviations: MSCs = mesenchymal stem cells; TNTs = tunneling nanotubes. Please click here to download this Video.

Discussion

Numerous studies have demonstrated that the phenomenon of TNT-mediated mitochondrial transfer is a prevalent physiological process in various types of tissue cells10,11,12,13. Functional mitochondrial donation from MSCs to cells with mitochondrial dysfunction exhibits strong therapeutic potential3,14,15,16,17,18.

Here, to present TNT-mediated bidirectional mitochondrial transfer between MSCs and ARPE19 cells, we constructed MSC-mito-GFP and ARPE19-mito-RFP cell lines. Next, the two kinds of cells were co-cultured for 24 h, and cytoskeletal staining was performed at the end of the co-culture. By confocal imaging, we observed and counted TNT formation and mitochondrial transfer rate. The outcomes of these observations may be influenced by factors such as the proportion of co-cultured cells and cell density. During co-culture, it is imperative to thoroughly mix the two cell types and maintain them in a single-cell state to minimize the formation of cell colonies by homotypic cells, thereby reducing the occurrence of mitochondrial transfer between the two cell types.

Flow cytometric analysis can similarly be used to analyze mitochondrial transfer rates, and this method is simpler than postimaging statistics. However, the mitochondrial transfer rate calculated using this method will be somewhat lower than the actual value, because small amounts of mitochondrial transfer may not be recorded. In this protocol, the counting of imaging results needs to be done very carefully to avoid omissions or incorrect counting of fluorescent spurious signals.

The study of bidirectional mitochondrial transfer by constructing two cell lines with mitochondrial fluorescence is an innovation of this protocol. The method avoids the interference of fluorescent dyes and makes the experimental results more credible. The transfer of mitochondria from retinal cells to MSCs deserves further study, for example, to explore changes in the proportion of mitochondria transferred in both directions under stressful conditions.

By adhering to the experimental procedures outlined in this protocol, we were able to ascertain that MSCs can transfer mitochondria to retinal pigment epithelial cells through tunneling nanotubes. This phenomenon may serve as a plausible mechanism underlying the advantageous impacts of MSCs on retinal tissues, thereby aiding in the elucidation of MSCs-driven regenerative therapies for ocular tissues.

Disclosures

The authors declare that they possess no conflicting interests.

Acknowledgements

We thank Guangzhou CSR Biotech Co. Ltd for imaging with their commercial super-resolution microscope (HIS-SIM), data acquisition, SR image reconstruction, analysis, and discussion. This work is partly supported by the National Natural Science Foundation of China (82125007,92368206) and the Beijing Natural Science Foundation (Z200014).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGibco25200-056
4% paraformaldehydeSolarbioP1110
6-well plateNEST703001
15 mL centrifuge tubeBD Falcon352097
24-well plateNEST702001
ARPE19 cellsATCCCRL-2302Cell lines
Bovine serum albumin (BSA)BeyotimeST025
CellTrace violetInvitrogenC34557
Cover slideNEST801007
DMSOsigmaD2650
DPBSGibcoC141905005BT
DMEM/F-12-GlutaMAXGibco10565-042
Fetal Bovine Serum (FBS)VivaCellC04002-500
FluorSave ReagentMillipore345789
MSCsNuwacellRC02003Cell lines
ncMissionShowninRP02010
Pen StrepGibco15140-122
pCT-Mito-GFPSBICYTO102-PA-1Plasmid; From  https://www.systembio.com/mitochondria-cyto-tracer-pct-mito-gfp-cmv
PuromycinMCEHY-B1743A
PipetteAxygenTF-1000-R-S
PhalloidinInvitrogenA22287
Triton X-100SolarbioT8200
Transwell plateCorning3470

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Bidirectional Mitochondrial TransferMitochondrial TracingConfocal MicroscopyCell Co CultureMito GFP LabelingMito RFP Labeling

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