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).

1. Generation of MSC-mito-GFP and ARPE19-mito-RFP cell lines
<ol>
	<li>Cell culture 
	NOTE: Only MSCs are used here as an example.
	<ol>
		<li>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.
		<ol>
			<li>Remove the original medium and wash the cells once with phosphate-buffered saline (DPBS) (see <...

Investigating Mitochondrial Transfer in MSC and ARPE19 Co-Cultures

<ol>
	<li>Caprara, C., Grimm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+oxygen+to+erythropoietin:+Relevance+of+hypoxia+for+retinal+development,+health+and+disease.">From oxygen to erythropoietin: Relevance of hypoxia for retinal development, health and disease.</a> Prog Retin Eye Res. 31 (1), 89-119 (2012).</li><li>Ferrington, D. A., Fisher, C. R., Kowluru, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+defects+drive+degenerative+retinal+diseases.">Mitochondrial defects drive degenerative retinal diseases.</a> Trends Mol Med. 26 (1), 105-118 (2020).</li><li>Spees, J. L., Olson, S. D., Whitney, M. J., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transfer+between+cells+can+rescue+aerobic+respiration.">Mitochondrial transfer between cells can rescue aerobic respiration.</a> Proc Natl Acad Sci U S A. 103 (5), 1283-1288 (2006).</li><li>Wei, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+transfer+from+bone+mesenchymal+stem+cells+protects+against+tendinopathy+both+in+vitro+and+in+vivo.">Mitochondrial transfer from bone mesenchymal stem cells protects against tendinopathy both in vitro and in vivo.</a> Stem Cell Res Ther. 14 (1), 104 (2023).</li><li>Borcherding, N., Brestoff, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+power+and+potential+of+mitochondria+transfer.">The power and potential of mitochondria transfer.</a> Nature. 623 (7986), 283-291 (2023).</li><li>Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+mitochondrial+transfer+as+a+means+of+tissue+revitalization.">Intercellular mitochondrial transfer as a means of tissue revitalization.</a> Signal Transduct Target Ther. 6 (1), 65 (2021).</li><li>Rustom, A., Saffrich, R., Markovic, I., Walther, P., Gerdes, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotubular+highways+for+intercellular+organelle+transport.">Nanotubular highways for intercellular organelle transport.</a> Science. 303 (5660), 1007-1010 (2004).</li><li>Cordero Cervantes, D., Zurzolo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peering+into+tunneling+nanotubes-the+path+forward.">Peering into tunneling nanotubes-the path forward.</a> Embo j. 40 (8), e105789 (2021).</li><li>Qin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions,+methods,+and+mobility+of+mitochondrial+transfer+between+cells.">The functions, methods, and mobility of mitochondrial transfer between cells.</a> Front Oncol. 11, 672781 (2021).</li><li>Jiang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+crosstalk+between+mesenchymal+stem+cells+and+various+ocular+cells+through+the+intercellular+trafficking+of+mitochondria.">Bioenergetic crosstalk between mesenchymal stem cells and various ocular cells through the intercellular trafficking of mitochondria.</a> Theranostics. 10 (16), 7260-7272 (2020).</li><li>Domhan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+communication+by+exchange+of+cytoplasmic+material+via+tunneling+nano-tube+like+structures+in+primary+human+renal+epithelial+cells.">Intercellular communication by exchange of cytoplasmic material via tunneling nano-tube like structures in primary human renal epithelial cells.</a> PLoS One. 6 (6), e21283 (2011).</li><li>Zhang, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+nanotubes:+Novel+communication+between+distant+cells.">Membrane nanotubes: Novel communication between distant cells.</a> Sci China Life Sci. 56 (11), 994-999 (2013).</li><li>Cheng, X. 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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</sup...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>NEST</td>
			<td>703001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>NEST</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>ARPE19 cells</td>
			<td>ATCC</td>
			<td>CRL-2302</td>
			<td>Cell lines</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Beyotime</td>
			<td>ST025</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace violet</td>
			<td>Invitrogen</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C141905005BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12-GlutaMAX</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VivaCell</td>
			<td>C04002-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FluorSave Reagent</td>
			<td>Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCs</td>
			<td>Nuwacell</td>
			<td>RC02003</td>
			<td>Cell lines</td>
		</tr>
		<tr>
			<td>ncMission</td>
			<td>Shownin</td>
			<td>RP02010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pCT-Mito-GFP</td>
			<td>SBI</td>
			<td>CYTO102-PA-1</td>
			<td>Plasmid; From&#160; https://www.systembio.com/mitochondria-cyto-tracer-pct-mito-gfp-cmv</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>MCE</td>
			<td>HY-B1743A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Axygen</td>
			<td>TF-1000-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22287</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell plate</td>
			<td>Corning</td>
			<td>3470</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial transfer via tunneling nanotubes between mesenchymal stem cells and retinal pigment epithelium in vitro

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.

Author Spotlight: Bidirectional Mitochondrial Transfer between MSCs and Retinal Pigment Epithelium Cells &#8212; Pathways and In Vivo Challenges

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

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...

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.

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important ...

mitochondrial-transfer-via-tunneling-nanotubes-between-mesenchymal

Research

JoVE Journal

Biology

322 vistas.  Capital Medical University. 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. 