Method Article

Standardized Method to Detect Tunneling Nanotubes in Human Skin Cells for Tissue Engineering Applications

790 views

⸱

DOI:

10.3791/69883

⸱

January 13th, 2026

 ,  ,  ,  ,  ,  , 
1Savaid Stomatology School, Hangzhou Medical College, 2Engineering Laboratory for Biomaterials and Tissue Regeneration, Ningbo Stomatology Hospital, 3Ningbo Savaid Stomatology and Otolaryngology Medical Institute, Ningbo ENT Hospital, 4Department of Urology, the Affiliated People's Hospital of Ningbo University

In This Article

Summary

Here, a standardized protocol to visualize tunneling nanotubes between human epidermal keratinocytes and dermal fibroblasts using membrane and F-actin labeling with z-stack confocal imaging is shown, with optional α-tubulin co-staining, enabling reproducible detection and cytoskeletal characterization for tissue-engineering applications.

Abstract

Tunneling nanotubes (TNTs) are thin, actin-based intercellular conduits that enable long-range transfer of organelles and signaling cargo. Although widely reported across multiple cell types, their presence in human skin cells has not been well described. This article describes a standardized protocol to detect and characterize TNTs in vitro between human epidermal keratinocytes and dermal fibroblasts. The method involves preparing a co-culture of primary cells, gentle fixation to preserve fragile TNTs, membrane labeling with wheat germ agglutinin, F-actin staining with phalloidin, and systematic z-stack imaging by inverted confocal microscopy to distinguish TNTs suspended above the substratum from adherent filopodia. Optional immunostaining for α-tubulin allows assessment of microtubule incorporation. TNTs are defined by three features: thin, straight protrusions connecting two or more cells, the presence of F-actin, and continuity across cell pairs in serial optical sections. Representative results demonstrate TNTs linking dermal-dermal, epidermal-epidermal, and dermal-epidermal pairs, with variable cytoskeletal composition (F-actin alone or F-actin plus α-tubulin). Critical steps include gentle fixation, use of fresh reagents, and acquisition of sufficient z-planes to avoid misclassification, while common artifacts include TNT breakage and incomplete staining. Together, these optimized steps enable reproducible TNT detection in skin cell systems and offer a methodological basis for future investigation of TNT-mediated communication in skin biology and regeneration.

Introduction

Tunneling nanotubes (TNTs) are nanoscale, actin-based channels that enable direct intercellular transfer of organelles and signaling molecules. They are typically 6-100 µm in length and 50-500 nm in diameter, with some reaching up to 700nm thick1. Unlike empty membrane protrusions, TNTs contain cytoskeletal filaments, most commonly F-actin, which provides structural rigidity and supports mitochondrial transport2,3,4. In two-dimensional culture, TNTs are defined by three features: thin (20-700 nm), straight protrusions suspended above the substratum that connect two or more cells; the presence of F-actin filaments; and the ability to transfer cargo.

Since their first identification in PC-12 cells by Rustom et al.5, TNTs have been studied in multiple systems, including the cardiovascular6, immune7, and respiratory8 systems, corneal epithelium9, tumors10, and the nervous system11,12. They contribute to physiological and pathological processes such as immune signaling, apoptosis, material transport, and angiogenesis13,14,15. TNTs can mediate long-range transfer of diverse cargos, including mitochondria, endoplasmic reticulum, nucleic acids, ions, and even viruses5,16,17.

The skin is a structurally complex organ, with the epidermis and dermis relying on constant communication for homeostasis, wound healing, and appendage regeneration18,19,20. Although TNTs have been described in many tissues, including the immune system, the neuronal system, and the corneal epithelium7,9,12, their presence in human skin cells has not been reported in the published literature. Building on the methodological framework described by Sáenz-de-Santa-María et al.21, we present a standardized and optimized protocol for detecting TNTs between epidermal keratinocytes and dermal fibroblasts22 using immunofluorescence staining combined with inverted confocal microscopy.

Compared with alternative TNT-detection approaches, this protocol offers a practical balance between sensitivity and accessibility. Live-cell imaging can capture TNT dynamics but is limited by phototoxicity and the short-lived nature of TNTs1,23. Electron microscopy provides ultrastructural detail yet often fails to preserve long TNT spans and is not practical for routine analysis of primary skin cells24,25. Reporter-based nanotube systems enable real-time tracking but require genetic manipulation, which is typically inefficient in primary keratinocytes and fibroblasts24,26. In contrast, the present protocol uses standard immunofluorescence and confocal microscopy, allowing reproducible TNT detection across independently prepared skin cell cultures while maintaining compatibility with fragile primary cells.

This method is particularly suitable for fixed-sample analysis where cytoskeletal composition and TNT prevalence are of interest. As TNTs are highly fragile, fixation and handling steps remain key limitations, and the approach cannot capture real-time TNT dynamics. Nevertheless, for laboratories working with primary human skin cells, this standardized workflow provides an accessible and consistent way to visualize TNTs and supports downstream studies of their potential functions in tissue engineering and regeneration.

Access restricted. Please log in or start a trial to view this content.

Protocol

All skin cells used in this study were previously frozen primary cells isolated from human skin tissues obtained as discarded foreskin samples from circumcision procedures, and were fully de-identified22. The acquisition of human skin samples was conducted at the Department of Urology, Ningbo University Affiliated People's Hospital, and the study protocol was approved by the institutional ethics committee (Protocol No. 2024104; approval date: October 30, 2024).

1. Identification of TNTs in human primary skin epidermal keratinocytes and dermal fibroblasts

  1. Solution preparation
    1. Prepare fixative solution 1 (200 µL) by adding 4% PFA (100 µL), 2.5% glutaraldehyde (4 µL), 1 M HEPES (40 µL), and 1x PBS (56 µL). Prepare fresh and prewarm to 37 °C before use.
    2. Prepare fixative solution 2 (200 µL) by adding 8% PFA (100 µL), 1 M HEPES (0.4 µL), 1x PBS (99.6 µL). Prepare fresh and prewarm to 37 °C before use.
    3. Prepare fixative solution 3 (200 µL) by adding 8% PFA (100 µL), 2.5% glutaraldehyde (4 µL), 1x PBS (96 µL). Prepare fresh and prewarm to 37 °C before use.
  2. Cell culture
    1. Thaw and culture human epidermal keratinocytes and dermal fibroblasts in 60 mm dishes using the appropriate growth medium. Supplement keratinocyte cultures with Y-27632 at a final concentration of 5 µM during resuscitation. Maintain cultures at 37 °C in a humidified incubator until cells reach ~80% confluency.
    2. Remove the medium and briefly wash the cells with 1 mL of 1x PBS (used to maintain physiological pH and osmolarity during washing). Aspirate the PBS.
    3. Add 1 mL of 0.05% trypsin-EDTA, distribute evenly, and incubate at 37 °C for 1.5-2.5 min.
    4. Gently resuspend cells by pipetting. Add 2 mL of growth medium, transfer to a 15 mL tube, and centrifuge at 200 x g for 5 min at room temperature.
    5. Discard the supernatant and resuspend the pellet in 1-2 mL of medium. Count cells using an automated counter.
    6. Seed 12,000 dermal fibroblasts or 20,000 keratinocytes in each well of an 8-well chamber slide in a final volume of 400 µL. After seeding, cross-shake the chamber well to distribute the cells evenly. Incubate for ~24 h at 37 °C.
    7. Following approximately 24 h of incubation, observe the cell confluence under a microscope. If it reaches ~80%, proceed to the subsequent fixation steps.
  3. Fixation
    1. When cultures reach ~80% confluency, gently add several drops of Fixative Solution 1 directly to the chamber well to pre-fix the cells. Incubate for 4 min at room temperature. Ensure the fixative spreads evenly without disturbing the cells.
      CAUTION: Handle carefully to avoid disruption of TNTs. Because the fixative solutions contain toxic components like PFA and glutaraldehyde, always wear protective gloves, protective clothing, and a mask, and handle them in a fume hood. Be sure to wash hands thoroughly afterwards. Collect PFA and glutaraldehyde fixative waste separately in labeled containers for hazardous chemical disposal.
    2. Aspirate the pre-fixative and replace it with 140 µL of fresh Fixative Solution 1, ensuring the entire well is covered. Incubate for 15 min at 37 °C.
    3. Remove Fixative Solution 1 and add 140 µL of Fixative Solution 2. Incubate for 15 min at 37 °C. Make sure the solution fully covers the cells to maintain uniform fixation.
    4. Remove the fixative, then add 140 µL of 100 mM NH₄Cl to quench residual aldehydes. Incubate for 10-30 min at room temperature.
    5. Rinse the samples 3x with 1x PBS for 30 s each at room temperature, pipetting gently along the chamber wall to avoid washing away cells or breaking TNTs.
  4. Membrane and cytoskeletal staining
    1. Incubate cells with 140 µL of 1:300 (v/v) WGA (Wheat Germ Agglutinin)-Alexa Fluor conjugate, which labels plasma membrane glycoproteins for visualization of membrane-derived TNTs, in 1x PBS for 20 min in the dark at room temperature.
      NOTE: The subsequent steps need to be carried out in a dark environment.
    2. Wash 3x with 1x PBS (30 s each). Incubate with 140 µL of phalloidin conjugate diluted 1:250 (v/v) in 1% BSA for 30 min in the dark at room temperature.
    3. Wash 3x with 1x PBS (30 s each). Incubate with 140 µL of DAPI solution for 5 min at room temperature in the dark.
    4. Wash 3x with 1× PBS. Add 2-3 drops of mounting medium, incubate for 20 min at room temperature, and store at least 20 min at 4 °C in the dark until imaging.
  5. Imaging
    1. Acquire images using an inverted confocal microscope with a 40x objective. Select random fields and collect Z-stacks (10-20 layers) from the bottom to the top of the cells. The specific microscope parameters used for image acquisition are listed below:
      Confocal laser scanning microscope: ZEISS LSM 900
      Laser lines: Track 1 (561 nm, 4.0%), Track 2 (488 nm, 16.0%), Track 3 (405 nm, 60.0%)
      Detector gain: 525 V
      Z-step size: 20 optical sections (total depth: 5.32 µm)

2. Co-culture of epidermal and dermal cells

  1. Prepare keratinocytes and fibroblasts as in steps 1.2.1-1.2.5.
  2. Seed 10,000 keratinocytes and 6,000 fibroblasts per chamber well in 400 µL of medium. After seeding, cross-shake the chamber well to distribute the cells evenly. Incubate for 24 h at 37 °C.
  3. Following approximately 24 h of incubation, observe the cell confluence under a microscope. If it reaches ~80%, proceed to the subsequent fixation steps.
  4. Fix and stain membranes as described in steps 1.3-1.4.
  5. Permeabilize cells with 0.1% Triton X-100, to allow antibody access to intracellular targets, for 3 min at room temperature, then wash 3x with 1x PBS.
    NOTE: 0.1% Triton X-100 should be freshly prepared before use.
    CAUTION: Triton X-100, harmful to aquatic life, must also be disposed of as chemical waste and never poured down the drain.
  6. Block with 140 µL of 2% BSA in PBS to reduce non-specific antibody binding for 20 min at room temperature.
    NOTE: 2% BSA should be freshly prepared before use.
  7. Incubate overnight at 4 °C with anti-cytokeratin 5 antibody diluted 1:200 (v/v) in 2% BSA.
  8. Wash 3x with 1x PBS, then incubate with Alexa Fluor 594-conjugated secondary antibody for 1-2 h at room temperature in the dark.
  9. Wash 3x with 1× PBS. Stain nuclei with DAPI and image as in steps 1.4.5-1.5.2.

3. Identification of cytoskeletal components of TNTs

  1. Cell culture
    1. Culture and fix cells as in steps 1.2-1.3. Permeabilize with 0.1% Triton X-100 for 3 min at room temperature, then wash with 1x PBS.
    2. Block with 2% BSA for 20 min at room temperature. Incubate overnight at 4 °C with anti-α-tubulin antibody.
    3. Wash 3x with 1x PBS and incubate with Alexa Fluor-conjugated secondary antibody for 1-2 h at room temperature in the dark.
      NOTE: The subsequent steps need to be carried out in a dark environment.
    4. Wash 3x with 1x PBS. Label F-actin with phalloidin and image as in steps 1.4.3-1.5
  2. Co-culturing of cells
    1. Prepare co-cultures as in steps 2.1-2.2. Fix with fixative solution 3 for 15 min at room temperature.
    2. Replace with 140 µL of 100 mM NH4Cl and incubate for 10-30 min at room temperature. Wash 3x with 1x PBS.
    3. First, identify epidermal cells by performing permeabilization, blocking, and CK5 immunostaining exactly as described in steps 2.4-2.8.
    4. Next, perform α-tubulin immunostaining following steps 3.1.2-3.1.4.
    5. Finally, stain F-actin with phalloidin and proceed with imaging as described in steps 1.4.2-1.5.2.

Access restricted. Please log in or start a trial to view this content.

Results

Using this standardized protocol, tunneling nanotubes (TNTs) were successfully identified in human skin cells under both single-culture and co-culture conditions. To validate antibody specificity, both positive and negative control staining were performed for all antibodies prior to the experimental analyses, as shown in Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, and Supplementary Figure 4. Representative images demonstrate the reproducible detection of TNTs ...

Access restricted. Please log in or start a trial to view this content.

Discussion

A critical step in this protocol is the preservation of TNTs during fixation and staining, as these structures are extremely fragile and easily disrupted. Gentle handling during fixation, appropriate timing, and the use of fresh fixatives are essential to maintain TNT integrity. The use of Z-stack confocal imaging is equally important to distinguish TNTs suspended above the substratum from filopodia or other adherent protrusions, which may otherwise lead to misinterpretation. These considerations underscore the need for ...

Access restricted. Please log in or start a trial to view this content.

Disclosures

The authors have no competing financial interests to declare.

Acknowledgements

This project was funded by the National Natural Science Foundation of China (Grant No. 82273554). The authors acknowledge the Biomaterials and Tissue Regeneration Engineering Laboratory at Ningbo Stomatology Hospital for continuous support and thank both current and past team members for their valuable discussions. The authors also thank Teacher Feng Li from the Research and Experiment Center of Hangzhou Medical College for generous assistance and strong support in facilitating access to the confocal microscope.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
15-mL tubeKIRGENKG2611Sterile, RNase/Dnase and pyrogenic free
37 °C incubatorThermo Scientific51032877The 37-degree CO2 incubator provides an ideal in vitro environment.
Actin-Tracker Red-RhodamineBeyotimeC2207sActin-Tracker Red-Rhodamine probe is a phalloidin labeled with the fluorescent dye Rhodamine, with a maximum excitation wavelength of 540nm and a maximum emission wavelength of 565nm.
Alexa Fluor conjugate of wheat germ agglutinin (WGA)Molecular ProbesW11261 Alexa Fluor 488 Conjugate
Anti-alpha Tubulin antibodyAbcamab7291Anti-alpha Tubulin antibody - Loading Control (ab7291) is a mouse monoclonal antibody detecting alpha Tubulin. Suitable for Human, Mouse, Rat.
Anti-cytokeratin 5 antibody Abcamab52635Anti-Cytokeratin 5 antibody - Cytoskeleton Marker (ab52635) is a rabbit monoclonal antibody detecting Cytokeratin 5. Suitable for Human, Mouse, Rat.
Aqua-Poly/MountPolysciences18606Water-Soluble, Non-Fluorescing Mounting Medium formulated for mounting sections from aqueous solutions. Useful for immunofluorescent techniques as it enhances and retains fluorescent stains.
Automated cell counterCountstarIC1000Automated cell counter for fast, precise cell counting
Benchtop centrifugeBIORIDGETD5Benchtop centrifuge for quick and efficient sample separation in laboratory settings
Biological safety cabinetThermo Scientific1379Biological Safety Cabinet is a sterile containment device that protects operators, samples, and environments from biohazards by filtering airborne pathogens and preventing cross-contamination during microbiological work.
Bovine serum albumin(BSA)YOBIBIOU5010-10gBSA is a globulin in bovine serum containing 583 amino acid residues with a molecular weight of 66.430 kDa and an isoelectric point of 4.7
Cell Culture Dish (60X15 mm)eppendorf30701119Tissue culture treated Sterile, free of detectable pyrogens, RNase & DNase, DNA.
Cellvis Chamber Slide-8 ChamberCellvisC8-1.5H-NDesigned for high resolution imaging such as confocal microscopy.
DAPI staining solutionBeyotimeC1006-10 mLDAPI Staining Solution is suitable for nuclear staining of all common cells and tissues.
Goat Anti-Mouse IgG H&L (Alexa Fluor 405) Abcamab175660Goat Anti-Mouse IgG H&L (Alexa Fluor 405) is a secondary antibody with a maximum absorption wavelength of 401nm and a maximum emission wavelength of 421nm.
Goat Anti-Rabbit IgG H&L conjugated to Alexa Fluor 405Abcamab175652Goat Anti-Rabbit IgG H&L (Alexa Fluor 405) is a secondary antibody with a maximum absorption wavelength of 401nm and a maximum emission wavelength of 421nm.
Goat Anti-Rabbit IgG H&L conjugated to Alexa Fluor 594Abcamab150080Goat anti-rabbit IgG H&L (Alexa Fluor 594) is a secondary antibody with a maximum absorption wavelength of 590nm and a maximum emission wavelength of 617nm.
Inverted confocal fluorescence microscopeLeicaTCS SP8 XLeica SP8 X laser scanning confocal microscope delivers unparalleled high-resolution imaging with advanced fluorescence detection and live-cell analysis capabilities.
Inverted confocal fluorescence microscopeAndorBC43The Andor BC43 is a compact, high-speed confocal microscope offering 10x faster imaging than traditional systems with one-click 2D/3D imaging capability
NH4Cl MedChemExpressHY-Y1269 Ammonium chloride as a polar compound for regulating pH.
Phalloidin Conjugates Oregon GreenTM488Life TechnologiesO7466Colour: Green. Excitation wavelength range: 496/520.
Phosphate Buffered Saline (PBS)Viva Cell BiosciencesC3582-0500Its function is to keep the pH of the medium within physiological range and maintain the osmotic pressure balance inside and outside the cell.
Triton-X 100SolarbioT8200-100mlIt is a versatile nonionic surfactant used to restore membrane components under mild denaturing conditions.
Trypsin-EDTA SolutionGIBCO25300054Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation.
Y-27632MedChemExpressHY-10071Y-27632 is an orally active, ATP-competitive inhibitor of ROCK-I and ROCK-II

References

  1. Austefjord, M. W., Gerdes, H. H., Wang, X. Tunneling nanotubes: Diversity in morphology and structure. Commun Integr Biol. 7 (1), e27934(2014).
  2. Liu, Z., Sun, Y., Qi, Z., Cao, L., Ding, S. Mitochondrial transfer/transplantation: an emerging therapeutic approach for multiple diseases. Cell Biosci. 12 (1), 66(2022).
  3. Yang, F., et al. Tunneling Nanotube-Mediated Mitochondrial Transfer Rescues Nucleus Pulposus Cells from Mitochondrial Dysfunction and Apoptosis. Oxid Med Cell Longev. 2022, 3613319(2022).
  4. Yang, C., et al. Mitochondria transfer from early stages of erythroblasts to their macrophage niche via tunnelling nanotubes. Br J Haematol. 193 (6), 1260-1274 (2021).
  5. Rustom, A., Saffrich, R., Markovic, I., Walther, P., Gerdes, H. H. Nanotubular highways for intercellular organelle transport. Science. 303 (5660), 1007-1010 (2004).
  6. Alarcon-Martinez, L., et al. Pericyte dysfunction and loss of interpericyte tunneling nanotubes promote neurovascular deficits in glaucoma. Proc Natl Acad Sci U S A. 119 (7), 2110329119(2022).
  7. Zhu, C., Shi, Y., You, J. Immune Cell Connection by Tunneling Nanotubes: The Impact of Intercellular Cross-Talk on the Immune Response and Its Therapeutic Applications. Mol Pharm. 18 (3), 772-786 (2021).
  8. Zhang, F., et al. TFAM-Mediated mitochondrial transfer of MSCs improved the permeability barrier in sepsis-associated acute lung injury. Apoptosis. 28 (7-8), 1048-1059 (2023).
  9. Jiang, D., et al. Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage. Cell Death Dis. 7 (11), e2467(2016).
  10. Zheng, Y., et al. Tunneling nanotubes mediate KRas transport: Inducing tumor heterogeneity and altering cellular membrane mechanical properties. Acta Biomater. 185, 312-322 (2024).
  11. Dilsizoglu Senol, A., et al. α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes. PLoS Biol. 19 (7), e3001287(2021).
  12. Scheiblich, H., et al. Microglia rescue neurons from aggregate-induced neuronal dysfunction and death through tunneling nanotubes. Neuron. 112 (18), 3106-3125.e8 (2024).
  13. Solimando, A. G., et al. Tunneling nanotubes between bone marrow stromal cells support transmitophagy and resistance to apoptosis in myeloma. Blood Cancer J. 15 (1), 3(2025).
  14. Korenkova, O., et al. Tunneling nanotubes enable intercellular transfer in zebrafish embryos. Dev Cell. 60 (4), 524-534 (2025).
  15. Ma, L., et al. Dynamic three-dimensional culture enhances tunneling nanotubes-mediated mitochondrial transfer in mesenchymal stromal cells to accelerate wound healing. J Nanobiotechnol. 23 (1), 559(2025).
  16. Sahinbegovic, H., et al. Intercellular Mitochondrial Transfer in the Tumor Microenvironment. Cancers (Basel). 12 (7), 12071787(2020).
  17. Gurke, S., Barroso, J. F., Gerdes, H. H. The art of cellular communication: tunneling nanotubes bridge the divide. Histochem Cell Biol. 129 (5), 539-550 (2008).
  18. Kobielak, K., Pasolli, H. A., Alonso, L., Polak, L., Fuchs, E. Defining BMP functions in the hair follicle by conditional ablation of BMP receptor IA. J Cell Biol. 163 (3), 609-623 (2003).
  19. Nowak, J. A., Polak, L., Pasolli, H. A., Fuchs, E. Hair follicle stem cells are specified and function in early skin morphogenesis. Cell Stem Cell. 3 (1), 33-43 (2008).
  20. Woo, W. M., Zhen, H. H., Oro, A. E. Shh maintains dermal papilla identity and hair morphogenesis via a Noggin-Shh regulatory loop. Genes Dev. 26 (11), 1235-1246 (2012).
  21. Sáenz-de-Santa-María, I., Henderson, J. M., Pepe, A., Zurzolo, C. Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. Curr Protoc. 3 (11), e939(2023).
  22. Liu, Z., et al. A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue. J Vis Exp. (138), e57784(2018).
  23. Dupont, M., Souriant, S., Lugo-Villarino, G., Maridonneau-Parini, I., Vérollet, C. Tunneling Nanotubes: Intimate Communication between Myeloid Cells. Front Immunol. 9, 43(2018).
  24. Gerdes, H. H., Rustom, A., Wang, X. Tunneling nanotubes, an emerging intercellular communication route in development. Mech Dev. 130 (6-8), 381-387 (2013).
  25. Sartori-Rupp, A., et al. Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells. Nat Commun. 10 (1), 342(2019).
  26. Dagar, S., Pathak, D., Oza, H. V., Mylavarapu, S. V. S. Tunneling nanotubes and related structures: molecular mechanisms of formation and function. Biochem J. 478 (22), 3977-3998 (2021).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Keratinocyte Fibroblast CocultureF Actin StainingWheat Germ AgglutininConfocal MicroscopyGentle FixationAlpha Tubulin ImmunostainingSkin Cell Communication

Related Articles