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

We developed methods to reliably detect and analyze the tunneling nanotubes between human skin cells and studying their potential role in skin regeneration. Our protocol is designed for fixed samples or for primary human skin cells. It's useful in research related to skin biology and the tissue regeneration.

To begin, thaw and culture human epidermal keratinocytes and dermal fibroblasts in 60 millimeter dishes containing the appropriate growth medium. Supplement the keratinocyte cultures with Y-27632 to a final concentration of five micromolar during resuscitation. Maintain all cultures at 37 degrees Celsius in a humidified incubator until they reach approximately 80%confluency.

Then remove the medium from the culture dish. Rinse the cells briefly with one milliliter of PBS to maintain physiological pH and osmolarity during washing. Aspirate the PBS completely.

Add one milliliter of 0.05%trypsin EDTA and distribute it evenly across the cell surface. Incubate the dish at 37 degrees Celsius for 1.5 to 2.5 minutes. Gently resuspend the cells by pipetting up and down.

Add two milliliters of growth medium to neutralize the trypsin. Transfer the cell suspension into a 15 milliliter tube and centrifuge at 200 G for five minutes at room temperature. After the centrifugation, discard the supernatant from the centrifuge tube.

Resuspend the cell pellet in one to two milliliters of medium and count the cells using an automated counter. Next, seed 12, 000 dermal fibroblasts or 20, 000 keratinocytes in each well of an eight-well chamber slide in a final volume of 400 microliters. After seeding, cross-shake the chamber well to distribute the cells evenly and incubate the cells at 37 degrees Celsius for approximately 24 hours.

Following incubation, confirm if the cell confluency is around 80%under a microscope. When cultures reach 80%confluency, gently add several drops of fixative solution one directly to the chamber well to prefix the cells. Incubate the cells at room temperature for four minutes, ensuring that the fixative spreads evenly without disturbing the cells.

Then aspirate the pre-fixative from the well and add 140 microliters of fresh fixative solution one, ensuring complete coverage of the well surface. Incubate the slide at 37 degrees Celsius for 15 minutes. Next, remove the fixative from the chamber well and add 140 microliters of 100 millimolar ammonium chloride to quench residual aldehydes.

Incubate the slide at room temperature for 10 to 30 minutes. Rinse the samples three times with PBS for 30 seconds each at room temperature. During each rinse, gently pipette the buffer along the chamber wall to avoid washing away cells or damaging tunneling nanotubes.

Incubate the cells with 140 microliters of a 1:300 volume-to-volume dilution of wheat germ agglutinin Alexa Flour conjugate in PBS. Perform the incubation in the dark at room temperature for 20 minutes to label plasma membrane glycoproteins for visualization of membrane-derived tunneling nanotubes. Then wash the cells three times with PBS for 30 seconds each.

Then incubate the cells with 140 microliters of phalloidin conjugate diluted one to 250 times in 1%BSA for 30 minutes in the dark at room temperature. After washing the cells three times with PBS, incubate them with 140 microliters of DAPI solution for five minutes at room temperature in the dark. Finally, add two to three drops of mounting medium to each well and incubate the sample at room temperature for 20 minutes.

Store the slides at four degrees Celsius in the dark for at least 20 minutes until imaging. In dermal fibroblast cultures, tunneling nanotubes were observed as long thin projections connecting adjacent cells with membranes stained by wheat germ agglutinin and F-actin labeled by phalloidin. In epidermal keratinocyte cultures, tunneling nanotubes with characteristic F-actin positive protrusions were also observed bridging neighboring cells.

In co-cultures of dermal fibroblasts and epidermal keratinocytes, tunneling nanotubes were consistently detected between dermal fibroblasts and epidermal keratinocytes, visualized by membrane and cytokeratin 5 staining. The tunneling nanotubes in the co-cultures exhibited diverse cytoskeletal organization with examples containing only F-actin, both the filaments, F-actin with partial alpha-tubulin, or with low alpha-tubulin levels. The foremost challenge is preserving the tunnel in nanotubes.

This requires gentle fixation, appropriate timing, and the use of freshly prepared fixed additives. Using our protocol, the of the tunneling nanotubes can be measured under varying cell conditions to explore their potential functions. In the future, we will fluorescently label mitochondria to study their transfer via tunneling nanotubes.