Sodium hydroxide (NaOH), trypsin inhibitor (from Glycine max), 3,3′,5-triiodo-L-thyronine sodium salt, hydrocortisone, cholera toxin, epidermal growth factor (EGF, recombinant human protein), insulin, transferrin (human), Hoechst 33258, sodium dodecyl sulfate (SDS), glycine, adenine hydrochloride hydrate, uranyl acetate, propylene oxide and l-ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). PrestoBlue cell viability reagent, SuperFrost Plus microscope slides, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (no phenol red), E-cadherin monoclonal antibody (4A2C7, cat. 33-4000), collagen IV monoclonal antibody (1042, cat. 14-9871-82), occluding polyclonal antibody (6HCLC, cat. 710192), goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (alexa fluor 546 and alexa fluor 488), goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (alexa fluor 546 and alexa fluor 488), ProLong diamond antifade mountant, tris(hydroxymethyl)aminomethane (Tris), SuperSignal West Dura Extended Substrate and MEM non-essential amino acids solution (100x) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum superior (FBS), 10x DMEM solution, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer (1 M) and Collagen G (0.4% solution in 15 mmol/l HCl, type I, 4 mg/ml) were purchased from Biochrom (Berlin, Germany). Triton X-100, urea and dimethyl sulfoxide were purchased from Carl Roth (Karlsruhe, Germany). ThinCert cell culture inserts for 12-well plates (translucent membrane (polyethylene terephthalate), pore diameter: 8 µm) and 12-well ThinCert cell culture plates (deep-well) were purchased from Greiner Bio-One (Kremsmünster, Austria). GA-1000 (gentamicin sulfate amphotericin-B, 1000 x) was purchased from Lonza (Basel, Switzerland). Dulbecco's phosphate-buffered saline (DPBS), trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05%/0.02%) in PBS, l-glutamine (200 mM) and penicillin/streptomycin (100 U/ml) were purchased from PAN-Biotech GmbH (Aidenbach, Germany). Tissue-Tek cryomolds (15 mm × 15 mm × 5 mm) and Tissue-Tek O.C.T. compounds were purchased from Sakura Finetek (Torrance, CA, USA). Dispase (1 U/ml) in DMEM/F-12 was purchased from Stemcell Technologies (Cologne, Germany). Keratinocyte Growth Medium 2 (kit, growth medium supplemented with final concentrations of: 0.004 ml bovine pituitary extract per ml final medium, 0.125 ng/ml EGF (recombinant human), 5 μg/ml insulin (recombinant human), 0.33 μg/ml hydrocortisone, 0.39 μg/ml epinephrine, 10 μg/ml transferrin (recombinant human), 0.06 mM CaCl2) was purchased from PromoCell (Heidelberg, Germany). Tenascin C monoclonal antibody (EB2, cat. ab88280) was purchased from Abcam (Cambridge, UK). Filaggrin monoclonal antibody (FLG/1562, cat. NBP2-53245-20 μg) was purchased from Novus Biologicals (Littleton, CO, USA). Glutaraldehyde and osmium tetroxide solution were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Gelatin capsules were purchased from Parke Davis (Detroit, MI, USA). EPON resin mixture was purchased from Agar Scientific (Stansted, UK). Lead citrate was purchased from Ferak Berlin GmbH (Berlin, Germany).
The pigments used in this study were Irgalite Orange D2895 (P.O.13, C.I. 21110, 4,4′-[(3,3′-dichloro[1,1′-biphenyl]-4,4′-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one)]) provided from BASF (Ludwigshafen, Germany), TiO2 nanopowder rutile (high purity, 99.9%, 30 nm, stock #: US3520) and anatase (high purity, 99.98%, 30 nm, stock #: US3498) purchased both from US Research Nanomaterials Inc. (Houston, TX, USA). ENSACO® 250P (carbon black, C.I. 77266, polycyclic aromatic hydrocarbons content below 1 ppm) was provided from Imerys Graphite & Carbon (Bironico, Switzerland).
Isolation of primary human skin cells from neonatal foreskin
Normal human keratinocytes (NHK) and NHDF were isolated from neonatal foreskin derived from therapeutically indicated circumcisions after written informed consent of the legal guardian (ethical approval EA2/104/18, Charité Berlin and ethical approval Eth-17/19, Berlin Chamber of Physicians).
Human foreskin tissue was transported in DPBS at 4 °C to the BfR. The foreskin tissue was then washed with DPBS. Vascular, mucosal and adipose tissue was removed using scalpels and/or scissors. Afterwards, the foreskin tissue was stretched on autoclaved metal gaze, the dermal side facing toward the metal gaze, and placed in a 6 cm dish containing dispase solution with the epidermis faced downwards and incubated overnight at 4 °C.
The next day, the epidermis was removed using forceps and incubated in trypsin/EDTA solution for 20 min at 37 °C to isolate NHK. Trypsin reaction was stopped using trypsin inhibitor (1 mg/ml) in DPBS. NHK were then cultured in keratinocyte growth medium 2.
To obtain NHDF, residual dermal tissue was cut into 3 mm pieces and put face down into 6 cm dishes containing minimal medium (DMEM/F12 without phenol red supplemented with 10% FBS, 1% penicillin/streptomycin and 0.1% GA-1000). NHDF were allowed to grow out of the tissue for 1 week. Both NHK and NHDF were passaged once followed by cryopreservation in DMEM/F12 with 10% FBS and 10% DMSO (passage = 2) until usage.
Isolated cells and dermal pieces in minimal medium were cultured at 37 °C, 5% CO2 atmosphere and 90% relative humidity.
Production of TatS
To produce tattooed reconstructed human skin equivalents (TatS) with and without tattoo pigments, we modified an existing protocol described elsewhere (Zoschke et al. 2016). We included tattoo pigments into the buffer used to create the dermal part of TatS. Several concentrations of pigments in the dermis were tested, starting with 0.4 mg/ml per pigment and decreased, if dermis formation was prevented, as seen by inhibited dermis shrinking. In detail, tattoo pigments were suspended in tenfold concentrated dermis buffer (50% 10 × DMEM, 25% NaOH in H2O, 25% HEPES). The pigment suspensions were then vortexed rigorously and ultra-sonicated for 25 min in a Sonopuls HD 2200 from Bandelin electronic (Berlin, Germany). Afterwards, 3 × 105 NHDF were embedded in a collagen matrix (80% collagen G solution, 10% dermis buffer with suspended tattoo pigments and 10% MM). 900 µl of this mixture was poured into a THINCERT trans-well placed in a THINCERT 12-deep-well-plate (both from Greiner Bio-One International GmbH, Kremsmünster, Austria) on top of the cell-free collagen and incubated for 45 min in the cell CO2 incubator. Subsequently, additional 250 µl of pigment-free collagen matrix with NHDF were given on top of the pigmented layer, forming a second pigment-free layer. After another incubation for 2 h, 500 µl dermal growth medium (minimal medium supplemented with 40 µM adenine hydrochloride monohydrate, 30 µg/l amphotericin B, 0.1 nM cholera toxin, 10 µg/l EGF, 3.5 mg/l hydrocortisone, 4.4 mg/l insulin, 0.5% non-essential amino acids, 4.4 mg/l transferrin, and 2 nM triiodothyronine) were given on top of the dermal layers and 4 ml into the deep well. Medium was changed twice before seeding the keratinocytes.
One week after construction of the dermal layers, medium in the trans-well was aspirated. 1 × 106 NHK (passage = 3) was poured onto each dermal layer. On the consecutive day, an airlift was performed: the remaining medium in the trans-wells was aspirated and medium in the wells was changed. Dermal growth medium was replaced by epidermal growth medium (dermal growth medium supplemented with 0.25 mM ascorbic acid and 1.8 mM calcium chloride). Epidermal growth medium was changed three times a week. TatS were incubated for two additional weeks until full differentiation of the epidermal layer.
Histological characterization of TatS
To compare general histological features and especially epidermal differentiation, we used haematoxylin–eosin (H&E) stain and immunofluorescence staining. At the end of culture, TatS were snap-frozen in Tissue-Tek cryomolds containing Tissue-Tek O.C.T. compound using liquid nitrogen. Consecutively, the embedded tissue sections were packed with laboratory film and aluminum foil and stored at − 80 °C until sectioning.
Cryopreserved TatS were sectioned using the cryostat Microm HM 550 (Thermo Fisher Scientific, Waltham, MA, USA) with a thickness of 5–7 µm and mounted on SuperFrost slides (Thermo Fisher Scientific). In the cryo block, each TatS was oriented vertically toward the microtome knife to prevent translocation of the pigments to other skin layers due to sectioning and in a way that a depth profile from stratum corneum to dermis is given. Mounted sections were stored at − 80 °C till H&E or immunofluorescence staining.
For immunofluorescence staining, the slides were fixed in 100% ice-cold methanol. Methanol was allowed to vaporize for 5 min at room temperature (RT) and the slides were consecutively washed and rehydrated using DPBS. TatS sections were then permeabilized using 0.5% Triton X-100 in DPBS for 15 min. Afterwards, the slides were washed again two times with DPBS and then blocked with the blocking solution containing 5% FBS and 0.1% Triton X-100 in DPBS (PBST) for 30 min at RT. Afterwards, the respective primary antibody solution was poured onto the slides and incubated in a dark chamber at 4 °C over night. TatS sections were stained with the following primary antibodies: E-cadherin monoclonal antibody (5 µg/ml), occludin recombinant polyclonal antibody (5 µg/ml) and filaggrin monoclonal antibody (4 µg/ml) to visualize epidermal development; collagen IV monoclonal antibody (5 µg/ml) and tenascin C (0.1 µg/ml) to visualize basal membrane development. After staining with primary antibodies, slides were washed two times with PBST followed by 45 min incubation with the correspondent secondary antibody (1:400 in PBST). For TatS sections with incorporated P.O.13 pigments, secondary antibodies coupled with alexa flour 488 instead of 546 were used to avoid interferences with the auto-fluorescence of P.O.13. Afterwards, slides were washed two times with PBST and once with DPBS for 5 min each. Sections were then incubated for 15 min with Hoechst 33258 (1 µg/ml in DPBS) and then washed again two times with DPBS. Residual DPBS was carefully removed. Slides were then covered with ProLong diamond antifade mountant and analyzed using an LSM700 confocal microscope from Carl Zeiss (Oberkochen, Germany). Negative controls were processed omitting the primary antibody.
Transmission electron microscopy
To analyze the shape and primary particle size of tattoo pigments and to investigate the possible uptake of the pigments by fibroblasts, we performed transmission electron microscopy (TEM).
At the end of culture, TatS were sectioned into 2 × 4 mm cuboids containing only the dermal layer with incorporated pigments. The cuboids were then placed in a 1.5 ml reaction tube and fixed with glutaraldehyde for 24 h. Following fixation, the cuboids were transferred into gelatin capsules and washed three times with HEPES buffer (0.2 M HEPES, pH 7.2). The cuboids were then incubated for 2 h in osmium tetroxide solution, followed by a 30 min wash in HEPES buffer. The blocks were then contrasted with uranyl acetate (0.6% w/v in H2O). Following dehydration using increasing amounts of ethanol, the blocks were transferred into a mixture of 96% ethanol, 4% propylene oxide for 30 min followed by 1 h in propylene oxide–Epon resin mixture. The solution was then removed, fresh Epon resin added to the gelatin capsule, and polymerized for 24 h each at 30 ºC, 40 ºC and 60 ºC. The blocks were cut into 70–80 nm ultrathin sections and transferred onto 200 mesh copper grids (Plano GmbH, Wetzlar, Germany). The sections on copper grids were further stained with lead citrate to achieve dual contrast.
Grids were then observed in a Jeol 1400plus TEM (Jeol, Japan) operated at 120 kV. Imaging was performed using a Veleta G2 camera and iTEM software (both from Olympus Europa SE & Co. KG, Hamburg, Germany).
Treatment of NHDF with tattoo pigments
NHDF were seeded in a 24-well plate at a concentration of 5 × 104 cells/well and treated with tattoo pigments at concentrations comparable to those used in TatS in terms of the amount of pigments per cell (NHDF). Pigments per cell ratio in TatS and the 2D experiment were 1.33 ng per cell for TiO2 anatase and TiO2 rutile, 0.67 ng per cell for P.O.13, and 0.067 ng per cell for carbon black, respectively. Final concentrations used to treat NHDF with pigments were 66 µg/ml for TiO2 anatase and TiO2 rutile, 33 µg/ml for P.O.13 and 3.3 µg/ml for carbon black.
Tattoo pigment suspensions in minimal medium were prepared as twofold concentrated with ultra-sonication as described above. In case of P.O.13, minimal medium with 0.01% Triton X-100 was used for the stock suspension (with a final Triton X-100 concentration below 0.001%). NHDF suspensions at a concentration of 1 × 105 cells/ml and pigment suspension were mixed 1:1 and then seeded in a 24-well plate.
PrestoBlue viability assay
Viability of TatS and NHDF was assessed using the PrestoBlue assay. PrestoBlue assay is a resazurin-based non-disruptive cell viability assay that bears the advantage to use TatS after assessment of viability for other purposes, like immunofluorescence (Young and Reed 2016). Three TatS with pooled cells from three donors were used. For NHDF, each replicate was done with cells from a different donor.
In case of TatS, 500 µl PrestoBlue solution (10% in MM) was put on TatS and incubated at 37 °C for 1.5 h. In case of NHDF, 300 µl of a 10% PrestoBlue solution in fibroblast growth medium was put on NHDF and incubated at 37 °C for 2 h. Afterwards, fluorescence in supernatants was measured using a Synergy HT-reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.
To analyze cytokine release from TatS and NHDF, we quantified G-CSF, GM-CSF, IL-1α, IL-6, IL-8, IL-18 and TGF-α using a custom LEGENDplex Multi-Analyte Flow Assay Kit (Biolegend, San Diego, CA, USA). TatS made of cells from different donors were used for each replicate.
Briefly, NHDF were treated with tattoo pigments as described above and cultured overnight for 16 h. Medium was changed and samples for cytokine quantifications were taken 8 h after. To reach comparable conditions for cytokine detection between TatS and NHDF, 666 µl medium was used, resulting in comparable amounts of medium per NHDF.
For TatS, samples were taken at the end of the culture period at day 21. At day 19, medium was changed from keratinocyte growth medium to minimal medium. Medium was then changed once more before cytokine analysis. At day 20, medium was changed again, and samples were taken 8 h afterwards.
150 µl of medium was transferred into a 1.5 ml reaction tube and centrifuged with 16,100×g for 10 min at 4 °C in a 5415 R micro-centrifuge (Eppendorf, Hamburg, Germany) with the rotor FA-45-24-11 to get rid of tattoo pigments and cell debris. The supernatant was then stored at − 80 °C until the bead-based cytokine assay was performed according to the manufacturer’s instructions. The flow cytometer BD FACSARIA III (Becton Dickinson, Franklin Lakes, NJ, USA) was used to read the beads. Blanks and standard curves were included in each experiment. Three independent experiments were carried out for TatS and 2D cells, respectively.
Microscopy image processing
Microscopic images were processed using ZEN Black 2012 software (Carl Zeiss, Oberkochen, Germany). Pairwise stitching of H&E pictures was performed in Fiji (Schindelin et al. 2012) as described elsewhere (Preibisch et al. 2009).
Data processing was performed with Microsoft Excel (Microsoft, Redmond, WA, USA). Data analysis and illustration were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Presto blue and cytokine data were normalized by dividing each value by the value of the respective pigment-free control of the same donor. Data were assumed to be normally distributed and statistically analyzed by one-way ANOVA with Dunnett's test. A value of p ≤ 0.05 was accepted as statistically significant. No experimental data were excluded from statistical analysis.