2.1.

Primary Cell Culture

Skin keratinocytes and human skin dermal fibroblasts were harvested from split thickness skin grafts (STSGs) obtained from specimens following routine breast reductions and abdominoplasties (Ethical Committee of the Northern General Hospital Trust, Sheffield, United Kingdom, Ref: 06/Q2306/25). Oral keratinocytes and oral fibroblasts were extracted from buccal tissue obtained from consenting patients during routine dental surgeries [Sheffield Research Ethics Committee approval (Ref: 07/H1309/105)]. Cell extraction was performed as previously described in Refs. 13, 14.

Skin and oral keratinocytes were grown in Green's medium consisting of Dulbecco's modified eagles medium (DMEM) and Ham's F12 medium in a 3:1 ratio supplemented with 10% (v/v) foetal calf serum (FCS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 0.625 μg/ml amphotericin B, 6.25 μg/ml adenine, 10 ng/ml epidermal growth factor, 1.36 ng/ml triidothyronine, 5 μg/ml transferrin, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, and 8.5 ng/ml cholera toxin. Dermal and oral fibroblasts were grown in fibroblast culture medium (FCM) consisting of DMEM supplemented with 10% (v/v) foetal calf serum (FCS), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.625 μg/ml amphotericin B. All reagents were purchased from Sigma-Aldrich, United Kingdom, except FCS, which was purchased from Biowest Biosera, United Kingdom. All cells were cultured at 37°C in a 5% CO₂/95% air humidified incubator. Passage 1 to 3 keratinocytes and passage 4 to 9 fibroblasts were used in all experiments.

2.2.

Human Melanoma Cell Culture

Three human metastatic melanoma cell lines were used, HBL, A375-SM, and C8161. The HBL cell line was derived from a lymph node metastasis of a nodular melanoma. Cells were maintained in Ham's F10 medium supplemented with 5% (v/v) FCS, 5% (v/v) newborn calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, plus 100 μg/ml streptomycin. The A375-SM cell line was a kind gift from Professor M. J. Humphries (University of Manchester, United Kingdom). The A375 cell line was established in culture from a lymph node metastasis of a 54-year-old female. The C8161 cell line was derived from an abdominal wall metastasis from a post-menopausal woman with recurrent malignant melanoma and was donated by M. Edwards (University of Glasgow, United Kingdom). Both A375-SM and C8161 cells were cultured in Eagle's modified essential media supplemented with 10% (v/v) FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1.2 μg/ml amphotericin B, 1.5% (v/v) (100 × stock) vitamin concentrate, 1 mM sodium pyruvate, and 10% (v/v) nonessential amino acids.

2.3.

Oral Cancer Cell Lines

The D20 dysplastic cell line was kindly donated by Dr. Keith Hunter (University of Sheffield, United Kingdom).^{15, 16} D20 cells were cultured in Green's medium (as previously described for keratinocytes). The SCC9 cell line was originally isolated from the tongue of a patient with squamous cell carcinoma (SCC) and cultured in a 1:1 ratio of DMEM: Ham's F12, supplemented with 10% FCS, 0.4 μg/ml hydrocortisone, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.625 μg/ml amphotericin B. Cal27 is a squamous cell carcinoma human cell line isolated from the tongue of a patient with squamous cell carcinoma (ATCC, Manassas, Virginia).

2.4.

Production of TE Models

STSGs were obtained during routine plastic surgery breast reduction and abdominoplasty operations or obtained from the Euro-Skin Bank (Netherlands). Glycerol was removed from Euro-Skin by extensive washing in phosphate buffered saline (PBS) and then both the STSG and Euro-Skin were immersed in sterile 1 M sodium chloride for 18 h at 37°C, resulting in an acellular de-epidermized human dermis (DED). The DED was thoroughly washed with PBS and placed into FCM. The DED was cut into squares approximately 2 × 2 cm and the papillary surface was orientated uppermost in Corning Costar 3516 6-well plates (Corning Inc., United Kingdom). Tissue-engineered skin and oral mucosa models were produced using a modified version of the method of Chakrabarty ¹⁷ In brief, 1 × 10⁵ dermal or 5 × 10⁵ oral fibroblasts and 1 × 10⁶ epithelial or 5 × 10⁵ oral keratinocytes were seeded in Green's media into a medical stainless steel ring placed onto the papillary surface of the DED. After 48 h in submerged culture, the constructs were raised to an air-liquid interface and cultured in Green's medium for up to 22 days. For models containing melanoma cells in addition to the 1 × 10⁶ keratinocytes, 5 × 10⁵ melanoma cells were also added to the models. For models of oral carcinoma, 2.5 × 10⁵ oral cancer cells were added to the model instead of 5 × 10⁵ oral keratinocytes.

Media changes were performed every three to four days, or more frequently if the media was visibly depleted, indicated by a color change from red-purple to orange-yellow. For routine histology, constructs were transferred to 10% phosphate-buffered formaldehyde at room temperature. Samples were processed, embedded in paraffin wax, sectioned to a thickness of 4 μm, mounted, and stained using haematoxylin and eosin (H&E).

2.5.

OCT Imaging of TE Models

Imaging of all of the TE models was performed using a SS-OCT system that is based on a Michelson Diagnostics Ltd. swept-source OCT data acquisition system (Michelson Diagnostics, Kent, United Kingdom) coupled to a Thorlabs LSM03 scan lens and in-house fiber interferometer (Thorlabs, Cambridgeshire, United Kingdom) with the models maintained in their standard six-well tissue culture plastic dishes. Imaging was performed through the tissue culture plastic lid to keep the TE models sterile and viable. The SS-OCT system was a Fourier domain OCT device, meaning that the images could be taken faster than with traditional time domain OCT. The light source was a Santec HSL-2000-10 wide sweep-laser with a wavelength of 1305 nm ± 15 nm with a sweep range of 150 nm. The OCT system uses an imaging engine (light source, detectors, PC/DAQ and software) adapted from a Michelson Diagnostics Ltd. EX1301 system. The interferometer and sample arm optics are built in-house using an SMF-28 beam splitter and circulator and a Thorlabs OCT objective and galvo scanners. The lateral resolution, in the plane of best focus, of the OCT system is defined by the Thorlabs LSM03 objective in the sample arm. In air, we measured a modulation depth of 45% using Element 1 of Group 5 in the USAF 1951 test target. For a Gaussian beam, this implies a full width half maximum (FWHM) spot size of around 15 μm. With the culture dish lid inserted, the modulation fell to 30%, implying a degradation of the FWHM spot size to 18 μm. This is most likely due to spherical and higher-order beam aberrations. The axial point spread function FWHM in air was measured to be 6.4 μm, degrading to 7.5 μm with the lid inserted. Mostly, this was due to uncompensated optical dispersion as it could be restored to 6.7 μm by inserting a matching lid into the reference arm (however, this was not done for these experiments). The overall dynamic range of this system was measured at 94 dB using a 10-kHz A-scan rate. The tissue culture plastic lids were left in place to ensure sterility of these experiments and also gently heated to remove any condensation before imaging and as required during imaging. Three TE models of each type were imaged in multiple sites toward the middle of the models. Only representative images are shown.

3.1.

Ability of OCT to Detect Changes in Reconstructed Skin Due to the Presence of Melanoma

Figure 1 shows representative SS-OCT images of the TE skin as it develops over 22 days after being raised to an air-liquid interface. The upper panel shows the controlled TE skin throughout 22 days of culture. The three lower panels show experiments in which melanoma cells were added, imaging the constructs at days 1, 8, 15, and 22. HBL cells are minimally invasive cells and as seen in Fig. 1 appear to cause minor changes to the structure of the epidermis. The A375-SM cells are moderately invasive and appear to cause a marked amount of disruption to the epidermis. The C8161 cells are highly invasive but in Fig. 1 do not appear to affect the reconstructed skin.

Fig. 1

Representative SS-OCT images of reconstructed skin developing over 22 days. HBL melanoma, A375-SM, and C8161 melanoma cells were also added to the reconstructed skin. Changes to the epidermis can be seen from day 8 onward when the HBL and A375-SM cells are in the reconstructed skin. There do not appear to be any differences when the C8161 cells are added.

Figure 2 compares the images obtained from reconstructed skin with and without melanoma after 22 days of culture to the accompanying H&E histology. In the controlled reconstructed skin, the SS-OCT [Fig. 2a] and histology images [Fig. 2b] both show a progressively differentiated epidermis that is attached to the underlying dermis. This also appears to be the case when the HBL cells are added to the reconstructed skin model. When A375-SM cells are added to the reconstructed skin model, there are significant changes to the SS-OCT image [Fig. 2e] and these are mirrored in the histology [Fig. 2f]. There is an additional darker, less scattered layer above the epidermis. The histology suggests that these are lightly attached melanoma cells that have not entered the epidermis. Moving on to C8161 cells, these are highly metastatic and invade into the papillary dermis [as can be seen in the inset to Fig. 2g] that cannot be seen in the OCT image [Fig. 2g].^{18, 19} This invasion of melanoma cells into the dermis does not cause any visible disruption of the epidermal layer.

Fig. 2

(a) Representative SS-OCT image of reconstructed skin and (b) accompanying histology. (c) Representative SS-OCT image of reconstructed skin + HBL melanoma cells and (d) accompanying histology. (e) Representative SS-OCT image of reconstructed skin + A375-SM melanoma cells and (f) accompanying histology. (g) Representative SS-OCT image of reconstructed skin + C8161 melanoma cells and (h) accompanying histology. All images were taken after the models had been at an air-liquid interface for 22 days. All histology stained with H&E. Scale bar = 1 mm.

3.2.

Ability of OCT to Detect Changes in Reconstructed Oral Epithelia Due to the Presence of Oral Squamous Cell Carcinoma Cell Lines

Figure 3 shows the appearance of three oral squamous cell carcinoma (OSCC) models and our TE oral mucosa when imaged using OCT and traditional histological methods. Figure 3a shows a complete epithelium that is confirmed in the accompanying histology [Fig. 3b]. In all four OCT images, the epithelium can be distinguished from the connective tissue component as there is a difference in brightness due to an increase in backscattered light (with the connective tissue appearing brighter). This gives information about the thickness of the epithelium, which is often altered in pre-cancerous lesions (dysplasia). More subtle features and individual cells within the epithelia could not be distinguished using OCT. In Fig. 3c, variations in the levels of brightness can be seen in the epithelium. This could potentially be explained by the accompanying histology [Fig. 3d], where islands of terminally differentiated cells are surrounded by highly proliferative cells. In Fig. 3e, the epithelium in the OCT images is too thin to make out much detail, however, there does appear to be some variation in the brightness of the epithelium. This may again be explained by the islands of terminally differentiated cells in the accompanying histology [Fig. 3f]. In Fig. 3h, the model cultured from Cal27 cells had a very distinct appearance with the epithelium appearing in two layers. The most superficial of these layers was highly keratinized with an irregular surface topology that split on sectioning from a lower layer of more proliferative-looking/less-differentiated epithelial cells. This two-layer epithelium and the irregular surface topology is just visible in Fig. 3g with the two layers of the epithelium showing slightly different brightness intensities; again, the less-differentiated cells appearing darker (less backscattering) than the more-differentiated cells.

Fig. 3

(a) Representative SS-OCT image of TE oral mucosa and (b) accompanying histology. (c) Representative SS-OCT image of D20 model of invasive carcinoma and (d) accompanying histology. (e) Representative SS-OCT image of SCC9 model of severe dysplasia and (f) accompanying histology. (g) Representative SS-OCT image of Cal27 model of invasive carcinoma and (h) accompanying histology. All images were taken after the models had been at an air-liquid interface for 14 days. All histology stained with H&E. Scale bar = 1 mm.

3.3.

Ability of OCT to Image Skin and Oral Mucosa In Vivo

Figure 4 shows OCT images of a human fingertip [Fig. 4a] and the mucosal surface of the lower lip [Fig. 4c] obtained in vivo from a healthy volunteer, along with corresponding histology of human skin [Fig. 4b] and oral mucosa [Fig. 4d]. This was imaged using the SS-OCT system. In these images, features within the connective tissues can be distinguished including blood vessels and minor sweat and salivary glands. These features were more clearly imaged in the lip than in the skin. The OCT was able to obtain information over 500 μm deep in both the oral mucosa and skin, highlighting its potential to clinically diagnose both epithelial disorders, such as OSCC and SCC, but also sub-mucosal conditions such as Sjögren's syndrome, which affects the salivary glands.

Fig. 4

(a) SS-OCT image of the skin of the finger tip in vivo. (b) Example histology of human skin. (c) SS-OCT image of the lower lip imaged in vivo. (d) Example histology of human oral mucosa. All histology stained with H&E. Scale bar = 1 mm.