The basal layers of the epidermis are most likely still attached to the dermis while portions of the apical layers may detach. For visualization of the epidermis, a nuclear stain is helpful in observing this since dense nuclei is a characteristic of lower and mid layers of the epidermis. Confocal imaging of the VHSE post-fixation has been discussed in the protocol, but it is also possible to image the VHSEs throughout the culture via upright based optical coherence tomography. VHSE are stable enough to withstand imaging without incubation or humidification for at least two hours without noticeable effects. As OCT is label free and noninvasive, it is possible to track the epidermal thickness during maturation. Other noninvasive imaging modalities can likely be employed as well. Volumetric imaging of the combined dermal and epidermal structures can be challenging due to laser attenuation deeper in the VHSE. This can be mitigated by imaging the construct in two orientations, from the epidermal side and from the dermal side , allowing for good resolution of dermal vascular structures and the epidermis. Additionally, the sample can be cleared, allowing for volumetric images of the entire structure with minimal attenuation. Several clearing methods were attempted, however, the methanol/methyl salicylate method described yielded the best results. Researchers interested in optimizing other clearing methods are directed towards these reviews . If clearing, it is suggested to fully image the sample prior to clearing, as the method can damage the fluorophores and/or the structure. Further,drainage collection pot the imaging should be completed as soon as possible after clearing, as the fluorescence may fade within days.
For simplicity and accessibility, this protocol utilized the simplest media blends found in previous literature. Although there are many advantages to using simple media blends, the limitations of this choice are also recognized. Other groups have studied the effects of specific media components on epidermal and dermal health and found that other media additives, such as external free fatty acids/lipids, enhance the stratum corneum of the epidermis and improve the skin barrier function. Although our immunofluorescent markers show appropriate differentiation and stratification in the epidermis, depending on the studies being conducted, additional media optimization may be needed. Further, an extensive analysis of the epidermal BM was not conducted when evaluating the VHSEs presented here. The integrity of the BM is an important indication of skin equivalents; various groups have done research on the culture duration and its effect on BM markings20 as well as analysis of fibroblast presence and added growth factor effects on BM expression. It is important to note that analysis of the BM component should be evaluated and optimized when using this protocol. In this protocol is described a procedure for VHSE generation, demonstrating results after 8 weeks at ALI. VHSE cultures have been cultured up to 12 weeks at ALI without noticeable change or loss of viability, and it is possible that they may be viable longer. Importantly, this protocol is readily adaptable to commonly available cell types, as demonstrated by the replacement of dermal fibroblasts with IMR90 lung fibroblasts. Depending on the researcher’s need and available resources, the cell types and media blends on the culture can be adjusted, although more dissimilar cell types may require media optimization. In summary, these procedures are meant to provide clarity on the culture of VHSEs for the study of skin biology and disease.
To maximize accessibility, the protocol was developed this simple and robust using common equipment, cell lines, and reagents as a minimal effective approach that can be further customized to the specific needs of research studies.Tissue engineered human skin equivalents , also referred to as organotypic skin cultures or full-thickness models, have been used to study skin development, cytotoxicity, healing, and disease. Typically, they are analyzed post-culture through sectioning and histology. These methods of analysis conceal the three dimensionality of the in vitro constructs and do not allow for real-time assessment of epidermal thickness during the long culture periods that HSE require . Increased availability of volumetric imaging provides an opportunity for HSE research; however, the three-dimensional analysis can be laborious. As an example, optical coherence tomography has been used before to analyze native human skin and a few in vitro skin models non-invasively. OCT and other non-invasive live imaging techniques allow for time-tracking of culture health and progression which is especially important when using HSEs for wounding, development, and aging studies. Further, as a non-invasive technique, OCT avoids disruption of sample morphology, which can occur during histology. Typically, when prior OCT studies that have investigated epidermal thickness in humans and in HSE models, the common thickness calculation is acquired by manually measuring then averaging a few pre-defined OCT line scans , and it is unclear how many OCT images are taken into account. Further, due to this manual selection, there is potential for bias introduction from the investigator. Here we present a technique for automated analysis of epidermal thickness in tissue engineered HSE using live, non-invasive OCT imaging, including optimized OCT imaging procedures and automated image analysis algorithms.
These results were further validated against postculture fluorescent confocal imaging and epidermal thickness quantification.Briefly, to generate the dermal portion of the VHSE, 120 µL of acellular Type I collagen was seeded onto the insert membrane and allowed to gel for 30 minutes. IMR90s and HMEC1 cells were suspended in a Type I collagen matrix at 75,000 and 750,000 cells/mL, respectively. 250 µL of cellular collagen was seeded onto the acellular component and allowed to gel for 30 minutes, then submerged in growth media . 24 h after collagen seeding, media was changed to 5% FBS complete IMR90 media and supplemented with 100 µg/ml Lascorbic acid . The dermal component was cultured for 7 days with the 5% FBS media blend. To establish the epidermis, on day 7 of dermal culture, N/TERT-1s were seeded onto the dermal surface and cultured to confluence for 3 days. On day 8 of culture media was changed to 1% FBS HSE differentiation media48 with a supplement of100 µg/mL L-AA. On day 9 of culture, media was changed to serum free HSE media with 100 µg/mL L-AA, this media blend was used until culture endpoint as maintenance media. Surrounding wells were filled with PBS to promote controlled humidity throughout culture and during OCT imaging. At day 10 of culture, VHSEs were brought to air-liquid interface and cultured with routine media supplemented with L-AA at 100 µg/mL and maintained for 8 weeks. All media and timepoints for media changes are as previously described.Imaging occurred at weeks 2, 3, 4, 5, 7, 8 of the air-liquid interface culture period. To ensure sterility during OCT imaging and prevent optical artifacts caused by the plastic well plate lid, residual media at the air-liquid interface, and condensation, specific procedures were followed. Culture plates were equilibrated at room temperature with the lid off in a sterile bio-safety cabinet for 20 minutes after being removed from the incubator, reducing condensation. Immediately prior to imaging,square plastic pot the constructs were checked for any residual media at the air interface of the VHSE, and it was removed with aspiration if necessary while maintaining media below the culture insert. Even with the removal of visible media from the epidermal surface, the sample remained moist and reflective. To minimize reflective effect of the epidermal surface and well plate lid, the sample arm of the OCT system was tilted at 15°, reducing reflection while maintaining an adequate sample illumination for structural imaging. During OCT imaging, constructs were at room temperature for a maximum of two hours; no loss of sample viability was observed. OCT imaging was conducted with a FC/APC fiber optics based spectral domain optical coherence tomography system centered at 1310 nm. Through SD-OCT, the reference arm remains stationary, and the interference pattern is detected in a spectrally resolved manner on a line scan camera. Light is sent into a circulator and then to an optical splitter that splits the light between the reference and sample arms. The reflected light from each arm travels back through the splitter where it interferes and is redirected by the circulator to the detection arm. Here, the light is collimated, dispersed by a grating, and focused onto a line scan camera. A galvo mirror system in the sample arm is controlled by the image acquisition program and performs raster scanning of the optical beam for 2D and 3D imaging. Example MATLAB code is included in the appendix. Briefly, the OCT algorithm was as follows. First, areas of high reflectance were removed by performing an initial intensity calculation, identifying pixels of outlier intensity, then updating the reference spectrum before recalculating the final intensity.
To reduce speckle noise, a single volume of each HSE was obtained by averaging 10 volume scans, resulting in a single 16-bit 4096 x 512 x 512 voxel volume. From the single volume per sample, custom algorithms were used to detect the skin epidermis by identifying regions of high intensity. To smooth small gaps in the image, a morphological closing with a two-dimensional disk followed by a Gaussian filter was applied to the image volume. To segment the epidermis, hysteresis thresholding was applied with a high threshold of 16000 and a low threshold of 8000 or 4200 . The resulting binary volume was cleaned via area opening to remove small artifacts and a morphological closing with a . The bottom and top surfaces of the binary object were detected. Streaking artifacts, common in OCT imaging, presented as sharp peaks on the surfaces, and were removed via automatic detection of high gradients and local averaging. Volumetric thickness was calculated from the voxel difference between the top and bottom surfaces scaled by the acquisition size . For each volume, 30 A-lines were removed from left and right sides for each frame due to edge effects. Thickness average was calculated from imaging volumes of 3.9 x 2 x 3.9 mm. For confocal, epidermal thickness was calculated from imaging volumes of ~3.3 mm x 370 µm x 250-400 µm . A similar custom algorithm was applied to calculate epidermal thickness from confocal volumes. Briefly, noise was removed via a median filter was applied to each XY-plane and intensities were scaled by linear image adjustment. Background auto-fluorescence was removed using a 20 voxel radius rolling ball filter on each XY-plane and the epidermis was segmented using hysteresis thresholding with values empirically determined for each fluorescent channel . Small artifacts and gaps in the epidermal binary volume were removed through morphological closing and opening with a disk structuring element for epidermal stains and a sphere structuring element for nuclear stain.OCT imaging was performed at ALI week 2, 3, 4, 5, 7, and 8 . Confocal imaging was completed on three of the same samples that were OCT imaged, after fixation and staining . ANOVA followed by Tukey’s HSD post-hoc test was used to test for statistically significant differences between time points; and pairwise comparisons of 8 week confocal and OCT data were completed through two-tailed t-test. Significant differences are represented by a single asterisk.Human skin provides essential physical protection, immune barrier function, and thermal regulation. As humans age, there is a decline of skin function, including loss of barrier function and healing capacity. This correlates with structural changes including decreased vasculature, decreased dermal elasticity and collagen organization, stiffening, lower hydration, reduced dermal and hypodermal volumes. These detrimental effects of natural aging are compounded by extrinsic aging factors such as Ultraviolet A photoaging that occurs with sun exposure. With normal aging, the skin has fine wrinkles, is smooth and pale, and has lower elasticity but with photoaging skin is course, rough, even lower elasticity, and has changes in pigmentation. Particularly, UVA sun exposure mainly damages by generation of reactive oxygen species and effects the dermis and hypodermis but can also interact with epidermal keratinocytes to induce mRNA and protein expression of inflammatory cytokines, including IL-6201. Further, UVA exposure to human skin has demonstrated decreased expression of subcutaneous adipokines such as adiponectin. These effects are harmful since adipokines have been found to benefit wound healing and antiinflammatory skin properties and the hypodermis as a whole contributes to thermal regulation, skin elasticity and regeneration.