Anatomically, skin is the largest organ in the human body and is made up of three main layers and possesses a complex system of stromal, vascular, glandular, and immune/nervous system components in addition to epidermal cells. The epidermis itself is composed of four layers of cells that are continuously renewed to maintain barrier function and other structures of native skin.Skin physiology is important in immune function, wound healing, cancer biology, and other fields, leading researchers to use a wide range of models, from in vitro monocultures to in vivo animal models. Animal models offer the ability to study the full complexity of skin physiology, however, commonly used animal models such as mice have significant physiological differences when compared to humans. These limitations, and the increased cost of animal models, have led many researchers to focus on developing in vitro models that more closely reflect the physiology of human skin. Of these, one of the simpler model types is the human epidermal equivalent which are composed of only epidermal keratinocytes on an acellular dermal matrix, but capture epidermal differentiation and stratification seen in vivo. Building on this, models containing dermal and epidermal components are often referred to as human skin equivalents , full-thickness skin models, or organotypic skin constructs . Briefly,pot with driange holes these models are generated by encapsulating dermal cells within gel matrices and seeding epidermal cells on top.
Epidermal differentiation and stratification can then be achieved via specialized media and air exposure. Skin equivalents have most often been generated through self-assembly techniques using dermal gels made of collagen type I, but similar models have incorporated other matrix components such as fibrin, fibroblast derived, cadaveric de-epidermized membranes, commercially available gels and others. Currently, there are skin equivalents commercially available . However, these are primarily developed for therapeutic purposes and cannot be readily customized to specific research questions. HSEs have been applied in studies of wound healing, grafting, toxicology, and skin disease/developement. Although 3D culture more comprehensively models functions of human tissue compared to 2D cultures, the inclusion of diverse cell types that more accurately reflect the in vivo population enables studies of cell-cell coordination in complex tissues. Most HSEs only include dermal fibroblasts and epidermal keratinocytes, although the in vivo skin environment includes many other cell types. Recent studies have started including more cell populations; these include endothelial cells in vasculature, adipocytes in sub-cutaneous tissue, nerve components, stem cells, immune cells, and other disease/cancer specific models. Particularly important among these is vasculature; while some HSEs include vascular cells, overall they still lack comprehensive capillary elements with connectivity across the entire dermis, extended in vitro stability, and appropriate vessel density.
Further, HSE models are typically assessed through post-culture histological sectioning which limits analysis of the three-dimensional structure of HSEs. Three dimensional analysis allows for volumetric assessment of vascular density as well as regional variation of epidermal thickness and differentiation. Although HSEs are one of the most common organotypic models, there are many technical challenges in generating these constructs including identification of appropriate extracellular matrix and cell densities, media recipes, proper air liquid interface procedures, and post-culture analysis. Further, while HEE and HSE models have published protocols, a detailed protocol incorporating dermal vasculature and volumetric imaging rather than histological analysis does not exist. This work presents an accessible protocol for the culture of vascularized human skin equivalents from mainly commercial cell lines. This protocol is written to be readily customizable, allowing for straight-forward adaptation to different cell types and research needs. In the interest of accessibility, availability, and cost, the use of simple products and generation techniques was prioritized over the use of commercially available products. Further, straightforward volumetric imaging and quantification methods are described that allow for assessment of the three-dimensional structure of the VHSE.
Translating this procedure into a robust and accessible protocol enables non-specialist researchers to apply these important models in personalized medicine, vascularized tissue engineering, graft development, and drug evaluation.Here is presented a protocol for generation of in vitro vascularized human skin equivalents using telomerase reverse transcriptase immortalized keratinocytes , adult human dermal fibroblasts , and human microvascular endothelial cells . Additionally, the customizable nature of this protocol is highlighted by also demonstrating VHSE generation and stability when using commonly available lung fibroblasts instead of hDF. Generation of the VHSE is completed in steps, while steps are optional end point processing and imaging techniques that were optimized for these VHSEs. It is important to note that the VHSEs can be processed according to specific research questions and steps are not required to generate the construct. Volumetric imaging, analysis, and 3D renderings were completed to demonstrate a volumetric analysis method. These volumetric construct preparation and imaging protocols preserve VHSE structure at both the microscopic and macroscopic levels, allowing for comprehensive 3D analysis. Characterization of the epidermis and dermis show appropriate immunofluorescent markers for human skin in the VHSE constructs . Cytokeratin 10 is an early differentiation keratinocyte marker which usually marks all suprabasal layers in skin equivalents . Involucrin and filaggrin are late differentiation markers in keratinocytes and mark the uppermost suprabasal layers in skin equivalents. A far-red fluorescent nuclear dye was used to mark nuclei in both the epidermis and dermis, with Col IV marking the vasculature of the dermis . Epidermal basement membrane components are not always properly expressed in HSE cultures; and Col IV staining of the BM is not consistently observed using this protocol. Research focused BM components and structure would benefit from additional media, cell, and imaging optimization. Though confocal imaging through the bulk of the VHSE cultures often yields high resolution images that are sufficient for computational analysis of the dermis and epidermis, the clearing method described allows for deeper tissue imaging. Clearing improves confocal laser penetration depth, and effective imaging in VHSEs can be achieved to over 1 mm for cleared samples . The described clearing technique sufficiently matches refractive index throughout VHSE sample tissue.
Clearing the VHSE allowed for straightforward imaging through the entire construct without manipulation.Volumetric images allow for generation of 3D rendering to map vasculature throughout each construct . Briefly,large pot with drainage confocal image sets were taken in dermal to epidermal orientation of several sub-volumes of VHSEs to detect Collagen IV stain and nuclei . Image stacks are loaded into computational software and a custom algorithm is used for 3D rendering and quantification as described previously . This algorithm automatically segments the vascular component based on the Col IV stain. The volumetric segmentation is passed to a skeletonization algorithm based on fast marching. Skeletonization finds the definitive center of each Col IV marked vessel and the resulting data can be used to calculate vessel diameter as well as vascular fraction . Wide field fluorescent microscopy is an accessible option if laser scanning microscopy is not available; the vascular network and epidermis can be imaged with wide field fluorescent microscopy . Three-dimensional quantification is possible using wide field imaging of VHSEs rather than laser scanning microscopy, although it may require more filtering and deconvolution of images due to out of-plane light.This protocol has demonstrated a simple and repeatable method for the generation of VHSEs and their three-dimensional analysis. Importantly, this method relies on few specialized techniques or equipment pieces, making it accessible for a range of labs. Further, cell types can be replaced with limited changes in the protocol, allowing researchers to adapt this protocol to their specific needs. Proper collagen gelation is a challenging step in establishing skin culture. Especially when using crude preparations without purification, trace contaminants could influence the gelation process. To help ensure consistency, groups of experiments should be performed with the same collagen stock that will be used for VHSE generation. Further, the gelation should ideally occur at a pH of 7-7.4, and trace contaminants may shift the pH. Before using any collagen stock, a practice acellular gel should be made at the desired concentration and the pH should be measured prior to gelation. Completing this collagen quality check before beginning dermal component seeding will identify the problems with proper gelation and collagen homogeneity prior to setting up a complete experiment. Instead of seeding acellular collagen directly onto a culture insert, seed some collagen onto a pH strip that evaluates the whole pH scale and verify a pH of 7-7.4. Gelation can be evaluated by applying a droplet of the collagen gel solution onto a coverslip or tissue culture plastic well plate . After gelation time, the collagen should be solid, i.e., it should not flow when the plate is tilted. Under phase contrast microscopy, the collagen should look homogeneous and clear. Occasional bubbles from collagen seeding are normal but large amorphous blobs of opaque collagen within the clear gel indicates a problem-likely due to insufficient mixing, wrong pH, and/or failure to keep the collagen chilled during mixing. The cell seeding amounts and media may be adjusted. In the protocol above, the encapsulated cell amounts have been optimized for a 12-well insert at 7.5 x 104 fibroblasts and 7.5 x 105 endothelial cells per mL of collagen with 1.7 x 105 keratinocytes seeded on top of the dermal construct. Cell densities have been optimized for this VHSE protocol based on the preliminary studies and the previous research investigating 3D vascular network generation in various collagen concentrations144 and HSE generation. In similar systems, the published endothelial cell densities are 1.0 x 106 cells/mL collagen; the fibroblast concentrations often range from 0.4 x 105 cells/mL of collagen,175 to 1 x 105 cells/mL of collagen; and the keratinocyte concentrations range from 0.5 x 105 [cells/cm2] 173 to 1 x 105 [cells/cm2].Three-dimensional cultures with contractile cells, such as fibroblasts, can contract leading to viability reduction and culture loss. Preliminary experiments should be completed to test contraction of the dermal compartment and to test epidermal surface coverage. Additionally, the number of days in submersion and the rate of tapering the serum content can also be customized if excessive dermal contraction is occurring or a different rate of keratinocyte coverage is required. For example, if contraction is noticed during the period of dermal submersion or while keratinocytes are establishing a surface monolayer, moving more quickly through the serum tapering process and raising VHSEs to ALI can aid in preventing additional contraction. Similarly, if keratinocyte coverage is not ideal, changing the number of days that the VHSE is submerged without serum may help increase the epidermal monolayer coverage and mitigate the contraction since serum is left out. Changes in cell densities or other suggestions above must be optimized for the specific cultures and research goals. To establish a proper stratification of the epidermis during the air liquid interface period, it is critical to regularly check and maintain fluid levels in each well so that ALI and appropriate hydration of each construct is kept throughout the culture length. Media levels should be checked and tracked daily until consistent ALI levels are established. The epidermal layer should look hydrated, not dry, but there should not be pools of media on the construct. During ALI, the construct will develop an opaque white/yellow color which is normal. The epidermal layer will likely develop unevenly. Commonly, the VHSEs are tilted due to the collagen seeding or dermal contraction. It is also normal to observe a higher epidermal portion in the middle of the construct in smaller constructs and a ridge formation around the perimeter of the VHSE in 12 well size. Contraction of the constructs may change these topographical formations, and/or may not be observed at all. Staining and imaging of VHSEs introduces mechanical manipulation to the VHSEs. It is very important to plan and limit manipulation of each culture. When manipulation is necessary, maintain gentle movements when removing VHSEs from the insert membranes, when adding staining or wash solutions to the construct surface, and when removing and replacing VHSEs in their storage/imaging wells during imaging preparation. Specifically, the apical layers of the epidermal component may be fragile and are at risk of sloughing off the basal epidermal layers. Apical layers of the epidermis are fragile and go through desquamation even in native tissue, but for accurate analysis of epidermal structure it is important to minimize damage or loss. If epidermal layers lift off the construct, they can be imaged separately.