# Applications of Tumor Cells in an _In Vitro_ 3D Environment
## 7 Applications of 3D Models of Cancer Cells
### ALI Systems of the Skin  
Historically, the ALI technique was primarily used to mimic respiratory tract epithelia _in vitro_[23]. The first ALI method was developed in the 1980s, which introduced a model of epidermal and respiratory tract epithelial tissues _in vitro_. Since then, routine approaches to growing fully differentiated _in vitro_ skin models have been established, even for clinical applications of wound healing [85; 86]. [PERSON] et al. illustrated this point clearly by elegantly reviewing different ALI systems, including skin [87]. To establish and propagate cells in an ALI culture, epithelial cells are seeded in compartmentalized systems on porous filter supports under submerged conditions for 3-7 days [87]. After an initial attachment, proliferation phase, and formation of a confluent monolayer, the apical medium of the epithelial cells is removed. The cells \"interface\" with the surrounding air, differentiating and generating an apical microenvironment [87]. Since the basal surface of the cells has access to the culture medium, its nutrients and other additives can nourish cells via diffusion through the porous membrane. The full differentiation of the _in vitro_ structure is completed in approximately 2 weeks [88; 89] (Figure 1A). The different barrier functions of human skin and their role in maintaining tissue homeostasis are important to understand when designing 3D reconstructed skin models [90; 91]. The epidermis is the outer compartment of the skin, and it contains keratinocytes, which comprise around 95% of all the cells in the epidermis [92]. These cells are essential for epidermis development as they differentiate and migrate upward to create four strata that facilitate skin barrier function [93; 94]. The _stratum basale_ is the bottom layer of the epidermis and is responsible for constantly renewing the cells of the epidermis. This layer contains just one row of undifferentiated epidermal cells, known as basal keratinocytes, that divide frequently [95]. The skin protects against chemical exposure, ultraviolet (UV) radiation, and penetration of pathogens and also regulates the diffusion of molecules. In cases of any barrier defect, Langerhans cells, keratinocytes, and skin-resident immune cells provide a secondary immunological barrier [91; 95]. In both the epidermis and the dermis, cells and molecules from the immune system are present to protect against pathogens and induce an immune response. Langerhans cells are a type of dendritic cell in the epidermal layer of the skin [95]. Those cells, which are the main skin-resident immune cells, patrol and protect the epidermis together with T cells. Keratinocytes are also producers of cytokines and chemokines, contributing to the immune defense. There are twice as many T cells present in the skin compared to in the bloodstream, indicating the importance of the skin as an immunological reservoir [91]. The epidermis also harbors the melanocytes, which produce the pigment melanin [95]. Examples of _in vitro_ 3D models to study different aspects of the skin and its function in a controlled laboratory environment are shown in Figure 1.  
Melanoma is the most aggressive form of skin cancer and is caused by a malignant transformation of melanocytes [96]. While melanoma has a positive outlook if it does not metastasize, it is associated with the majority of skin-cancer-related deaths. However, stage IV malignant melanoma carries a worse prognosis [97]. Multicellular tumor spheroids (MCTSs) are composed of tumor cells forming sphere-like 3D structures. The MCTS structures of melanoma cells can feature _in vivo_-like cell-cell adhesion, barriers to mass transport, extracellular matrix deposition, as well as cell-matrix adhesion [40]. The development of engineered human skin equivalents (hSEs) has recently progressed significantly and serves as an important link between animal models, conventional 2D cell culture systems, and human skin biopsies [98; 99; 100]. Figure 1: _In vitro_ 3D models of skin. (**A**) Epidermis model.