# Applications of Tumor Cells in an _In Vitro_ 3D Environment
## 7 Applications of 3D Models of Cancer Cells
### ALI Systems of the Skin
A thin layer of keratinocytes is grown on a porous filter support. After culture at the air–liquid interface (ALI), the keratinocytes are arranged in a multi–layered structure, with the outermost layer consisting of flattened cells resembling the stratum corneum. Below this layer, several layers of keratinocytes are present, forming the stratum granulosum, stratum spinosum, and stratum basale, mimicking the epidermis layer of the skin. (**B**) Epidermis/dermis model (full–thickness model). The top layer is the epidermis, similar to (**A**), and the bottom layer is the dermis. The dermis layer consists of fibroblasts cultured in collagen or a polymeric scaffold for support. (**C**) A full–thickness model with melanocytes and immune cells. In addition to the keratinocytes and fibroblasts, melanocytes and/or immune cells can also be added to the epidermal layer. These cells are important for studying the immune response of the skin and how it reacts to different stimuli. Created with BioRender.com. Moreover, more and more companies are commercializing skin analogues composed of fibroblasts and keratinocytes that replicate the skin anatomy to some extent. Phenion(r) (Dusseldorf, Germany) FT, StrataTest(r) (Stratatech, Madison, WI, USA), VUmc skin equivalent, EpidermFT(tm) (MatTek Life Sciences, Ashland, MA, USA), and organotypic skin cultures (ORGs) are a few examples of reconstructed bi-layered hSEs that are commercially available to date [101; 102; 103; 104; 105]. Those models are a quick, direct, and cheaper alternative to animal testing since they are considered safe and provide a human system, although _in vitro_[100]. Further advantages with hSE systems are that cellular components can be added and evaluated to better mimic human skin. Currently, two main types of engineered 3D tissue models exist, viz., scaffold-free and scaffold-based models [98]. The division dictates how the model's dermal part is reconstructed, and it has far-reaching consequences that determine further advantages and disadvantages of the models [92; 93]. Hence, scaffold-free 3D skin models are based on the idea of stimulating native ECM production, which is advantageous because of the low cost and high reproducibility owing to the batch-to-batch independence of the applied scaffold. Moreover, such models are instrumental in high-throughput cellular function and cytotoxicity studies or biochemical analyses. However, a full-thickness stratified epidermis is required to obtain proper tissue architecture. Scaffold models of 3D tissues are cells grown in the presence of support scaffolds, either hydrogel-based or polymeric fiber-based support [98]. Such scaffold-based models exhibit superior biocompatibility and cell adhesion but are limited by their short lifespan and weakened mechanical strength. In addition, 3D hSEs can also be assembled using de-epidermized dermis (DED), where the donated human samples undergo the process of de-cellularization. This allows for the creation of a dermal substitute that is rich in ECM scaffolds, facilitating keratinocyte attachment and proliferation [93].