# Applications of Tumor Cells in an _In Vitro_ 3D Environment
## 3 Scaffolds and Matrices  
There are several commonly used methods for 3D cultures, including scaffold-free spheroid formation, such as the hanging drop method, non-adherent surface culture, suspension culture, and nanoimprinted scaffolds [9; 16; 17]. Scaffold-free 3D cell cultures are preferred when high numbers of spheroids are needed. Hanging drop plates allow the formation of spheroids via self-aggregation by relying on gravity. The spheroids hang in open, bottomless wells in order to regulate the environmental humidity of the cells. Hanging drop plate methods have a wide range of uses due to their replicability [2]. The hanging drop method can be easily applied to a wide range of cell lines, and the efficiency of spheroid formation relies on the inherent ability of the cells to self-aggregate. Indeed, the advantages of scaffold-free 3D cell cultures lie in the simplicity of the models, the reproducibility of results, the ease of preparation, scalability, and applicability to various cell lines [18]. The scaffolds used for 3D cultures are based on either natural or synthetic hydrogels, or hybrids of both, and are of animal or plant origin. The particular type is chosen depending on the biocompatibility advantage or the physicochemical nature, respectively [9]. Natural gels (natural polymers) are, for example, fibrinogen, hyaluronic acid (HA), collagen, Matrigel(Corning, Corning, NY, USA), gelatin, chitosan, and alginate [19; 20], whereas most synthetic hydrogels are synthesized by modification of synthetic polymers, which exhibit versatile biophysical, mechanical, and biological properties [9]. Polymer scaffolding offers a favorable 3D setting for cellular performance. Collagen-based hydrogels are the most often used scaffolding models since collagen is the major constituent of the basement membrane [9]. Hydrogels are cross-linked networks formed of hydrophilic polymers attached through physical, electrostatic, or covalent interactions. Indeed, hydrogels are an attractive scaffolding material because of their mechanical properties that can mimic those of natural tissues [19]. Collagen-based hydrogels are expensive, difficult to prepare reproducibly, and also require extensive handling and specific equipment [9]. Nonetheless, they offer excellent support regarding cellular heterogeneity and spontaneous cell organization. The stiffness of the 3D culture can be controlled by adjusting the hydrogel concentration. This system can also be applied to study the interaction between any type of neoplastic cell, even using more than two different cell types [21]. Cellular adhesion directly to the gel can be favored over suspension within the scaffold by the incorporation of various peptide domains into the hydrogel structure, thus dramatically increasing the tendency for cellular attachment [19]. A successful strategy to mediate cellular attachment is the inclusion of an RGD adhesion peptide sequence (arginine-glycine-asparatic acid). Fibroblasts, endothelial cells, smooth muscle cells, osteoblasts, and chondrocytes are cell types that have been shown to bind to RGD. This way, cellular migration, proliferation, growth, and tissue regeneration were enhanced. Hydrogels are unique because of their ability to mimic the ECM while allowing soluble factors, such as cytokines and growth factors, to migrate through the tissue-like gel. The ECM is rich in growth factors and metabolic precursors [9]. In addition, the ECM plays a basic role in the development and maintenance of epithelial tissues. For example, the ECM of human breast tissue is composed of protein fibril complexes intertwined in a network of glycosaminoglycan (GAG) chains. Natural polymers include the commercially available \"golden standard\" Matrigel(r), which is a mixture of gelatinous proteins derived from the basement membrane isolated from the mouse sarcoma Engelleth-Holm-Swarm [9].