*2.3. Research Applications of Hydrogels*

Research applications of hydrogels are varied in vitro. They are often used as scaffolds in 3D cultures for the study of cellular physiology and characteristics, as they interact with tissue-mimetic hydrogels. While 2D culturing methods have provided valuable information on cellular characteristics, recent studies have undertaken a shift toward preference for 3D-culturing methods for more advanced and sensitive studies that imitate native tissues and cells more closely [126,197]. These 3D platforms provide more physiologically relevant information on cell-environment interactions biochemically and mechanically, including: morphology, cell and environmental stiffness, motility, and signaling [117]. Some 3D platforms that have been used for cell culture include microporous and nanofibrous scaffolds encapsulating cells. However, these either have pore sizes that are too large, which negate the 3D structure and act as a 2D scaffold, or are too weak for mechanical studies [198,199]. Hydrogels are useful as mechanically and biochemically tunable matrices for 3D cultures, which simulate soft tissue structure and have potential for translation and clinical applications [184]. For instance, Jiang et al. found that the formation of a hybrid hydrogel with ultralong hydroxyapatite nanowires and sodium alginate allows for improved mechanical properties of the hydrogels and enhanced biocompatibility for in vitro studies [200].

Drug screening or efficacy assays are prone to showing promising effects in vitro, only to fail or be substantially less effective in animal models and clinical trials [201]. In the search for in vitro cell-culture systems that can provide more accurate and relevant results, 3D scaffolds such as hydrogels have been gaining attention, in part due to their potential to mimic the ECM and inhibit drug delivery [119]. Huber et al. compared the response of 2D cultures of non-small cell lung cancer cultured to 3D microtissues of the same cell line to various drugs and found that drug efficacy was significantly different between the models [127]. Singh et al. developed a hydrogel microarray assay to generate uniform microtumors and subsequently study tumor response to epidermal growth factor (EGF) and cetuximab treatments [202]. Such systems increase the chances of success for translation because they enable studies of treatment response more closely aligned to clinical response.
