# Applications of Tumor Cells in an _In Vitro_ 3D Environment
## 4 Organoids  
Tissue fragments from the stroma were originally separated, and cells were cultured within 3D gels to form organoids [30; 31]. Today, organoid cultures include techniques that result in self-organizing, self-renewing 3D cultures derived from a variety of tissues,including primary tissues, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) (reviewed in [30]). ESC- or iPSC organoids make it possible to generate the complex structure of adult organs in which all cells are fully differentiated [14]. They may contain mesenchymal, epithelial, or endothelial components but tend to also constitute other types of cells. Adult stem cell (ASC)-derived organoids can mimic both physiological and pathological adult tissues, including cancer.  
Organoids can be produced from individual patient tumor samples, meaning that the biodiversity is similar to that of _in vivo_ tumors [16]. With more experience and knowledge of the precise culture techniques of organoids, screening platforms for drug discovery that are more cost-effective than animal models will be developed. Today, transcriptome profiling is one of the most common downstream applications of organoids, together with applications in drug discovery and precision therapy [30].  
The selection of appropriate media, growth factors, and morphogens is also fundamental to preserving the functional characteristics of cells and obtaining the desired 3D construct [23]. Many different technologies have promoted the development of specific 3D models applicable in various biomedical and medical fields. According to [PERSON] et al., the definition of organoids can be summarized in the following four points: (i) two or more interacting cell types; (ii) 3D multicellular structure; (iii) self-organization of multiple distinct cell phenotypes into specific supracellular structures found in organs; and (iv) functional properties resembling the corresponding native tissue [23]. Moreover, self-organization is a key feature of organoids due to multicellular structures with similar architecture as the _in vivo_ organs evolving from stem and progenitor cells [23; 32].  
[PERSON] et al. discussed the challenges of 3D cell cultures versus _in vivo_ tumors in a recent review [33]. The spheroids are designed to more accurately reproduce the gradients of nutrients and oxygenation found in the tumor microenvironment. The added complexity also requires consideration of the experimental design as well as monitoring and acquisition. For example, some reagents will reach the center of a larger 3D cell culture, whereas for others, lytic assays disrupting the cells within these structures are necessary [33].  
Imaging spheroids is challenging due to light scattering, and fluorescent imaging of fixed spheroids is limited to the outer layers of cells. Live imaging requires compatible _in vitro_ culture chambers attached to the microscope. A solution for imaging 3D cultures could be using digital holographic (DH) microscopy, a convenient noninvasive method to analyze living cells [34; 35; 36; 37]. With this technique, _in vitro_ cell cultures can be imaged in 2D and pseudo-3D, and measurements of the size and morphology of the cells will be provided. Recently, novel methodologies utilizing DH microscopy for the analysis of spheroids were presented [34; 38]. In addition, light-sheet-based fluorescence microscopes can be used to study spheroid development over longer time scales [33].  
Three-dimensional cell culture systems and organoids both provide excellent artificial microenvironments in which cells grow, differentiate, and interact with each other and with the encapsulating biomaterial in 3D, mimicking the _in vivo_ microenvironment.