**1. Introduction**

The growth of cells in a controlled artificial environment isolated from their natural habitat is referred to as cell culture [1]. It is a significant tool used widely to study cell and molecular biology, screening drugs and toxicity analysis, the role of a particular gene in a disease, and cancer research. Due to their unique properties, they also have been tuned for screening and developing biopharmaceutical compounds such as vaccines and recombinant proteins. One of the major advantages of using cell culture is the homogenous and reproducible data generated [2].

Drug discovery is a lengthy and time-consuming process that undergoes several stages of testing and optimization. This encompasses identification of the target, lead discovery, pre-clinical validation, and clinical trials [3]. Therefore, it is very pertinent to obtain information about the biological activity, biochemical mechanisms, toxicity, and off-target interactions of drug molecules leading to the early stages of drug discovery. *In vitro*, cell-based assays prove futile to understand the effects of drugs on the cells at an early stage of drug discovery which attributes an increased chance of development of drugs with good efficacy and safety [4].

Two-dimensional (2D) cell culture was introduced many decades ago that has been the major type of cell culture technique in numerous fields. This traditional approach has been extensively used for drug screening due to its relatively inexpensive feature and convenience to use. However, the issue of mimicking the *in vivo* environment restricts its use [5]. The 2D cell cultures grow as a monolayer in controlled flat environments, such as a glass or polystyrene flask that comprises live proliferating cells because of the detachment of dead cells from the surface (**Figure 1**). As a result, this leads to uniformity in nutrients and growth factors present in the medium to which the cells get access and proliferate at a faster rate than they would *in vivo* [6]. Thus, the morphology of the cells is completely changed as they appear flattered and stretched as compared to the *in vivo* environment. Besides this, the cell–cell interactions and cell-extracellular interactions become different in comparison to the tumor [7].

Recently there has been an upsurge of interest towards three-dimensional (3D) cell culture in biomedical research and drug development processes due to its highthroughput accuracy and refined *in vitro* models [8]. They have been broadly used in understanding the cell shape, cell–cell interaction, and the cellular environment that efficiently mimics the *in vivo* environment. 3D cultures grow as clusters or aggregates called spheroids either with a matrix or without a matrix [9] (**Figure 1**). There is a gradient of nutrients across these spheroids due to which the cells at the surface of spheroids proliferate more as compared to the cells that are present in the interior [10]. As a result of the difference in the proliferation rate, the cells in the spheroids are usually in different stages of the cell cycle such as proliferating, quiescent, hypoxic,

**Figure 1.** *Simplified sketch of 2D cell culture (a) and 3D cell culture (b).*

#### *Two-Dimensional and Three-Dimensional Cell Culture and Their Applications DOI: http://dx.doi.org/10.5772/intechopen.100382*

and necrotic cells. In the 3D culture, the cells have uniform access to nutrients as in the case of a tumor. Also, the shape of the cells, cell–cell interactions, and cell-environment interactions are well defined in 3D culture [7]. It has also been observed that 2D cell cultures are more sensitive to drugs as compared to 3D cells [11]. This chapter intends to provide the true meaning and significance of both cultures. It starts by making a clear distinction between the two and proceeds further to discuss their different applications *in vitro*. The significance of 2D culture is projected through different assays and therapeutic treatment to understand cell motility and treatment of diseases, while 3D culture includes different models and spheroid structures consisting of multiple layers of cells, and puts a light on its use in drug discovery and development. The chapter is concluded with a detailed account of the production of therapeutic proteins by the use of cells.
