Spheroids as 3D Cell Models for Testing of Drugs

The process of cell culture was developed in 1907 by Harrison while investigating the origin of nerve fibers. This method allowed continuous observation of growth and differentiation of tissues and demonstrated an environment in which the desired cells could be maintained outside the body of origin and observed over time (Breslin and O’Driscoll, 2013). Currently, experiments can be performed using isolated primary cell banks from donor material or established cell-stored culture banks that offer characterized models of different types of cell lines that are commonly used in research (Kapałczyńska et al., 2018). Cell cultures make it possible to understand cell biology, tissue morphology, disease mechanisms, action of drugs, protein production, and tissue engineering development. Selection of the most suitable methods of cell culture in disease and tumor research can allow us to better understand the biology of tumors, thereby optimizing radiotherapy and chemotherapy or even finding new therapeutic approaches and strategies (Aggarwal et al., 2009). Cells can be cultured under adherent conditions when they are attached to glass, plastic bowl, or in suspension. A common type of cell culture used is a two-dimensional (2D) model, and in recent years, the 3D cultivation method has started to be used (Pampaloni et al., 2007).

Conventional 2D cell culture relies on cell adhesion to a flat surface of Petri dishes made of glass or polystyrene to provide mechanical support to the cells (Breslin and O’Driscoll, 2013). 2D cultured cells do not mimic natural tissue structures or tumors. Cell–cell and cell–extracellular cell interactions found in a tumor mass are absent in this culture method. These interactions are responsible for cell differentiation, proliferation, vitality, expression of protein genes, sensitivity to stimuli, drug metabolism, and other cellular functions (Baker and Chen, 2012). After isolation from the tissue and transition to 2D conditions, the cell morphology as well as the mode of dividing cells are changed. Loss of distinct phenotype also results from 2D cultivation. Cells cultured in 2D cultures are typically flatter and stretched than would appear in vivo. Changed morphology of cells can affect their function, including proliferation, differentiation, apoptosis, gene and protein expression, organization of intracellular structures, secretion, and cell signaling. As a result of disruption of interaction with the external environment, growing cells lose their polarity, which changes the response of these cells to various phenomena, for example, apoptosis (Kapałczyńska et al., 2018). Another disadvantage of 2D culture is that the cells are in a monolayer and they have unlimited access to media such as oxygen, nutrition, metabolites, signal molecules, and growth factors (Pampaloni et al., 2007). The monolayer mainly consists of proliferating cells because necrotic cells are usually separated from the surface and easily removed during media exchange. Furthermore, the 2D system was observed to alter the gene expression and splicing, topology, and cellular biochemistry (Edmondson et al., 2014). For more than a century, 2D cell cultures were used as in vitro models for studying cellular responses to biophysical and biochemical stimuli (Duval et al., 2017). Due to the many disadvantages of 2D systems, it was necessary to find alternative models. The shift from cell monolayers to three-dimensional (3D) cultures was motivated by the need to obtain a cellular model that mimics the functions of living tissues that naturally occur in 3D (Amelian et al., 2017). As a potential link between monolayer cultures and studies in animal models, 3D cell cultures have been proposed (Edmondson et al., 2014).

To improve the physiological equivalence of in vitro experiments, a 3D cell culture was developed. It is a culture of living cells inside micro-assembled devices with a 3D structure mimicking tissue- and organ-specific microarchitecture. In 3D cell culture, growing cells in their 3D physical shape allows for better cell-to-cell contact and cell-to-cell signaling. The 3D environment also facilitates developmental processes that allow cells to differentiate into more complex structures (Joseph et al., 2018). 3D cell cultures are characterized by strong cell–cell and cell–extracellular matrix (ECM) interactions that more closely mimic the natural in vivo environment, that is, the cell morphology more closely resembles its natural shape in the body (Li et al., 2007). 3D culture conditions are more similar to the natural environment of cells, and are thus able to provide more physiologically useful information that improves the accuracy and flexibility of cell culture. To facilitate the development of cells in three dimensions, a container, a culture medium, and, in many cases, a scaffold are needed, along with proper cell nutrition and incubation conditions. 3D cell cultures allow modeling and investigation of “flows.” The flow of fluids such as urine or blood affects cells that come into contact with each other in the body. These conditions can only be replicated in 3D, with the intention of creating target cells or testing a growth environment that is more similar to the body. 3D cell culture also helps stimulate or evaluate barrier tissues and reduces the use of animal models by more reliably mimicking the conditions of the cells’ natural environment. During the years 1870–1959, biologist Ross Granville tested the first 3D method called the hanging drop, which was advanced in several areas of biology as well as oncology and genetics. But only much later did the technique become more popular due to new cell lines, more accessible media, and increased stimulation, computing, and imaging capabilities. There are two techniques for 3D cell culture, namely, scaffold-based and scaffold-free techniques. Scaffold-based techniques include hydrogel-based carriers, polymeric hard material-based carriers, hydrophilic glass fiber, and organoids, each of which has its own advantages and applications. Techniques for cell culture in a scaffold-free manner include hanging drop microplates, magnetic levitation, and spheroid microplates with an ultra-low adhesion coating (Jensen and Teng, 2020). One of the multicellular 3D structures, 3D spheroids, is composed of cells in different stages, usually proliferating, quiescent, apoptotic, hypoxic, and necrotic cells (Khaitan et al., 2006). Nuclear cells receive lesser nutrients, oxygen, and growth factors from the medium, due to which they are in a quiescent or hypoxic state (Mehta et al., 2012). The added dimensionality of 3D cultures is a crucial feature leading to different cellular responses, as it not only affects the spatial organization of cell surface receptors involved in interactions with surrounding cells, but also induces physical cell constraints. These spatial and physical aspects in 3D cultures affect signal transduction from the outside to the inside of cells and ultimately affect the gene expression and cell behavior. Cell responses in 3D cultures have been shown to be more similar to in vivo behavior, compared to 2D culture (Figs 1 and 2) (Lee et al., 2008).

Figure 1.

Figure 2.

Among the most common and widely used methods of 3D cell culture are spherical cell clusters called spheroids. Compared to 2D cell culture, in which cells are grown as a monolayer on glass or, more commonly, on tissue cultures from polystyrene plastic bottles, 3D cell cultures are cultured into 3D aggregates or spheroids in a matrix, on a matrix, or in the suspension medium (Fig. 3) (Edmondson et al., 2014).

Spheroids are composed of two layers, the outer and the inner. The outer layer consists of proliferating cells that have enough nutrients and oxygen. Conversely, resting cells located in the inner layer of the spheroid have a limited transport of nutrients and oxygen (Mehta et al., 2012). However, a critical situation occurs in the spheroid nucleus, in which lack of oxygen (hypoxia), depletion of nutrients, and accumulation of waste lead to cell necrosis (Lazzari et al., 2017). Cells in the hypoxic region are resistant to drugs promoting cell apoptosis through reactive oxygen species. On the other hand, the existence of necrotic and quiescent cells reduces the therapeutic efficacy of drugs that are active against proliferative ones (Fig. 4) (Costa et al., 2016).

Figure 3.

Figure 4.

There are different methods to create spheroids. The first method used is the so-called hanging drop, which works on the basis of the culture of suspended droplets of a cell line (Landecker, 2010). Originally, this method was used to study bacteria in a limited and controlled environment. Drops of cell suspension are placed on the underside of the Petri dish lid. The lid, where the cells hang due to surface tension, is placed on a Petri dish containing PBS to prevent dehydration of the droplets. Cells accumulate at the tip of the droplet at the liquid–air interface, spontaneously aggregate, and eventually form spheroids (Hoarau-Véchot et al., 2018). This method was first used by Robert Koch to grow anthrax bacilli in a suspended drop of fluid taken from an ox’s eye (Landecker, 2010). A few years later, the hanging drop method was appropriated by Harrison, who used it to observe nerve growth (Harrison, 1907). Furthermore, a method was discovered based on overlaying 3D microfabrics with a liquid attached to nonstick surfaces. Based on this technique, a large number of heterogeneous spheroids were generated using random interactions (Landry et al., 1985).

Currently available approaches allow the formation of spheroids in reproducible conditions using simple tools and cost-effective methodologies. Liquid overlay technique (LOT) is one of the simplest and cheaper techniques that can be used to create spheroids. It is relatively simple, although more labor-intensive than a traditional 2D monolayer. The liquid overlay technique of 3D cell culture enables the creation of one spheroid per well of a 96-well plate. Because this technique enables the creation of spheroids in isolated wells, individual monitoring, and easy handling for further analysis. In addition, this technique was studied in detail in recent decades to increase its potential (Costa et al., 2014, 2017). Before assembling the spheroids, the wells of the culture plates were coated with 1% agarose. This technique consists in inhibiting the adhesion of cells to culture surfaces. These surfaces have nonadhesive properties, and therefore, intercellular interactions are more pronounced than cell–surface interactions. As a result, the cells aggregate, leading to spheroid formation in 1–4 days of culture for most cell lines. The medium is a mixture of essential growth factors, nutrients, and proteins in the serum. It is necessary for development, cell differentiation, and growth. The purpose of the medium is to provide an isotonic environment with optimal pH, and that is why it must be changed regularly to replenish depleted nutrients; also, the accumulation of toxic secreted molecules was prevented (Costa et al., 2014, 2017). Furthermore, many studies have demonstrated the suitability of LOT for spheroid formation aimed at evaluating the effect of various therapeutic substances and compounds (Fig. 5) (Costa et al., 2014).

Figure 5.

Cell culture is a widely used model in cell and molecular biology that plays an important role in vaccine production, cancer treatment, genetic counseling, gene therapy, and tissue engineering. It is also used to study the interaction between a cell and disease agents such as bacteria or viruses. Cell cultures are considered a simpler and less-expensive way to demonstrate viral proliferation and still provide a desirable medium for the detection and identification of many human viral pathogens. The most commonly used type of cell culture is the 2D model, but at the beginning of the 21st century, the 3D culture model is becoming increasingly popular. While 2D cell cultures grow in monolayers on culture flasks, 3D cultures grow as spheroids on special nonadherent surfaces. It is proven that 3D cell cultures differ from 2D cell cultures not only morphologically, but also physiologically. Cells cultured in 3D conditions better simulate the in vivo environment and provide physiologically relevant information suitable for further prognoses. 2D cells lack cell–cell and cell–ECM interactions, and are therefore unable to mimic the tumor microenvironment in vivo. Since understanding the connection between cells and the ECM in which these cells interact is central to drug discovery, 3D models have begun to be used. The 3D models have many features that resemble tumors in vivo, including hypoxia and central necrosis, as well as drug resistance. In addition to molecular resistance, 3D spheroid cultures can also help predict drug penetration, an important cause of resistance in tumors. Precisely because of these properties, the spheroid model of 3D cell culture is considered a more representative platform for in vitro drug screening.