**3. 3D cell cultures**

Cell cultures that are 3D involve cells that are combined and shaped into a 3D form using a surrounding medium or specialized condition to help maintain the shape [7]. The equipment used for 2D cell cultures such as a biosafety hood, cell incubator with a temperature of 37°C and 95% O2 , refrigerator for culture medium storage and other supplies [2, 3] may also be used for 3D cell cultures. Supplies used for 3D cultures which differ from those in 2D cell cultures include the matrices, scaffolds, and proprietary plasticware for aggregate formation (e.g., AlgiMatrix, microplates, and multidishes) [21]. Other supplies used in 3D cell cultures may include cell reagents for determining cell health and viability, such as the live/dead Viability/Cytotoxicity Kit and CellTracker Deep Red Dye by ThermoFisher Scientific [21].

The techniques used in creating 3D cell cultures are typically divided into methods that use a scaffold and methods that do not (**Figure 1**) [22]. Methods that do not use a scaffold are typically considered to be a better representation of *in vivo* activity as the cells in this method aggregate on their own [22]. When cells come together and aggregate, they are referred to as

gelatin, collagen, agarose, and fibronectin, amongst other materials [27]. The idea of scaffolds and matrices is to provide an imitation of the extracellular matrix, allowing for cells to grow and differentiate using these materials [27]. The extracellular matrix consists of essential substances, such as proteins, and surrounds cells in a fluid form that operates as a natural scaffold for cellular growth and differentiation [28, 29]. As discussed previously, fibroblasts in the body are the cells responsible for secreting extracellular matrix and the substances within

2D vs. 3D Cell Culture Models for *In Vitro* Topical (Dermatological) Medication Testing

http://dx.doi.org/10.5772/intechopen.79868

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Another type of 3D cell culture method is the microfabricated systems, also referred to as cells on chips, which uses microtechnology to provide cells with the required growth environment that can be easily controlled and uses microfluidics for providing nutrients [30]. Microfluidics is the handling of fluids within the microliters range [31]. Microfabricated systems have the advantage of being highly reproducible and provide a better control of the cell culture environment [30]. Another advantage of microfabricated systems is greater cell-to-extracellular

Bioprinting is another method of developing 3D cell cultures. Bioprinting involves the printing of 3D systems by placing living cells in layers [33]. Bioprinting involves both a pre-processing stage as well as the printing stage [34]. The use of imaging diagnostics such as magnetic resonance imagining (MRI), computed tomography (CT), and X-rays to scan an image of the organ or tissue of interest is the first step involved in bioprinting and is part of the pre-processing stage [33, 34]. Selecting the materials such as biomaterials (i.e., biopolymers) as well as cell selection is also part of the preprocessing stage [33, 34]. The printing stage is then completed using different methods such as inkjet printing [33, 35, 36]. Inkjet printing uses bioink for the

As discussed previously, the results obtained from *in vitro* 2D monolayer cell culture models have not translated to *in vivo* successes [7]. Thus, alternative methods for testing dermatological products have been sought out. According to an article by Bergers et al., [39] there are various 3D models that exist for a number of different dermatological conditions such as psoriasis and other autoimmune diseases. Skin cell cultures that are 3D have the advantages of containing a stratum corneum and thus the ability of testing pharmaceutical products on the stratum corneum as a skin barrier [40]. Another advantage to the use of 3D skin cell cultures is the longer cell culture use, which is usually between 10 and 30 days [40]. More recent human skin equivalent (HSE) cultures can even be used for up to 20 weeks [41]. The main disadvantage to the use of 3D skin cell cultures however is the cost associated with developing these cultures [40]. The different types of 3D skin cell cultures that will be discussed in detail are histocultures, human skin equivalents, on-chip skin cultures, and pigmented cell cultures. A

Histocultures are cultures of intact tissues that were developed to better mimic *in vivo* skin responses [42]. The process of hair growth is an example of the ability of histocultures to

process of bioprinting and may include the use of cells as the bioink [37, 38].

discussion on bioprinting of skin constructs is also included.

it such as collagen [17].

**3.1. 3D skin cell cultures**

*3.1.1. Histocultures*

matrix interactions while requiring less cell culture [32].

**Figure 1.** Illustration of general 3D tissue culture methods (floating and matrix-based). Commercially available wellplates and cell-support matrices are often used.

a spheroid in 3D cell cultures [22, 23]. Some of the methods used in 3D cell cultures that will be further explained include the hanging drop method, agitation-based approaches, forcedfloating method, and the use of scaffolds [11, 24, 25]. Microfabricated 3D culture systems and bioprinting will also be discussed.

The hanging drop method provides a suspension of cells on a surface which is then inverted, allowing for the cells to hang and form cell-to-cell interactions and aggregate, forming spheroids [24, 25]. Another method is the utilization of continuous mixing methods in agitationbased approaches, which allows cells to aggregate to one another to form a 3D cell culture, as a container that is moving will not allow the cells to stick to the walls of that container [11]. Another method of preventing cells from sticking to the container in which they are placed in is using the forced-floating method which involves the placement of a nonadhesive coating such as poly-2-hydroxyethyl methacrylate (poly-HEMA) or agar, which will in turn force the cells to form interactions and aggregate as opposed to sticking to the surface of the container [11].

The use of scaffolds is another method of forming 3D cell cultures [11]. Scaffolds are materials with pores that allow cells to form aggregates [24]. Scaffolds can be prepared through different methods such as weaving and freeze drying **[**26] and are typically composed of gelatin, collagen, agarose, and fibronectin, amongst other materials [27]. The idea of scaffolds and matrices is to provide an imitation of the extracellular matrix, allowing for cells to grow and differentiate using these materials [27]. The extracellular matrix consists of essential substances, such as proteins, and surrounds cells in a fluid form that operates as a natural scaffold for cellular growth and differentiation [28, 29]. As discussed previously, fibroblasts in the body are the cells responsible for secreting extracellular matrix and the substances within it such as collagen [17].

Another type of 3D cell culture method is the microfabricated systems, also referred to as cells on chips, which uses microtechnology to provide cells with the required growth environment that can be easily controlled and uses microfluidics for providing nutrients [30]. Microfluidics is the handling of fluids within the microliters range [31]. Microfabricated systems have the advantage of being highly reproducible and provide a better control of the cell culture environment [30]. Another advantage of microfabricated systems is greater cell-to-extracellular matrix interactions while requiring less cell culture [32].

Bioprinting is another method of developing 3D cell cultures. Bioprinting involves the printing of 3D systems by placing living cells in layers [33]. Bioprinting involves both a pre-processing stage as well as the printing stage [34]. The use of imaging diagnostics such as magnetic resonance imagining (MRI), computed tomography (CT), and X-rays to scan an image of the organ or tissue of interest is the first step involved in bioprinting and is part of the pre-processing stage [33, 34]. Selecting the materials such as biomaterials (i.e., biopolymers) as well as cell selection is also part of the preprocessing stage [33, 34]. The printing stage is then completed using different methods such as inkjet printing [33, 35, 36]. Inkjet printing uses bioink for the process of bioprinting and may include the use of cells as the bioink [37, 38].
