**3.1. 3D skin cell cultures**

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

**Figure 1.** Illustration of general 3D tissue culture methods (floating and matrix-based). Commercially available well-

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

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

bioprinting will also be discussed.

plates and cell-support matrices are often used.

container [11].

6 Cell Culture

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 discussion on bioprinting of skin constructs is also included.

#### *3.1.1. Histocultures*

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 mimic *in vivo* processes, as this process occurs in histocultures of skin cells and allows for the testing of pharmaceutical products aimed at inhibiting or improving hair growth [42]. The process of histocultures involves growing skin tissues on a growth medium on its own or with the support of collagen [42, 43]. Research has shown that both epidermal and dermal cells as well as hair follicles maintain their functions and physiology in skin histocultures [44]. Histocultures have thus successfully been used as *in vitro* models for testing dermatological products, particularly with respect to toxicity screening [42, 44].

culture model with the ability to mimic vasculature is the model developed by Mori et al. [51], which includes vascular channels attached to an external perfusion system and can be used to

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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A comparison of on-chip cell cultures to traditional transwell skin cultures was completed by Song et al., [52] in which a static (no flow) chip, dynamic (flow, based on a gravity flow system) chip, and transwell skin equivalents were compared. The comparison revealed that the static chip was not the ideal method as differentiation of skin did not occur and the epidermis was not attached by the end of 1 week [52]. It is thought that this is a result of lack of flow or perfusion in the static chip and thus insufficient flow of nutrients for the growth of the cells [52]. Thus an advantage to using microfabricated skin cell cultures vs. typical 3D cell cultures is the provision of microfluidics which can aid in the supply of essential nutrients for the growth and differentiation of skin cells as well as more closely mimicking *in vivo* processes

A reconstructed pigmented human skin model was developed by Duval et al. [53]. Using a dermal equivalent with a steel ring on top, seeding of human keratinocytes and melanocytes was completed [53]. The culture developed a monolayer after a period of 1 week in medium after which it was exposed to air for another week to allow differentiation of keratinocytes [53]. This 3D model allows for a better understanding of the interactions between melanocytes, keratinocytes, and fibroblasts [53] and also allows for a pigmented human skin model that could potentially be used for testing of pharmaceutical products on pigmented skin.

The process of bioprinting explained above is still the same process followed for bioprinting of skin constructs. An important consideration, however, in the pre-processing stage, is that the imaging equipment used ideally should be able to differentiate skin color [34]. Also, with respect to cell selection, keratinocytes are the primary cell types used for bioprinting of skin cells [34]. The advantages to using bioprinting of skin constructs includes greater accuracy in placement of cells and extracellular matrix as well as having the potential of imbedding vasculature in the skin construct as bioprinting of vasculature is also possible [54]. Skin constructs made through bioprinting are also considered to have great plasticity [54]. Skin bioprinting may also be used for developing 3D models for drug testing, such as diseased skin models, and are believed to provide more uniform models compared to manually developed skin models [55]. The main disadvantage of bioprinting for skin constructs is the high cost

Wound healing is a physiological process that consists of four phases: hemostasis, inflammation, proliferation, and remodeling [56]. In the first phase, after a wound injury, hemostasis,

test vascular absorption of topical products.

such as vasculature [51, 52].

*3.1.4. Pigmented skin cell cultures*

*3.1.5. Bioprinting of skin constructs*

associated with its use [54].

**4. Skin cell cultures for wound healing**

#### *3.1.2. Human skin equivalents*

In order to test pharmaceutical products on the skin *in vitro* and provide suitable skin replacement options for patients with various skin conditions, such as burn victims, HSEs were created [45]. HSEs are 3D cell culture models created from various human skin cells and materials that mimic the extracellular matrix [45] and are created as either epidermal equivalents, dermal equivalents or skin equivalents consisting of both layers [8, 45]. Skin equivalents that are used as replacement skin for patients, known as skin grafts, are useful in conditions where the skin cannot adequately heal on its own, such as in the case of a burn victim or chronic ulcers/wounds [10].

An example of the method involved in developing full thickness human skin equivalents includes the isolation of keratinocytes and fibroblasts from donor skin which are then cultured in medium at 37°C at 5% CO2 [46]. Keratinocytes are then incubated for 2 days following their transfer onto a de-epidermized dermal equivalent and then placed at air-liquid interface for the development of full thickness human skin equivalent [46].

Currently available HSEs are primarily epidermal substitutes composed of keratinocytes in 3D cell culture models such as Epiderm™, SkinEthic RHE™, and EpiSkin™, which are used for pharmaceutical and cosmetic testing of skin irritation from topical products [45, 47, 48]. As these skin substitutes are derived of only the epidermal layer and primarily keratinocytes, it limits their use for testing of products related to particular types of skin conditions that involve the immune system, including testing of products for wound healing [47]. Full thickness models consisting of both the epidermal and dermal layers are thus beneficial [8]. One such model is the full thickness model developed by MatTeck Corporation, the EpidermFT™, which includes both normal human keratinocytes and fibroblasts cultured into a 3D model of several layers of the epidermis and dermis to more closely mimic human skin [49].

### *3.1.3. On-chip skin culture*

Microfabricated systems, also referred to as cells on chips, are a type of 3D cell culture that uses microtechnology to provide cells with the required growth environment that can be easily controlled [30]. On-chip culturing also uses microfluidics for providing nutrients [30]. In order to mimic *in vivo* properties of the skin, an on-chip skin culture was created by Lee et al. [50] and is composed of a microfabricated cell culture of keratinocytes (HaCaT cells) and fibroblasts with microfluidic channels to mimic vasculature. This model provides an opportunity for dermatological medication testing on a microfabricated cell system as growth and differentiation of skin cells were made possible through this model [50]. Another on-chip skin culture model with the ability to mimic vasculature is the model developed by Mori et al. [51], which includes vascular channels attached to an external perfusion system and can be used to test vascular absorption of topical products.

A comparison of on-chip cell cultures to traditional transwell skin cultures was completed by Song et al., [52] in which a static (no flow) chip, dynamic (flow, based on a gravity flow system) chip, and transwell skin equivalents were compared. The comparison revealed that the static chip was not the ideal method as differentiation of skin did not occur and the epidermis was not attached by the end of 1 week [52]. It is thought that this is a result of lack of flow or perfusion in the static chip and thus insufficient flow of nutrients for the growth of the cells [52]. Thus an advantage to using microfabricated skin cell cultures vs. typical 3D cell cultures is the provision of microfluidics which can aid in the supply of essential nutrients for the growth and differentiation of skin cells as well as more closely mimicking *in vivo* processes such as vasculature [51, 52].
