*3.1.4. Pigmented skin cell cultures*

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

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

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 cul-

lowing their transfer onto a de-epidermized dermal equivalent and then placed at air-liquid

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

interface for the development of full thickness human skin equivalent [46].

several layers of the epidermis and dermis to more closely mimic human skin [49].

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

[46]. Keratinocytes are then incubated for 2 days fol-

products, particularly with respect to toxicity screening [42, 44].

*3.1.2. Human skin equivalents*

8 Cell Culture

chronic ulcers/wounds [10].

*3.1.3. On-chip skin culture*

tured in medium at 37°C at 5% CO2

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.

#### *3.1.5. Bioprinting of skin constructs*

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 associated with its use [54].
