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

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, platelets are activated and migrated to the site of injury [57]. The second phase, inflammation, begins about 1 day postinjury and inflammatory mediators such as histamine are released, providing the typical traits of inflammation such as heat and swelling [57]. Proliferation is the phase in which granulation tissue forms at the site of injury and after which re-epithelization occurs [58]. In the last phase, remodeling of the tissue occurs to improve the strength of the skin tissue, which is only ever within 80% of the original tissue's strength [58]. The normal wound healing process could be affected, however, leading to chronic ulcers or excessive wound healing resulting in hypertrophic scars [59]. For this reason, topical agents to improve wound healing or reduce scarring may be of interest and as such *in vitro* testing models for wound healing will be discussed.

in 24-well cell plates with transwell inserts [67]. This model allows application of topical medications to the uncovered epidermal layer and is thus useful for testing of pharmaceutical

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

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

11

The skin substitutes derived of only the epidermal layer and primarily keratinocytes, however, are limited in their use for testing of products related to particular types of skin conditions that involve the immune system, including the 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]. This kit can be purchased with a wound healing assay kit [49] and is thus a commercially available 3D

It is thus evident from the examples provided in this section that 3D wound healing models more closely mimic *in vivo* wound healing processes and thus may be a better choice for testing of pharmaceutical products aimed at wound healing. Wound healing models are also commercially available which may improve the use of *in vitro* models as a replacement for

As an inflammatory skin condition that involves the immune system, psoriasis has the characteristic appearance of silver scales that arise from increased proliferation of the keratinocytes in the epidermal layer [68, 69]. Psoriasis is typically treated with topical steroid medications or with vitamin D analogue medications such as calcipotriol as well as with moisturizing agents [68]. The treatment of psoriasis also includes systemic medications that suppress the immune system such as methotrexate and cyclosporine as well as biologic drugs [68, 70].

Psoriasis has been linked to various mental health illnesses such as anxiety and depression which are thought to result from having a chronic visible skin condition [71], and therefore the need for developing new pharmaceutical products and testing of these products is evident. As psoriasis is primarily treated with topical medications [70], having *in vitro* cell cultures or

Skin cell cultures that are 2D for dermatological conditions such as psoriasis and other autoimmune disorders exist [39]. 2D cell cultures of psoriasis initially consisted of psoriatic keratinocytes; however, this proved to be an ineffective model for psoriasis as the cells were not able to grow and lost their psoriatic genes with time [72]. As a result, alternative methods such as adding cytokines to normal human keratinocytes to induce psoriatic features were developed [73]. As stated previously, however, testing of medications on 2D cell culture models does not always translate to *in vivo* responses [7], and thus, 3D models to better mimic *in vivo* responses

Barker et al. [74] introduce an *in vitro* model of psoriatic human skin, whereby a monolayer cell culture of psoriatic keratinocytes on top of collagen and fibroblasts from the dermis was

models for testing the safety and efficacy of these topical medications is essential.

for testing of topical medications for psoriasis have been developed.

products, including those used for transdermal drug delivery [67].

model for testing of dermatological products aimed at wound healing.

animal testing.

**5. Skin cell cultures for psoriasis**

Both 2D and 3D skin cell cultures are available as wound healing models [60]. 2D wound healing models involve the creation of a site of injury in a monolayer of skin cells, either through mechanical or chemical means, in which cells then migrate to the site of injury [60, 61]. Cells in 2D monolayer cultures are thought to adhere to the flat environments, such as a Petri dish, in which they are cultured and will therefore migrate to areas of free space within the dish, an activity thought to mimic *in vivo* migration involved in cell differentiation [62]. One method of mechanical introduction of a wound to a 2D cell culture is through the scratch assay that utilizes materials such as pipette tips or needles to introduce a wound or scratch into the monolayer cell culture [63, 64]. Images of the wound are taken within set time frames to assess the migration of cells [63]. Typically, however, it is difficult to ensure wounds that are equal in size using this method [63], and thus for this reason, testing of pharmaceutical products on these types of cultures are not ideal. 2D skin cell cultures also lack essential functions that could mimic *in vivo* processes, such as immune functionality and blood perfusion [65]. 3D wound healing models have thus attempted to more closely mimic *in vivo* wound healing processes and are available as histocultures or HSEs.

Histocultures are cultures of intact tissues, consisting of more than one type of skin cell, such as neutrophils and other cells involved in wound healing, and are thus able to better mimic *in vivo* skin responses of wound healing [42, 43]. HSEs, on the other hand, 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]. Some examples of 3D wound healing skin cell cultures and HSEs are described below.

A 3D wound healing skin equivalent developed by Herman et al. [66] included capillary endothelial cells which were capable of creating a similar formation to *in vivo* capillaries in the skin. This wound healing model involved several different cells, such as keratinocytes and epithelial cells, and used 3D matrices composed of Matrigel™ and collagen [66]. As angiogenesis is involved in the process of wound healing [66], this model is an excellent example of improved 3D wound healing cell culturing protocols to more closely mimic *in vivo* wound healing processes.

Another example of a 3D skin cell culture model that could be used for wound healing was reported by Sidgwick et al. [67]. This model allows for the immersion of biopsies in Williams E culture media with the epidermal layer of the skin uncovered and uses whole tissue biopsies in 24-well cell plates with transwell inserts [67]. This model allows application of topical medications to the uncovered epidermal layer and is thus useful for testing of pharmaceutical products, including those used for transdermal drug delivery [67].

The skin substitutes derived of only the epidermal layer and primarily keratinocytes, however, are limited in their use for testing of products related to particular types of skin conditions that involve the immune system, including the 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]. This kit can be purchased with a wound healing assay kit [49] and is thus a commercially available 3D model for testing of dermatological products aimed at wound healing.

It is thus evident from the examples provided in this section that 3D wound healing models more closely mimic *in vivo* wound healing processes and thus may be a better choice for testing of pharmaceutical products aimed at wound healing. Wound healing models are also commercially available which may improve the use of *in vitro* models as a replacement for animal testing.
