**3.2.2 Collagen gels**

Despite the absence of a complete *in vitro* model allowing the observation of interactions between different cell types, such as keratinocytes and fibroblasts, some teams have developed specialized techniques which imply an exogenous matrix: the collagen gel.

#### **3.2.2.1 Organ culture**

Some teams decided to put down complete skin biopsies on collagen gel, containing fibroblasts, to observe cell proliferation. Total surface recovered by keratinocytes was used to calculate cell proliferation percentage (Saiag *et al.*, 1985). Higher keratinocyte proliferation values were obtained in the presence of psoriatic fibroblasts (Saiag *et al.*, 1985). Furthermore, this model led to the conclusion that normal fibroblasts are unable to suppress the hyperproliferative growth of psoriatic keratinocytes, and that hyperproliferation of normal epidermis can be induced both by uninvolved and involved psoriatic fibroblasts (Saiag *et al.*, 1985, Jean & Pouliot, 2010).

#### **3.2.2.2 Models using many cellular types**

Other teams developed skin substitutes composed of two cell types, in order to observe the effects of psoriatic keratinocytes on fibroblasts and *vice versa*. In a global way, these models consist of isolating normal and pathological cells from a small biopsy. Fibroblasts are extracted from dermis, expanded and seeded in collagen gel. Keratinocytes are extracted in a similar way and are placed on the pre-prepared collagen gel (Konstantinova *et al.*, 1996). Barker *et al.* developed and characterized an *in vitro* psoriatic skin model using collagen gel. This model was very representative of the pathology (Barker *et al.*, 2004). In fact, they have demonstrated that the model kept many characteristics of psoriasis such as hyperproliferation and abnormal differentiation of keratinocytes, augmentation of the interleukin 6 and 8 concentrations, as well as the overexpression or underexpression of some proliferation, differentiation and inflammatory markers observed in psoriatic skin. Researchers concluded that involved and uninvolved skins seem to have the same pathological characteristics as psoriatic human skin (Barker *et al.*, 2004, Jean & Pouliot, 2010). Barker, Konstantinova and Saiag models are interesting *in vitro* models for studying psoriasis, but they are produced with a contractile exogenous material (collagen gel).

#### **3.2.3 Self-assembly approach**

56 Psoriasis

By using only a small skin biopsy, monolayer techniques allow the attainment of a large number of cells (normal or pathological) supporting the production of many experiments. In monolayer models, only one cell type is studied. Thus, keratinocytes (or fibroblasts) can be used to test different conditions or to observe psoriatic skin features such as hyperproliferation or abnormal differentiation of keratinocytes. These models allow the isolation of one cell type for step by step dissection of the implied mechanisms. Even if it was not possible to observe direct interaction between cell types, these models allowed the discovery of many interesting facts about psoriasis, and favoured a better understanding of

Despite the absence of a complete *in vitro* model allowing the observation of interactions between different cell types, such as keratinocytes and fibroblasts, some teams have developed specialized techniques which imply an exogenous matrix: the collagen gel.

Some teams decided to put down complete skin biopsies on collagen gel, containing fibroblasts, to observe cell proliferation. Total surface recovered by keratinocytes was used to calculate cell proliferation percentage (Saiag *et al.*, 1985). Higher keratinocyte proliferation values were obtained in the presence of psoriatic fibroblasts (Saiag *et al.*, 1985). Furthermore, this model led to the conclusion that normal fibroblasts are unable to suppress the hyperproliferative growth of psoriatic keratinocytes, and that hyperproliferation of normal epidermis can be induced both by uninvolved and involved psoriatic fibroblasts (Saiag *et al.*,

Other teams developed skin substitutes composed of two cell types, in order to observe the effects of psoriatic keratinocytes on fibroblasts and *vice versa*. In a global way, these models consist of isolating normal and pathological cells from a small biopsy. Fibroblasts are extracted from dermis, expanded and seeded in collagen gel. Keratinocytes are extracted in a similar way and are placed on the pre-prepared collagen gel (Konstantinova *et al.*, 1996). Barker *et al.* developed and characterized an *in vitro* psoriatic skin model using collagen gel. This model was very representative of the pathology (Barker *et al.*, 2004). In fact, they have demonstrated that the model kept many characteristics of psoriasis such as hyperproliferation and abnormal differentiation of keratinocytes, augmentation of the interleukin 6 and 8 concentrations, as well as the overexpression or underexpression of some proliferation, differentiation and inflammatory markers observed in psoriatic skin. Researchers concluded that involved and uninvolved skins seem to have the same pathological characteristics as psoriatic human skin (Barker *et al.*, 2004, Jean & Pouliot, 2010). Barker, Konstantinova and Saiag models are interesting *in vitro* models for studying psoriasis, but they are produced with a contractile exogenous

**3.2** *In vitro* **models 3.2.1 Monolayer** 

**3.2.2 Collagen gels** 

**3.2.2.1 Organ culture** 

1985, Jean & Pouliot, 2010).

material (collagen gel).

**3.2.2.2 Models using many cellular types** 

the pathology (Jean & Pouliot, 2010).

Facing the absence of exogenous material-free models, our group developed a new pathological skin model to study psoriasis *in vitro* by using the self-assembly approach (Michel *et al.*, 1999) (Fig. 3). Briefly, normal and pathological fibroblasts are thawed and cultured with ascorbic acid for a period of time of four weeks. Then, dermal sheets are produced and removed from flasks. Two fibroblast sheets are superimposed to form a new dermal equivalent. Seven days later, normal or pathological keratinocytes are seeded on the dermal equivalent to obtain a new epidermal equivalent. After another 7 days of culture, the substitutes are raised to the air–liquid interface to favour cell differentiation and stratification. Finally, biopsies are taken after 21 days of culture at the air–liquid interface, and samples are analyzed using histological, immunohistochemical, physico-chemical or permeability techniques (Jean *et al.*, 2009).

Fig. 3. The self-assembly approach for the production of skin substitutes

Schematic representation of the various steps of skin substitutes production in function of time. Reproduced and modified from Jean *et al.*, 2010 according to the copyright policy of the publisher. 2010 InTech.

In 2009, Jean *et al.* showed that self-assembled skin substitutes partially maintained psoriasis-like features such as a thick epidermis, hyperproliferation as well as abnormal cell differentiation of epidermal cells (Jean *et al.*, 2009). In 2011, they demonstrated for the first time that pathological substitutes produced by the self-assembly approach can be treated with an anti-psoriatic molecule and react positively to the treatment such as observed in psoriatic skin *in vivo*. This functional study suggests that the self-assembled skin substitutes could be useful to better understand the mechanisms through which retinoic acid regulates cellular physiology in psoriatic skin, and could become an effective and innovative dermopharmaceutical tool for the screening of new treatments (Jean *et al.*, 2011).
