**2. Modified self-assembly methodology**

As mentioned previously, our team has developed a model of *in vitro* psoriatic skin substitutes using the self-assembly method. In this present research, the production of the tissue-engineered psoriatic skin substitutes was done according to the self-assembly method partially modified, using 6-well plates and 12-well plates [31, 35] (**Table 1** and **Figure 1**). All methods were also compared to the reconstruction of healthy skin substitutes. Briefly, pathological fibroblasts were cultured 28 days with Dulbecco-Vogt modification of Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 UI/ml penicillin G, 25 μg/ml gentamicin, and 50 μg/ml ascorbic acid until they form manipulable sheets. Then, these fibroblast sheets were detached, and two of them were superimposed to form a new dermal equivalent. Subsequently, they were incubated for 7 days to allow the fusion of the two sheets and thus form the new layer. After this period, pathological keratinocytes were seeded on the dermal equivalent to form a new epidermal layer. Seven days later, the substitutes were raised to the air-liquid interface to promote cell differentiation and obtain the different epidermal layers. Skin substitute biopsies were taken at 21 days after being raised to the air-liquid interface and analyzed by histology and immunohistochemistry.

#### **2.1. Results**

#### *2.1.1. Macroscopic results*

Healthy skin substitutes reconstructed using either the original or modified methods (**Figure 2A–C**) showed a uniform and opaque epidermis recovering all the seeding area within the anchoring paper (white contours). For substitutes produced with psoriatic cells,

> those reconstructed according to the original method and the 6-well plate modifications (**Figure 2D–E**) showed an irregular and contracted epidermis. Psoriatic skin substitutes produced using the 12-well plate modification (**Figure 2F**) demonstrated a more uniform epider-

> **Figure 1.** Schematic representation of the self-assembly method. (A) Original self-assembly method: fibroblasts were

iron, and two sheets were superimposed to form a dermal equivalent. After one week, a metal ring was deposited on substitutes, and keratinocytes were seeded into the ring. Then, substitutes were cultured one more week in submerged conditions. At day 42, substitutes were placed on an anchoring paper and were raised to the air-liquid interface for three weeks. (B) Modified self-assembly method: fibroblasts were seeded in 6-well or 12-well plates in which anchoring paper have been previously placed. After 28 days of culture, two fibroblast sheets were easily superimposed and solidified with Ligaclip®. Dermal equivalents were cultured seven days before keratinocytes seeding. At day 42, substitutes were

flasks for the production of fibroblast sheets. At day 28, flasks were opened with a soldering

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Healthy skin substitutes reconstructed according to the original method, and the 6-well plate modification (**Figure 3A** and **B**) demonstrated a well-differentiated epidermis and similar

mis compared to other methods.

raised to the air-liquid interface and cultured for three weeks.

*2.1.2. Histology*

cultured 28 days in 25 cm2


**Table 1.** Technical characteristics of the original method, the 6-well plate and the 12-well plate modifications.

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**Figure 1.** Schematic representation of the self-assembly method. (A) Original self-assembly method: fibroblasts were cultured 28 days in 25 cm2 flasks for the production of fibroblast sheets. At day 28, flasks were opened with a soldering iron, and two sheets were superimposed to form a dermal equivalent. After one week, a metal ring was deposited on substitutes, and keratinocytes were seeded into the ring. Then, substitutes were cultured one more week in submerged conditions. At day 42, substitutes were placed on an anchoring paper and were raised to the air-liquid interface for three weeks. (B) Modified self-assembly method: fibroblasts were seeded in 6-well or 12-well plates in which anchoring paper have been previously placed. After 28 days of culture, two fibroblast sheets were easily superimposed and solidified with Ligaclip®. Dermal equivalents were cultured seven days before keratinocytes seeding. At day 42, substitutes were raised to the air-liquid interface and cultured for three weeks.

those reconstructed according to the original method and the 6-well plate modifications (**Figure 2D–E**) showed an irregular and contracted epidermis. Psoriatic skin substitutes produced using the 12-well plate modification (**Figure 2F**) demonstrated a more uniform epidermis compared to other methods.

#### *2.1.2. Histology*

pathological skin complexity through the possibility of: (1) dissecting step by step the mechanisms of skin pathologies according to which kinds of cells are present in the model at that time and/or (2) using various cell combinations such as healthy fibroblasts and healthy keratinocytes, which can be compared with healthy fibroblasts and pathological keratinocytes. Although the self-assembly method is very effective for the reconstruction of substitutes used in basic mechanisms studies, it required an optimization of its original protocol to consider a productive capacity of it in the pharmaceutical industry. Thus, the aim of this work was to improve the original self-assembly method to allow the reconstruction of more reproducible

As mentioned previously, our team has developed a model of *in vitro* psoriatic skin substitutes using the self-assembly method. In this present research, the production of the tissue-engineered psoriatic skin substitutes was done according to the self-assembly method partially modified, using 6-well plates and 12-well plates [31, 35] (**Table 1** and **Figure 1**). All methods were also compared to the reconstruction of healthy skin substitutes. Briefly, pathological fibroblasts were cultured 28 days with Dulbecco-Vogt modification of Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 UI/ml penicillin G, 25 μg/ml gentamicin, and 50 μg/ml ascorbic acid until they form manipulable sheets. Then, these fibroblast sheets were detached, and two of them were superimposed to form a new dermal equivalent. Subsequently, they were incubated for 7 days to allow the fusion of the two sheets and thus form the new layer. After this period, pathological keratinocytes were seeded on the dermal equivalent to form a new epidermal layer. Seven days later, the substitutes were raised to the air-liquid interface to promote cell differentiation and obtain the different epidermal layers. Skin substitute biopsies were taken at 21 days after being raised to the air-liquid interface and analyzed by histology and immunohistochemistry.

Healthy skin substitutes reconstructed using either the original or modified methods (**Figure 2A–C**) showed a uniform and opaque epidermis recovering all the seeding area within the anchoring paper (white contours). For substitutes produced with psoriatic cells,

> **Volume of medium by fibroblast sheet (ml)**

**Anchoring papers**

**Final size of substitute** 

**)**

Original 25 3.87 5 Day 42 6-well plates 9.6 3.87 2 Day 0 12-well plates 3.8 1.27 2 Day 0

**Table 1.** Technical characteristics of the original method, the 6-well plate and the 12-well plate modifications.

**(usable) (cm2**

psoriatic skin substitutes that could be used for pharmacological testing.

**2. Modified self-assembly methodology**

**2.1. Results**

198 Cell Culture

**Self-assembly method**

*2.1.1. Macroscopic results*

**Culture surface area (cm2 )**

> Healthy skin substitutes reconstructed according to the original method, and the 6-well plate modification (**Figure 3A** and **B**) demonstrated a well-differentiated epidermis and similar

characteristics. A thickening of the living epidermis was observed in substitutes produced with psoriatic cells using the original method, as well as in those reconstructed according to the modified protocols (**Figure 3D–F**). A less differentiated epidermis was observed in psoriatic substitutes produced according to the original method and the 6-well plate modification (**Figure 3D–E**), compared with the substitutes produced according to the 12-well plate modification (**Figure 3F**). The substitutes reconstructed according to the 12-well plate modification (**Figure 3C** and **F**) demonstrated a greater cell differentiation which results in a thickening of the *stratum corneum*.

#### *2.1.3. Epidermal thickness*

Thickness of skin substitutes' living epidermis was measured with the AxioVision software. No significant differences were observed between the skin substitutes reconstructed with healthy cells according to the original method or the two new modifications (65.6 ± 13.9 vs. 69.0 ± 6.8 vs. 78.39 ± 18.69 μm; **Figure 4A**). Measurements of psoriatic substitutes thickness reconstructed according to the original method, and the 6-well plate modification did not show any significant differences between these two methods (93.50 ± 18.9 μm vs. 106.7 ± 23.7 μm; **Figure 4B**), while psoriatic substitutes produced with the 12-well plate modification demonstrated a significant difference compared with the original method (130.8 ± 18.8 vs. 93.50 ± 18.9 μm).

**Figure 2.** Macroscopic analyses. Macroscopic appearance of healthy substitutes (A–C) and psoriatic substitutes (D–F). Substitutes were produced according to the original method (A and D), 6-well plate modification (B and E), and 12-well plate modification (C and F). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were used (scale bar = 1 cm).

*2.1.4. Immunofluorescence analyses*

presented are means ± S.D. (\*\*) = *p*-value < 0.001.

(scale bar = 100 μm).

Immunofluorescent markers were used to compare protein expression. Late differentiation markers, such as filaggrin and loricrin, were observed in order to compare the different skin substitute models. The *stratum granulosum* of *in vivo* skin is characterized by the presence of keratohyalin granules and the expression of late differentiation markers, since their synthesis depends on keratohyalin granules [36, 37]. Moreover, late differentiation markers are found

**Figure 4.** Thickness of the living part of epidermis. (A) Measurements of healthy substitutes produced with the original method, the 6-well plate modification, and the 12-well plate modification. (B) Measurements of psoriatic substitutes produced according to the original method, the 6-well plate modification, and the 12-well plate modification. The statistical significance was determined using ANOVA test (*p* < 0.05, *n* = 3, *N* = 3, 90 measurements by condition). Data

**Figure 3.** Histological analyses. Masson's trichrome staining of healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced with the original method (A and D), 6-well plate modification (B and E), and 12-well plate modification (C and F). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were used

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**Figure 3.** Histological analyses. Masson's trichrome staining of healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced with the original method (A and D), 6-well plate modification (B and E), and 12-well plate modification (C and F). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were used (scale bar = 100 μm).

**Figure 4.** Thickness of the living part of epidermis. (A) Measurements of healthy substitutes produced with the original method, the 6-well plate modification, and the 12-well plate modification. (B) Measurements of psoriatic substitutes produced according to the original method, the 6-well plate modification, and the 12-well plate modification. The statistical significance was determined using ANOVA test (*p* < 0.05, *n* = 3, *N* = 3, 90 measurements by condition). Data presented are means ± S.D. (\*\*) = *p*-value < 0.001.

#### *2.1.4. Immunofluorescence analyses*

characteristics. A thickening of the living epidermis was observed in substitutes produced with psoriatic cells using the original method, as well as in those reconstructed according to the modified protocols (**Figure 3D–F**). A less differentiated epidermis was observed in psoriatic substitutes produced according to the original method and the 6-well plate modification (**Figure 3D–E**), compared with the substitutes produced according to the 12-well plate modification (**Figure 3F**). The substitutes reconstructed according to the 12-well plate modification (**Figure 3C** and **F**) demonstrated a greater cell differentiation which results in a thickening of the *stratum corneum*.

Thickness of skin substitutes' living epidermis was measured with the AxioVision software. No significant differences were observed between the skin substitutes reconstructed with healthy cells according to the original method or the two new modifications (65.6 ± 13.9 vs. 69.0 ± 6.8 vs. 78.39 ± 18.69 μm; **Figure 4A**). Measurements of psoriatic substitutes thickness reconstructed according to the original method, and the 6-well plate modification did not show any significant differences between these two methods (93.50 ± 18.9 μm vs. 106.7 ± 23.7 μm; **Figure 4B**), while psoriatic substitutes produced with the 12-well plate modification demonstrated a significant difference compared with the original method (130.8 ± 18.8 vs. 93.50 ± 18.9 μm).

**Figure 2.** Macroscopic analyses. Macroscopic appearance of healthy substitutes (A–C) and psoriatic substitutes (D–F). Substitutes were produced according to the original method (A and D), 6-well plate modification (B and E), and 12-well plate modification (C and F). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were

*2.1.3. Epidermal thickness*

200 Cell Culture

used (scale bar = 1 cm).

Immunofluorescent markers were used to compare protein expression. Late differentiation markers, such as filaggrin and loricrin, were observed in order to compare the different skin substitute models. The *stratum granulosum* of *in vivo* skin is characterized by the presence of keratohyalin granules and the expression of late differentiation markers, since their synthesis depends on keratohyalin granules [36, 37]. Moreover, late differentiation markers are found in the *stratum granulosum*. In healthy substitutes, filaggrin was expressed from the last layers of the *stratum granulosum* to the first ones of the *stratum corneum* (**Figure 5A–C**). In healthy substitutes produced according to the 6-well plate and 12-well plate modifications, a slight decrease of filaggrin was observed (**Figure 5B** and **C**). However, since keratohyalin granules are in lesser amount in psoriatic skin than normal skin due to abnormal keratinocytes differentiation [38], expression of late differentiation markers are downregulated. In our psoriatic substitutes, whether the original method was used or the two modifications to protocol were followed, filaggrin is reduced and almost missing (**Figure 5D–F**). Loricrin, another late differentiation marker found in *stratum granulosum*, was observed in healthy substitutes (**Figure 5G–I**). In the psoriatic substitutes, a decrease or an absence of loricrin was observed which is similar to *in vivo* psoriatic skin (**Figure 5J–L**).

Other markers, such as keratin 14, keratin 1, and laminin, were also observed. Keratin 14 (K14) is expressed in the basal layer of the epidermis and is gradually reduced until keratins 1 (K1) and 10 (K10) are synthesized. In healthy substitutes produced according to the original method and the 6-well plate modification, keratin 14 was normally expressed (**Figure 6A** and **B**), whereas in healthy substitutes produced according to the 12-well plate modification, this keratin was still present in the *stratum corneum* (**Figure 6C**). In psoriatic skin, this protein is expressed in all epidermis such as observed regardless of the method used in the production of psoriatic substitutes (**Figure 6D–F**). Keratin 1 staining showed no difference between healthy substitutes reconstructed according to the original method or the 6-well plate modification (**Figure 6G–H**), while this keratin was overexpressed in *stratum corneum* of healthy substitutes produced according to the 12-well plate modification (**Figure 6I**). In psoriasis, keratin 1 is decreased such as observed in the psoriatic substitutes produced according to the original method or the 6-well plate modification (**Figure 6J–K**), whereas psoriatic substitutes produced according to the 12-well plate modification showed a higher expression of this keratin in the *stratum corneum* compared to the other two (**Figure 6L**). Laminin expression was similar for all reconstructed healthy substitutes (**Figure 7A–C**), while for psoriatic substitutes, laminin expression seems more distributed through the dermis using the original method and the 6-well plate modification (**Figure 7D–E**). Laminin staining of psoriatic substitutes produced according to the 12-well plate modification (**Figure 7F**) was similar to the healthy substitute expression.

#### **2.2. Discussion**

With psoriasis etiology still unknown, several *in vivo* models of psoriasis have been generated for a better understanding of the pathology. Genetically modified, spontaneous mutations, cytokine injections, xenografts, and gene knockout mouse models are all examples [21, 39–41]. Although these models showed psoriasis-like lesions, psoriasis is a specific human disease, and mouse skin does not represent characteristics of human skin such as epidermal thickness and structure, rete ridges and differences in certain immune cells [40, 41]. Due to the advances that have been made in the field of tissue engineering in the past years, it has been possible to develop *in vitro* psoriatic skin models to understand the disease, improve the development of new treatments, and limit the use of animals [24, 25, 28, 42]. Our team has developed a representative psoriatic skin model reconstructed according to a self-assembly method showing *in vivo* features of psoriasis such as hyperproliferation and abnormal differentiation of

keratinocytes [31]. However, each model has limitations, and some of the model developed in our team is the large number of cells required for the production of skin substitutes, in addition to complex manipulations that may generate less reproducibility. Therefore, the optimization of the production of psoriatic skin substitutes with the aim of making them suitable

**Figure 5.** Filaggrin and loricrin staining. Expression of filaggrin (red) in healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced according to the original method (A and D), the 6-well plate modification (B and E), and the 12-well plate modification (C and F). Expression of loricrin (green) in healthy substitutes (G–I), psoriatic substitutes (J–L) and substitutes produced according to the original method (G and J), the 6-well plate modification (H and K), and the 12-well plate modification (I and L). The nuclei were stained with Hoechst (blue). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy

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models for their use in pharmacological testing remains a challenge.

patients and three different psoriatic patients were used (scale bar = 100 μm).

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in the *stratum granulosum*. In healthy substitutes, filaggrin was expressed from the last layers of the *stratum granulosum* to the first ones of the *stratum corneum* (**Figure 5A–C**). In healthy substitutes produced according to the 6-well plate and 12-well plate modifications, a slight decrease of filaggrin was observed (**Figure 5B** and **C**). However, since keratohyalin granules are in lesser amount in psoriatic skin than normal skin due to abnormal keratinocytes differentiation [38], expression of late differentiation markers are downregulated. In our psoriatic substitutes, whether the original method was used or the two modifications to protocol were followed, filaggrin is reduced and almost missing (**Figure 5D–F**). Loricrin, another late differentiation marker found in *stratum granulosum*, was observed in healthy substitutes (**Figure 5G–I**). In the psoriatic substitutes, a decrease or an absence of loricrin was observed

Other markers, such as keratin 14, keratin 1, and laminin, were also observed. Keratin 14 (K14) is expressed in the basal layer of the epidermis and is gradually reduced until keratins 1 (K1) and 10 (K10) are synthesized. In healthy substitutes produced according to the original method and the 6-well plate modification, keratin 14 was normally expressed (**Figure 6A** and **B**), whereas in healthy substitutes produced according to the 12-well plate modification, this keratin was still present in the *stratum corneum* (**Figure 6C**). In psoriatic skin, this protein is expressed in all epidermis such as observed regardless of the method used in the production of psoriatic substitutes (**Figure 6D–F**). Keratin 1 staining showed no difference between healthy substitutes reconstructed according to the original method or the 6-well plate modification (**Figure 6G–H**), while this keratin was overexpressed in *stratum corneum* of healthy substitutes produced according to the 12-well plate modification (**Figure 6I**). In psoriasis, keratin 1 is decreased such as observed in the psoriatic substitutes produced according to the original method or the 6-well plate modification (**Figure 6J–K**), whereas psoriatic substitutes produced according to the 12-well plate modification showed a higher expression of this keratin in the *stratum corneum* compared to the other two (**Figure 6L**). Laminin expression was similar for all reconstructed healthy substitutes (**Figure 7A–C**), while for psoriatic substitutes, laminin expression seems more distributed through the dermis using the original method and the 6-well plate modification (**Figure 7D–E**). Laminin staining of psoriatic substitutes produced according to the 12-well

plate modification (**Figure 7F**) was similar to the healthy substitute expression.

With psoriasis etiology still unknown, several *in vivo* models of psoriasis have been generated for a better understanding of the pathology. Genetically modified, spontaneous mutations, cytokine injections, xenografts, and gene knockout mouse models are all examples [21, 39–41]. Although these models showed psoriasis-like lesions, psoriasis is a specific human disease, and mouse skin does not represent characteristics of human skin such as epidermal thickness and structure, rete ridges and differences in certain immune cells [40, 41]. Due to the advances that have been made in the field of tissue engineering in the past years, it has been possible to develop *in vitro* psoriatic skin models to understand the disease, improve the development of new treatments, and limit the use of animals [24, 25, 28, 42]. Our team has developed a representative psoriatic skin model reconstructed according to a self-assembly method showing *in vivo* features of psoriasis such as hyperproliferation and abnormal differentiation of

**2.2. Discussion**

202 Cell Culture

which is similar to *in vivo* psoriatic skin (**Figure 5J–L**).

**Figure 5.** Filaggrin and loricrin staining. Expression of filaggrin (red) in healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced according to the original method (A and D), the 6-well plate modification (B and E), and the 12-well plate modification (C and F). Expression of loricrin (green) in healthy substitutes (G–I), psoriatic substitutes (J–L) and substitutes produced according to the original method (G and J), the 6-well plate modification (H and K), and the 12-well plate modification (I and L). The nuclei were stained with Hoechst (blue). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were used (scale bar = 100 μm).

keratinocytes [31]. However, each model has limitations, and some of the model developed in our team is the large number of cells required for the production of skin substitutes, in addition to complex manipulations that may generate less reproducibility. Therefore, the optimization of the production of psoriatic skin substitutes with the aim of making them suitable models for their use in pharmacological testing remains a challenge.

in both new protocols, a significant increase in the living epidermis thickness of psoriatic substitutes is observed compared with their respective healthy substitute counterparts as for the original method. Interestingly, the epidermal thickness of psoriatic substitutes produced according to the 12-well plate modification was significantly thicker than those produced according to the original method. This difference could be explained by the same seeding number of keratinocytes in a smaller seeding area for the 12-well plate protocol. Thus, this induces an increase of acanthosis. Considering histological analysis, our model stands out from other models of *in vitro* psoriatic skin that did not show acanthosis [24, 25]. However, based on these histological observations, the 6-well plate modification to protocol seems to be

**Figure 7.** Laminin staining. Expression of laminin (green) in healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced according to the original method (A and D), the 6-well plate modification (B and E), and the 12-well plate modification (C and F). The nuclei were stained with Hoechst (blue). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients

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Differentiation of psoriatic skin is characterized by the altered expression of several epidermal proteins [43]. In a normal differentiation process (approximately 28 days), the basal layer transit amplifying cells differentiate and migrate into upper epidermal layers and synthesize important proteins involved in the differentiation and the skin barrier function such as filaggrin, loricrin, and keratins [36, 37, 44]. Filaggrin is normally synthesized from a precursor, profilaggrin, found in the granular layer. This protein is a key role in the formation of the cornified envelope [37]. In skin disease, such as psoriasis, expression of filaggrin is decreased, such that it is sometimes even absent due to an altered differentiation process and a reduction or an absence of the granular layer (agranulose) [38, 45, 46]. Loricrin, a major component of the cornified envelope, is stored in granules of the *stratum granulosum*, and its expression is also decreased or absent in psoriatic skin [36, 47]. In agreements with these observations, these features were observed with the substitutes reconstructed according to the original method

the most representative compared with the original protocol.

and three different psoriatic patients were used (scale bar = 100 μm).

**Figure 6.** Keratin 14 and keratin 1 staining. Expression of keratin 14 (green) in healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced with the original method (A and D), the 6-well plate modification (B and E), and the 12-well plate modification (C and F). Expression of keratin 1 (green) in healthy substitutes (G–I), psoriatic substitutes (J–L) and substitutes produced according to the original method (G and J), the 6-well plate modification (H and K), and the 12-well plate modification (I and L). The nuclei were stained with Hoechst (blue). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were used (scale bar = 100 μm).

Previous studies of our group have shown that this psoriatic skin model demonstrated phenotypic characteristics of *in vivo* psoriasis [31–33]. In the present study, it is demonstrated that the improvements made to the original self-assembly method did not affect the psoriatic phenotype of the substitutes. *In vivo* psoriatic features are still expressed in the substitutes reconstructed according to the 6-well plate and 12-well plate modifications to protocol. Indeed,

**Figure 7.** Laminin staining. Expression of laminin (green) in healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced according to the original method (A and D), the 6-well plate modification (B and E), and the 12-well plate modification (C and F). The nuclei were stained with Hoechst (blue). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two different healthy patients and three different psoriatic patients were used (scale bar = 100 μm).

in both new protocols, a significant increase in the living epidermis thickness of psoriatic substitutes is observed compared with their respective healthy substitute counterparts as for the original method. Interestingly, the epidermal thickness of psoriatic substitutes produced according to the 12-well plate modification was significantly thicker than those produced according to the original method. This difference could be explained by the same seeding number of keratinocytes in a smaller seeding area for the 12-well plate protocol. Thus, this induces an increase of acanthosis. Considering histological analysis, our model stands out from other models of *in vitro* psoriatic skin that did not show acanthosis [24, 25]. However, based on these histological observations, the 6-well plate modification to protocol seems to be the most representative compared with the original protocol.

Differentiation of psoriatic skin is characterized by the altered expression of several epidermal proteins [43]. In a normal differentiation process (approximately 28 days), the basal layer transit amplifying cells differentiate and migrate into upper epidermal layers and synthesize important proteins involved in the differentiation and the skin barrier function such as filaggrin, loricrin, and keratins [36, 37, 44]. Filaggrin is normally synthesized from a precursor, profilaggrin, found in the granular layer. This protein is a key role in the formation of the cornified envelope [37]. In skin disease, such as psoriasis, expression of filaggrin is decreased, such that it is sometimes even absent due to an altered differentiation process and a reduction or an absence of the granular layer (agranulose) [38, 45, 46]. Loricrin, a major component of the cornified envelope, is stored in granules of the *stratum granulosum*, and its expression is also decreased or absent in psoriatic skin [36, 47]. In agreements with these observations, these features were observed with the substitutes reconstructed according to the original method

Previous studies of our group have shown that this psoriatic skin model demonstrated phenotypic characteristics of *in vivo* psoriasis [31–33]. In the present study, it is demonstrated that the improvements made to the original self-assembly method did not affect the psoriatic phenotype of the substitutes. *In vivo* psoriatic features are still expressed in the substitutes reconstructed according to the 6-well plate and 12-well plate modifications to protocol. Indeed,

different healthy patients and three different psoriatic patients were used (scale bar = 100 μm).

204 Cell Culture

**Figure 6.** Keratin 14 and keratin 1 staining. Expression of keratin 14 (green) in healthy substitutes (A–C), psoriatic substitutes (D–F) and substitutes produced with the original method (A and D), the 6-well plate modification (B and E), and the 12-well plate modification (C and F). Expression of keratin 1 (green) in healthy substitutes (G–I), psoriatic substitutes (J–L) and substitutes produced according to the original method (G and J), the 6-well plate modification (H and K), and the 12-well plate modification (I and L). The nuclei were stained with Hoechst (blue). Three substitutes of each condition were analyzed, and the results were confirmed with three independent experiments. Cells from two and the two modifications to protocol (6-well plate and 12-well plate modifications). Indeed, filaggrin and loricrin were detected in healthy skin substitutes, whereas their absence was observed in psoriatic skin substitutes. This therefore confirms that the characteristics associated with the psoriatic phenotype are preserved with these new methods.

fibroblasts and culture medium than the original method for the production of fibroblast sheets, which favorably reduce production costs. In addition, the use of plates and anchoring papers at the beginning of the production greatly facilitate the handling and the superposition of fibroblast sheets increasing the quality of the dermal equivalent. The anchoring papers allow the production of more reproducible and uniform size substitutes within the same experiment and between the different studies, which are essential for pharmaceutical studies. In conclusion, the modifications made to the original self-assembly method for the production of psoriatic substitutes are effective and demonstrate a comparable phenotype. However, the 6-well method is the one that leads to reconstructed substitutes with characteristics more similar to those seen *in vivo*. These modifications make it possible to obtain substitutes that are distinguished by better

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The authors acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) through their joint Collaborative Health Research Projects (CHRP) program. The "Fonds d'Enseignement et de Recherche (FER)" of the Faculty of Pharmacy, Université Laval, Québec, QC, Canada (Isabelle Gendreau scholarship), the "Fonds de Recherche du Québec—Santé (FRQS)" (Alexe Grenier scholarship) and the support of the "Réseau ThéCell du Québec" are

The authors state no conflict of interest. The authors have no relationship with a for-profit or a not-for-profit organization to disclose. There is no financial conflict with the subject or the

1 Centre de Recherche en Organogénèse Expérimentale de l'Université Laval/LOEX,

2 Centre de Recherche du CHU de Québec-Université Laval, Québec, QC, Canada

† These two authors contributed equally to the work and therefore should be considered

reproducibility making them new tools of choice for pharmacological analyses.

also acknowledged. Moreover, Dr. Pouliot is a FRQS career award scholar.

materials discussed in the manuscript apart from those disclosed.

Alexe Grenier1,2,3†, Isabelle Gendreau1,2,3† and Roxane Pouliot1,2,3\* \*Address all correspondence to: roxane.pouliot@pha.ulaval.ca

3 Faculté de Pharmacie, Université Laval, Québec, QC, Canada

**Acknowledgements**

**Conflict of interest**

**Author details**

Québec, QC, Canada

equivalent first authors.

Keratins are intermediate filaments highly involved in epidermal structure and different types are expressed in the varying differentiation stages [48]. K5 and K14 are normally found in the basal layer of the epidermis, and they are progressively replaced by K1 and K10 in suprabasal layers [49]. However, *in vivo* psoriatic skin shows K14 in all layers of the epidermis, including the *stratum corneum*. This therefore suggests that the degradation mechanism of this keratin is altered in psoriasis [48, 50]. Moreover, in such hyperproliferative diseases, a new pair of keratins, K6 and K16, is appearing, causing a decrease in the expression of K1 and K10 [48, 50–52]. In the present work, healthy and psoriatic skin substitutes reconstructed according to the original method and the 6-well plate modification to protocol have demonstrated the same K1 and K14 expression than *in vivo*. Thus, taking together, K1, K14, filaggrin, and loricrin results validated the conservation of psoriatic skin differentiation in the new 6-well plate modification. This suggests that the 6-well method would be a great alternative to the original method. In healthy substitutes produced following the 12-well plate modification, an abnormal presence of K14 and K1 was observed in the *stratum corneum*, showing a less efficient differentiation of keratinocytes. This can probably be explained by the higher number of cells seeded in the culture area. These observations demonstrated that the 6-well plate modification is more effective for the production of healthy substitutes than the 12-well plate modification.

Some studies suggested that alterations in the basal membrane of psoriatic skin play an important role in the abnormal proliferation and differentiation of psoriatic keratinocytes [53–55]. Indeed, the expression of proteins such as laminin, which is one of the main proteins that forms the basal membrane, is decreased and disrupted in psoriatic skin unlike in healthy skin. In this last, laminin forms a linear and continuous structure [54]. Laminin expression in our skin substitutes produced with healthy cells regardless of the method (original method and two new modifications) was intense, continuous and more restricted to the basal lamina, demonstrating a good structure of the basal membrane. For psoriatic skin substitutes (original method and 6-well plate modification), the expression of laminin was more distributed through the dermis compared to healthy substitutes, showing disorganization in the basal membrane. For the psoriatic skin substitutes reconstructed according to the 12-well plate modification, laminin staining was more compact and similar to healthy substitute expression. Interestingly, this observation is showing that the basal membrane was more organized, thus less similar to the psoriatic phenotype. These results showed that the 6-well plate modification is more representative of the *in vivo* psoriatic skin than the 12-well plate modification.

#### **2.3. Conclusion**

These new modifications to protocol provide several advantages in the production of skin substitutes. Indeed, the 6-well and 12-well plate modifications require almost 3 times fewer fibroblasts and culture medium than the original method for the production of fibroblast sheets, which favorably reduce production costs. In addition, the use of plates and anchoring papers at the beginning of the production greatly facilitate the handling and the superposition of fibroblast sheets increasing the quality of the dermal equivalent. The anchoring papers allow the production of more reproducible and uniform size substitutes within the same experiment and between the different studies, which are essential for pharmaceutical studies. In conclusion, the modifications made to the original self-assembly method for the production of psoriatic substitutes are effective and demonstrate a comparable phenotype. However, the 6-well method is the one that leads to reconstructed substitutes with characteristics more similar to those seen *in vivo*. These modifications make it possible to obtain substitutes that are distinguished by better reproducibility making them new tools of choice for pharmacological analyses.
