BACKGROUND\nMETHODS\nRESULTS\nCONCLUSION\nVulvar squamous cell carcinoma (VSCC) is a rare disease with a poor prognosis. To date, there's no proper in vitro modeling system for VSCC to study its... Show moreBACKGROUND\nMETHODS\nRESULTS\nCONCLUSION\nVulvar squamous cell carcinoma (VSCC) is a rare disease with a poor prognosis. To date, there's no proper in vitro modeling system for VSCC to study its pathogenesis or for drug evaluation.\nWe established healthy vulvar (HV)- and VSCC-like 3D full thickness models (FTMs) to observe the tumor-stroma interaction and their applicability for chemotherapeutic efficacy examination. VSCC-FTMs were developed by seeding VSCC tumor cell lines (A431 and HTB117) onto dermal matrices harboring two NF subtypes namely papillary fibroblasts (PFs) and reticular fibroblasts (RFs), or cancer-associated fibroblasts (CAFs) while HV-FTMs were constructed with primary keratinocytes and fibroblasts isolated from HV tissues.\nHV-FTMs highly resembled HV tissues in terms of epidermal morphogenesis, basement membrane formation and collagen deposition. When the dermal compartment shifted from PFs to RFs or CAFs in VSCC-FTMs, tumor cells demonstrated more proliferation, EMT induction and stemness. In contrast to PFs, RFs started to lose their phenotype and express robust CAF-markers α-SMA and COL11A1 under tumor cell signaling induction, indicating a favored 'RF-to-CAF' transition in VSCC tumor microenvironment (TME). Additionally, chemotherapeutic treatment with carboplatin and paclitaxel resulted in a significant reduction in tumor-load and invasion in VSCC-FTMs.\nWe successfully developed in vitro 3D vulvar models mimicking both healthy and tumorous conditions which serve as a promising tool for vulvar drug screening programs. Moreover, healthy fibroblasts demonstrate heterogeneity in terms of CAF-activation in VSCC TME which brings insights in the future development of novel CAF-based therapeutic strategies in VSCC. Show less
Mieremet, A.; Helder, R.W.J.; Nadaban, A.; Boiten, W.A.; Gooris, G.S.; El Ghalbzouri, A.; Bouwstra, J.A. 2021
In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to... Show moreIn vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin. Show less
Bouwstra. J.A.; Helder, R.W.J.; El Ghalbzouri, A. 2021
To advance drug development representative reliable skin models are indispensable. Animal skin as test model for human skin delivery is restricted as their properties greatly differ from human skin... Show moreTo advance drug development representative reliable skin models are indispensable. Animal skin as test model for human skin delivery is restricted as their properties greatly differ from human skin. In vitro 3D-human skin equivalents (HSEs) are valuable tools as they recapitulate important aspects of the human skin. However, HSEs still lack the full barrier functionality as observed in native human skin, resulting in suboptimal screening outcome. In this review we provide an overview of established in-house and commercially available HSEs and discuss in more detail to what extent their skin barrier biology is mimicked in vitro focusing on the lipid properties and cornified envelope. Further, we will illustrate how underlying factors, such as culture medium improvements and environmental factors affect the barrier lipids. Lastly, potential improvements in skin barrier function will be proposed aiming at a new generation of HSEs that may replace animal skin delivery studies fully. Show less
Helder, R.W.J.; Rousel, J.; Boiten, W.A.; Gooris, G.S.; Nadaban, A.; El Ghalbzouri, A.; Bouwstra, J.A. 2021
Full thickness models (FTM) are 3D in vitro skin cultures that resemble the native human skin (NHS) to a great extent. However, the barrier function of these skin models is reduced. The skin... Show moreFull thickness models (FTM) are 3D in vitro skin cultures that resemble the native human skin (NHS) to a great extent. However, the barrier function of these skin models is reduced. The skin barrier is located in the stratum corneum (SC) and consists of corneocytes embedded in a lipid matrix. In this matrix, deviations in the composition of the FTMs lipid matrix may contribute to the impaired skin barrier when compared to NHS. One of the most abundant changes in lipid composition is an increase in monounsaturated lipids for which stearoyl-CoA desaturase-1 (SCD-1) is responsible. To improve the SC lipid composition, we reduced SCD-1 activity during the generation of the FTMs. These FTMs were subsequently assessed on all major aspects, including epidermal homeostasis, lipid composition, lipid organization, and barrier functionality. We demonstrate that SCD-1 inhibition was successful and resulted in FTMs that better mimic the lipid composition of FTMs to NHS by a significant reduction in monounsaturated lipids. In conclusion, this study demonstrates an effective approach to normalize SC monounsaturated lipid concentration and may be a valuable tool in further optimizing the FTMs in future studies. Show less
Mieremet, A.; Helder, R.W.J.; Nadaban, A.; Gooris, G.S.; Boiten, W.A.; El Ghalbzouri, A.; Bouwstra, J.A. 2019
The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is... Show moreThe outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 μM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs. Show less
Helder, R.W.J.; Boiten, W.A.; Dijk, R. van; Gooris, G.S.; El Ghalbzouri, A.; Bouwstra, J.A. 2019
Full thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a... Show moreFull thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a reduced skin barrier. The most pronounced differences in lipid composition are a reduction in lipid chain length and increased monounsaturated lipids. The liver-X-receptor (LXR) activates the monounsaturated lipid synthesis via stearoyl-CoA desaturase-1 (SCD-1). Therefore, the aim was to improve the SC lipid synthesis of FTMs by LXR deactivation. This was achieved by supplementing culture medium with LXR antagonist GSK2033. LXR agonist T0901317 was added for comparison. Subsequently, epidermal morphogenesis, lipid composition, lipid organization and the barrier functionality of these FTMs were assessed. We demonstrate that LXR deactivation resulted in a lipid composition with increased overall chain lengths and reduced levels of monounsaturation, whereas LXR activation increased the amount of monounsaturated lipids and led to a reduction in the overall chain length. However, these changes did not affect the barrier functionality. In conclusion, LXR deactivation led to the development of FTMs with improved lipid properties, which mimic the lipid composition of NHS more closely. These novel findings may contribute to design interventions to normalize SC lipid composition of atopic dermatitis patients. Show less
Mieremet, A.; Dijk, R. van; Boiten, W.A.; Gooris, G.S.; Bouwstra, J.A.; El Ghalbzouri, A. 2019
Human skin equivalents (HSEs) are in vitro developed three-dimensional models resembling native human skin (NHS) to a high extent. However, the epidermal lipid biosynthesis, barrier lipid... Show moreHuman skin equivalents (HSEs) are in vitro developed three-dimensional models resembling native human skin (NHS) to a high extent. However, the epidermal lipid biosynthesis, barrier lipid composition, and organization are altered, leading to an elevated diffusion rate of therapeutic molecules. The altered lipid barrier formation in HSEs may be induced by standardized culture conditions, including a culture temperature of 37°C, which is dissimilar to skin surface temperature. Therefore, we aim to determine the influence of culture temperature during the generation of full thickness models (FTMs) on epidermal morphogenesis and lipid barrier formation. For this purpose, FTMs were developed at conventional core temperature (37°C) or lower temperatures (35°C and 33°C) and evaluated over a time period of 4 weeks. The stratum corneum (SC) lipid composition was analysed using advanced liquid chromatography coupled to mass spectrometry analysis. Our results show that SC layers accumulated at a similar rate irrespective of culture temperature. At reduced culture temperature, an increased epidermal thickness, a disorganization of the lower epidermal cell layers, a delayed early differentiation, and an enlargement of granular cells were detected. Interestingly, melanogenesis was reduced at lower temperature. The ceramide subclass profile, chain length distribution, and level of unsaturated ceramides were similar in FTMs generated at 37°C and 35°C but changed when generated at 33°C, reducing the resemblance to NHS. Herein, we report that culture temperature affects epidermal morphogenesis substantially and to a lesser extent the lipid barrier formation, highlighting the importance of optimized external parameters during reconstruction of skin. Show less
Mieremet, A.; Boiten, W.A.; Dijk, R. van; Gooris, G.S.; Overkleeft, H.S.; Aerts, J.M.F.G.; ... ; El Ghalbzouri, A. 2019
Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of... Show moreRelative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH. Show less
Mieremet, A.; Dijk, R. van; Gooris, G.; Bouwstra, J.A.; El Ghalbzouri, A. 2019
Human skin equivalents (HSEs) are three dimensional models resembling native human skin (NHS) in manyaspects. Despite the manifold similarities to NHS, a restriction in its applications is the... Show moreHuman skin equivalents (HSEs) are three dimensional models resembling native human skin (NHS) in manyaspects. Despite the manifold similarities to NHS, a restriction in its applications is the altered in vitro lipidbarrier formation, which compromises the barrier functionality. This could be induced by suboptimal cell culturingconditions, which amongst others is the diminished activation of the vitamin D receptor (VDR) signallingpathway. The active metabolite of this signalling pathway is 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). An interactingrole in the formation of the skin barrier has been ascribed to this pathway, although it remains unresolvedto which extent this pathway contributes to the (mal-)formation of the epidermal barrier in HSEs. Ouraim is to study whether cell culture medium enriched with 1,25(OH)2D3 affects epidermal morphogenesis andlipid barrier formation in HSEs. Addition of 20 nM 1,25(OH)2D3 resulted in activation of the VDR signallingpathway by inducing transcription of VDR target genes (CYP24A and LL37) in keratinocyte monocultures and inHSEs. Characterization of HSEs supplemented with 1,25(OH)2D3 using immunohistochemical analyses revealeda high similarity in epidermal morphogenesis and in expression of lipid processing enzymes. The barrier formationwas assessed using state-of-the art techniques analysing lipid composition and organization. Addition of1,25(OH)2D3 did not alter the composition of ceramides. Additionally, the lateral and lamellar organization ofthe lipids was similar, irrespective of supplementation. In conclusion, epidermal morphogenesis and barrierformation in HSEs generated in presence or absence of 1,25(OH)2D3 leads to a similar morphogenesis andcomparable barrier formation in vitro. Show less
Niehues, H.; Bouwstra, J.A.; El Ghalbzouri, A.; Brandner, J.M.; Zeeuwen, P.L.J.M.; Bogaard, E.H. van den 2018
The skin barrier is an important shield regulating the outside-in as well as inside-out penetration of water, nutrients, ions and environmental stimuli. We can distinguish four different barrier... Show moreThe skin barrier is an important shield regulating the outside-in as well as inside-out penetration of water, nutrients, ions and environmental stimuli. We can distinguish four different barrier compartments: the physical, chemical, immunological and microbial skin barrier. Well-functioning of those is needed to protect our body from the environment. To better understand the function and the contribution of barrier dysfunction in skin diseases, 3D skin or epidermal models are a valuable tool for in vitro studies. In this review, we summarize the development and application of different skin models in skin barrier research. During the last years, enormous effort was made on optimizing these models to better mimic the in vivo composition of the skin, by fine-tuning cell culture media, culture conditions and including additional cells and tissue components. Thereby, in vitro barrier formation and function has been improved significantly. Moreover, in this review we point towards changes and chances for in vitro 3D skin models to be used for skin barrier research in the nearby future. Show less
Mieremet, A.; Rietveld, M.; Dijk, R. van; Bouwstra, J.A.; El Ghalbzouri, A. 2018
Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These... Show moreOur in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements. Show less
Drongelen, V. van; Danso, M.O.; Out, J.J.; Mulder, A.A.; Lavrijsen, A.P.; Bouwstra, J.A.; El Ghalbzouri, A. 2015
Atopic dermatitis (AD) is a common inflammatory skin disorder characterised by various epidermal alterations. Filaggrin (FLG) mutations are a major predisposing factor for AD and much research has... Show moreAtopic dermatitis (AD) is a common inflammatory skin disorder characterised by various epidermal alterations. Filaggrin (FLG) mutations are a major predisposing factor for AD and much research has been focused on the FLG protein. Human skin equivalents (HSEs) might be useful tools for increasing our understanding of FLG in AD and to provide a tool for the screening of new therapies aimed at FLG replacement. Our aim is to establish an explant HSE (Ex-HSE) for AD by using non-lesional skin from AD patients wildtype for FLG or harbouring homozygous FLG mutations. These Ex-HSEs were evaluated as to whether they maintained their in vivo characteristics in vitro and whether FLG mutations affected the expression of various differentiation markers. FLG mutations did not affect the outgrowth from the biopsy for the establishment of Ex-HSEs. FLG expression was present in healthy skin and that of AD patients without FLG mutations and in their Ex-HSEs but was barely present in biopsies from patients with FLG mutations and their corresponding Ex-HSEs. AD Ex-HSEs and AD biopsies shared many similarities, i.e., proliferation and the expression of keratin 10 and loricrin, irrespective of FLG mutations. Neither KLK5 nor Lekti expression was affected by FLG mutations but was altered in the respective Ex-HSEs. Thus, Ex-HSEs established from biopsies taken from AD patients maintain their FLG genotype-phenotype in vitro and the expression of most proteins in vivo and in vitro remains similar. Our method is therefore promising as an alternative to genetic engineering approaches in the study of the role of FLG in AD. Show less
Danso, M.O.; Drongelen, V. van; Mulder, A.; Gooris, G.S.; Smeden, J. van; El Ghalbzouri, A.; Bouwstra, J.A. 2015
BACKGROUND\nExplant human skin equivalents (Ex-HSEs) can be generated by placing a 4mm skin biopsy onto a dermal equivalent. The keratinocytes migrate from the biopsy onto the dermal equivalent,... Show moreBACKGROUND\nExplant human skin equivalents (Ex-HSEs) can be generated by placing a 4mm skin biopsy onto a dermal equivalent. The keratinocytes migrate from the biopsy onto the dermal equivalent, differentiate and form the epidermis of 1(st) generation Ex-HSEs. This is especially suitable for the expansion of skin material from which only small fragments of skin can be harvested e.g. diseased skin.\nOBJECTIVE\nWe evaluated whether 2(nd) and 3(rd) generation Ex-HSEs can also be generated from a single skin biopsy whilst maintaining the epidermal properties of 1(st) generation Ex-HSEs and native human skin.\nMETHODS\n2(nd) generation Ex-HSEs were produced by placing a biopsy from the 1(st) generation Ex-HSE onto a new dermal equivalent. Likewise, the 3(rd) generation Ex-HSEs were generated from a 2(nd) generation Ex-HSE biopsy.\nRESULTS\nWe show for the first time that Ex-HSEs can be passaged to the 2(nd) and 3(rd) generation and display similar epidermal morphology and expression of differentiation markers as in native human skin and 1(st) generation Ex-HSEs except for involucrin. The 2(nd) and 3(rd) generation Ex-HSEs also show many similarities with 1(st) generation Ex-HSEs in lipid properties e.g. presence of all lipid classes, similar fatty acid chain length distribution and lamellar lipid organization. However, some differences arise in increased level of hexagonal lateral packing and a change in ceramide profiling. The changes in specific lipid classes were also accompanied by changes in the expression of the enzymes responsible for their synthesis.\nCONCLUSION\nThe expansion of skin biopsies to the 2(nd) and 3(rd) generation Ex-HSEs could be a promising method to expand valuable epidermal tissue to analyze morphological and differentiation parameters in the native epidermis. Show less
