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
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
Cell-based in vitro developed human skin equivalents facilitate screenings of compounds for therapeutic potential or toxicity and enable scientific research expanding knowledge on skin physiology... Show moreCell-based in vitro developed human skin equivalents facilitate screenings of compounds for therapeutic potential or toxicity and enable scientific research expanding knowledge on skin physiology and pathophysiology. Human skin equivalents resemble key features of native human skin, including the dermal and epidermal architecture. However, a limitation of human skin equivalents is the altered lipid barrier formation, which leads to a decreased barrier functionality. This could be induced by suboptimal cell culture conditions or the different cell microenvironment. The primary aim of this dissertational research was to enhance the morphogenesis and barrier formation of human skin equivalents to better mimic that of native human skin. The results indicate that modification of the dermal extracellular matrix by the biopolymer chitosan enhanced epidermal morphogenesis and barrier formation. Furthermore, by better resembling native skin conditions in vitro, primarily through a reduction in oxygen level, the epidermal morphogenesis and lipid barrier formation was improved. Finally, using a combinatory approach of optimized cell culture conditions and enhanced cell culture medium, the epidermal morphogenesis and barrier formation of human skin equivalents resembled that of native human skin more closely. 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.; Vázquez, García A.; Boiten, W.; Dijk, R. van; Gooris, G.; Bouwstra, J.A.; Ghalbzouri, A. El 2019
Human skin equivalents (HSEs) are three-dimensional cell models mimicking characteristics of native human skin (NHS) in many aspects. However, a limitation of HSEs is the altered in vitro... Show moreHuman skin equivalents (HSEs) are three-dimensional cell models mimicking characteristics of native human skin (NHS) in many aspects. However, a limitation of HSEs is the altered in vitro morphogenesis and barrier formation. Differences between in vitro and in vivo skin could have been induced by suboptimal cell culture conditions, of which the level of oxygen in vitro (20%) is much higher than in vivo (0.5-8%). Our aim is to study how external oxygen levels affect epidermal morphogenesis and barrier formation in HSEs. In the present study, fibroblast and keratinocyte monocultures, and HSEs were generated under 20% (normoxia) and 3% (hypoxia) oxygen level. In all cultures under hypoxia, expression of hypoxia-inducible factor target genes was increased. Characterization of HSEs generated under hypoxia using immunohistochemical analyses of morphogenesis biomarkers revealed a reduction in epidermal thickness, reduced proliferation, similar early differentiation, and an attenuated terminal differentiation program compared to normoxia, better mimicking NHS. The stratum corneum ceramide composition was studied with liquid chromatography coupled to mass spectrometry. Under hypoxia, HSEs exhibited a ceramide composition that more closely resembles that of NHS. Consequently, the lipid organization was improved. In conclusion, epidermal morphogenesis and barrier formation in HSEs reconstructed under hypoxia better mimics that of NHS. 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
Mieremet, A.; Rietveld, M.; Dijk, R. van; Bouwstra, J.A.; Ghalbzouri, A. el 2018
In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a... Show moreIn the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32 °C and 37 °C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37 °C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32 °C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37 °C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32 °C and 37 °C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37 °C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32 °C. Potentially, this model can be used for testing formulations for skin barrier repair. Show less
Drongelen, V. van; Danso, M.O.; Mulder, A.; Mieremet, A.; Smeden, J. van; Bouwstra, J.A.; Ghalbzouri, A. el 2014
