Application of Doxorubicin-loaded PLGA nanoparticles targeting both tumor cells and cancer-associated fibroblasts on 3D human skin equivalents mimicking melanoma and cutaneous squamous cell carcinoma.

Nanoparticle (NP) use in cancer therapy is extensively studied in skin cancers. Cancer-associated fibroblasts (CAFs), a major tumor microenvironment (TME) component, promote cancer progression, making dual targeting of cancer cells and CAFs an effective therapy. However, dual NP-based targeting therapy on both tumor cells and CAFs is poorly investigated in skin cancers. Herein, we prepared and characterized doxorubicin-loaded PLGA NPs (DOX@PLGA NPs) and studied their anti-tumor effects on cutaneous melanoma (SKCM)(AN, M14) and cutaneous squamous cell carcinoma (cSCC) (MET1, MET2) cell lines in monolayer, as well as their impact on CAF deactivation. Then, we established 3D full thickness models (FTM) models of SKCM and cSCC using AN or MET2 cells on dermis matrix populated with CAFs respectively, and assessed the NPs' tumor penetration, tumor-killing ability, and CAF phenotype regulation through both topical administration and intradermal injection. The results show that, in monolayer, DOX@PLGA NPs inhibited cancer cell growth and induced apoptosis in a dose- and time-dependent manner, with a weaker effect on CAFs. DOX@PLGA NPs reduced CAF-marker expression and had successful anti-tumor effects in 3D skin cancer FTMs, with decreased tumor-load and invasion. DOX@PLGA NPs also showed great delivery potential in the FTMs and could be used as a platform for future functional study of NPs in skin cancers using human-derived skin equivalents. This study provides promising evidence for the potential of DOX@PLGA NPs in dual targeting therapy for SKCM and cSCC.

The development of in vitro organotypic 3D vulvar models to study tumor-stroma interaction and drug efficacy.

BACKGROUND: Vulvar 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. METHODS: We 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. RESULTS: HV-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. CONCLUSION: We 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.

Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for proper lariat debranching and ectodermal differentiation.

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.

Mimicking fat grafting of fibrotic scars using 3D-organotypic skin cultures.

Wound healing of deep burn injuries is often accompanied by severe scarring, such as hypertrophic scar (HTS) formation. In severe burn wounds, where the subcutis is also damaged, the scars adhere to structures underneath, resulting in stiffness of the scar and impaired motion. Over the recent years, a promising solution has emerged: autologous fat grafting, also known as lipofilling. Previous clinical reports have shown that the anti-fibrotic effect has been attributed to the presence of adipose-derived stromal cells (ADSC). In the proposed study, we aim to investigate the effect of fat grafting in 3D organotypic skin cultures mimicking an HTS-like environment. To this end, organotypic skin cultures were embedded with normal skin fibroblasts (NF) or HTS-derived fibroblasts with or without incorporation of human adipose subcutaneous tissue (ADT) and one part was thermally wounded to examine their effect on epithelialization. The developed skin cultures were analysed on morphology and protein level. Analysis revealed that ADT-containing organotypic skin cultures comprise an improved epidermal homeostasis, and a fully formed basement membrane, similar to native human skin (NHS). Furthermore, the addition of ADT significantly reduced myofibroblast presence, which indicates its anti-fibrotic effect. Finally, re-epithelialization measurements showed that ADT reduced re-epithelialization in skin cultures embedded with NFs, whereas HTS-fibroblast-embedded skin cultures showed complete wound closure. In conclusion, we succeeded in developing a 3D organotypic HTS-skin model incorporated with subcutaneous tissue that allows further investigation on the molecular mechanism of fat grafting.

Human Papillary and Reticular Fibroblasts Show Distinct Functions on Tumor Behavior in 3D-Organotypic Cultures Mimicking Melanoma and HNSCC.

Human dermis can be morphologically divided into the upper papillary and lower reticular dermis. Previously, we demonstrated that papillary (PFs) and reticular (RFs) fibroblasts show distinct morphology and gene expression profiles. Moreover, they differently affect tumor invasion and epithelial-to-mesenchymal transition (EMT) in in vitro 3D-organotypic cultures of cutaneous squamous cell carcinoma (cSCC). In this study, we examined if these distinct effects of PFs and RFs can be extrapolated in other epithelial/non-epithelial tumors such as melanoma and head and neck squamous cell carcinoma (HNSCC). To this end, 3D-Full-Thickness Models (FTMs) were established from melanoma (AN and M14) or HNSCC cell lines (UM-SCC19 and UM-SCC47) together with either PFs or RFs in the dermis. The interplay between tumor cells and different fibroblasts was investigated. We observed that all the tested tumor cell lines showed significantly stronger invasion in RF-FTMs compared to PF-FTMs. In addition, RF-FTMs demonstrated more tumor cell proliferation, EMT induction and basement membrane disruption. Interestingly, RFs started to express the cancer-associated fibroblast (CAF) biomarker α-SMA, indicating reciprocal interactions eventuating in the transition of RFs to CAFs. Collectively, in the melanoma and HNSCC FTMs, interaction of RFs with tumor cells promoted EMT and invasion, which was accompanied by differentiation of RFs to CAFs.

The effect of TGFßRI inhibition on fibroblast heterogeneity in hypertrophic scar 2D in vitro models.

In burn patients, wound healing is often accompanied by hypertrophic scarring (HTS), resulting in both functional and aesthetic problems. HTSs are characterized by abundant presence of myofibroblasts (MFs) residing in the dermis. HTS development and MF persistence is primarily regulated by TGF-ß signalling. A promising method to target the transforming growth factor receptor I (TGFßRI; also known as activin-like kinase 5 (ALK5)) is by making use of exon skipping through antisense oligonucleotides. In HTS the distinguishing border between the papillary dermis and the reticular dermis is completely abrogated, thus exhibiting a one layered dermis containing a heterogenous fibroblast population, consisting of papillary fibroblasts (PFs), reticular fibroblasts (RFs) and MFs. It has been proposed that PFs, as opposed to RFs, exhibit anti-fibrotic properties. Currently, it is still unclear which fibroblast subtype is most affected by exon skipping treatment. Therefore, the aim of this study was to investigate the effect of TGFßRI inhibition by exon skipping in PF, RF and HTS fibroblast monocultures. Morphological analyses revealed the presence of a PF-like population after exon skipping in the different fibroblast cultures. This observation was further confirmed by the expression of genes specific for PFs, demonstrated by qPCR analyses. Further investigations on mRNA and protein level revealed that indeed MFs and to a lesser extent RFs are targeted by exon skipping. Furthermore, collagen gel contraction analysis showed that ALK5 exon skipping reduced TGF-ß- induced contraction together with decreased alpha-smooth muscle actin expression levels. In conclusion, we show for the first time that exon skipping primarily targets pro-fibrotic fibroblasts. This could be a promising step towards reduced HTS development of burn tissue.

Exon skipping of TGFßRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts.

BACKGROUND: In burn patients, wound healing is often accompanied by hypertrophic scar (HS) development, resulting in both functional and aesthetic problems. HSs are characterised by abundant presence of myofibroblasts that contribute to overproduction of extracellular matrix (ECM) that is regulated by the TGF-ß signalling pathway. Studies have shown that inhibition of TGF-ß receptors in fibrotic diseases reduces the fibrotic load. In the present study, we aim to inactivate ALK5, also known as TGF-ß receptor I, in human HS fibroblasts by exon skipping using antisense oligonucleotides (AONs). METHODS: HS biopsies were used to isolate and set up fibroblast monocultures. AONs targeting ALK5 were supplemented to the fibroblast cultures to induce exon skipping, while pharmacological ALK5 inhibition was induced using SB431542. AON delivery in HS fibroblasts was examined using immunofluorescence (IF), while TGF-ß signalling downstream targets, such as Smad2/3, PAI-1, ACTA2, COL1A1 and COL3A1, were analysed using touchdown polymerase chain reaction (PCR), quantitative PCR (qPCR), IF or western blotting. RESULTS: Our data clearly demonstrate that AONs were successfully delivered in the nuclei of HS fibroblasts and that functional exon skipping of ALK5 took place as confirmed with touchdown PCR and qPCR. In addition, exon skipping affected the expression of ECM-related genes, such as type I/III collagens, PAI-1 and CCN2. Moreover, AON treatment did not affect the migration of HS fibroblasts in a model for wound healing. CONCLUSION: Exon skipping is a promising tool to modulate the TGF-ß signalling pathway in HS. This would open a therapeutic window for the treatment of patients suffering from HSs.

Eradication of meticillin-resistant Staphylococcus aureus from human skin by the novel LL-37-derived peptide P10 in four pharmaceutical ointments.

Skin bacterial colonization/infection is a frequent cause of morbidity in patients with chronic wounds and allergic/inflammatory skin diseases. This study aimed to develop a novel approach to eradicate meticillin-resistant Staphylococcus aureus (MRSA) from human skin. To achieve this, the stability and antibacterial activity of the novel LL-37-derived peptide P10 in four ointments was compared. Results indicate that P10 is chemically stable and antibacterial in hypromellose gel and Softisan-containing cream, but not in Cetomacrogol cream (with or without Vaseline), at 4 °C for 16 months. Reduction in MRSA counts on Leiden human epidermal models (LEMs) by P10 in hypromellose gel was greater than that of the peptide in Cetomacrogol cream or phosphate buffered saline. P10 did not show adverse effects on LEMs irrespective of the ointment used, while Cetomacrogol with Vaseline and Softisan cream, but not hypromellose gel or Cetomacrogol cream, destroyed MRSA-colonized LEMs. Taking all this into account, P10 in hypromellose gel dose-dependently reduced MRSA colonizing the stratum corneum of the epidermis as well as biofilms of this bacterial strain on LEMs. Moreover, P10 dose-dependently reduced MRSA counts on ex-vivo human skin, with P10 in hypromellose gel being more effective than P10 in Cetomacrogol and Softisan creams. P10 in hypromellose gel is a strong candidate for eradication of MRSA from human skin.

Blocking negative effects of senescence in human skin fibroblasts with a plant extract.

There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris, which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

A shift from papillary to reticular fibroblasts enables tumour-stroma interaction and invasion.

BACKGROUND: Tumour stroma consists of a heterogeneous population of fibroblasts and related mesenchymal cells, collectively dubbed cancer-associated fibroblasts (CAFs). These CAFs are key players in cancer invasion of cutaneous squamous cell carcinoma (SCC). As we have shown earlier, papillary and reticular fibroblasts (Pfs and Rfs, respectively) have distinct effects on epidermal and dermal homeostasis, but their role in cancer invasion and epithelial-to-mesenchymal transition (EMT) remains to be determined. METHODS: We used 3D cultures of human skin equivalents (HSEs) to analyse the effects of Pfs and Rfs on the invasive behaviour of SCC and EMT. RESULTS: We reveal for the first time the importance of Pfs versus Rfs in SCC invasion and EMT. Cell lines from different stages of SCC showed significantly more extensive invasion into a dermis composed of Rfs than of Pfs. In addition, Rfs-based HSEs showed increased cell activation and stained positive for CAF biomarkers α-SMA and vimentin. Further analysis revealed that invasively growing cancer cells in Rf-HSEs express markers of EMT transition, like SNAIL2, N-cadherin and ZEB1. CONCLUSIONS: Conversely, our results show that Pfs contain cancer cells more within the epidermis. Rfs are clearly predisposed to differentiate into CAFs upon SCC signals, assisting invasion and EMT.

Recapitulation of Native Dermal Tissue in a Full-Thickness Human Skin Model Using Human Collagens.

OBJECTIVE: Full-thickness skin models comprise a three-dimensional dermal equivalent based on an animal-derived collagen matrix that harbors fibroblasts and an epidermal equivalent formed by keratinocytes. The functionality of both equivalents is influenced by many factors, including extracellular matrix composition and resident cell type. Animal-derived collagens differ in amino acid composition and physicochemical properties from human collagens. This composition could alter the functionality of the dermal equivalent and epidermal morphogenesis with the barrier formation in full-thickness models (FTMs). By replacement of animal-derived collagen for human collagen, we generated and characterized the animal material-free human collagen full-thickness models (hC-FTMs) that better mimic native dermal tissue. MATERIALS AND METHODS: An isolation procedure to obtain soluble collagen from human abdominal dermis was developed. Both FTMs and hC-FTMs were generated with primary human fibroblasts and keratinocytes. Immunohistochemical analyses with biomarkers for the dermal matrix composition, basement membrane (BM) formation, epidermal proliferation, differentiation, and activation were performed. The stratum corneum (SC) lipid composition was studied with liquid chromatography-mass spectrometry. Lipid lamellar organization was determined by small-angle X-ray diffraction. RESULTS: The FTMs and hC-FTMs exhibit many similarities, including the dermal matrix structure, BM formation, epidermal basal layer proliferation, and execution of differentiation programs. The SC contains a similar number of corneocyte layers and the same level of lipids. The ceramide chain length distribution and ceramide subclass profile showed only minor differences. Subsequently, this led to an unaltered lamellar organization. CONCLUSION: The animal material-free hC-FTM is generated successfully using collagens isolated from human abdominal dermis. Utilization of human collagens revealed that (epi-)dermal morphogenesis and lipid barrier formation resembled that of original FTMs. The hC-FTMs contain a dermal equivalent that mimics the native stromal tissue to a higher extent. Therefore these in vitro skin models can be used as promising tool for research purposes that contribute to animal-free experimentation.

Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

Improved epidermal barrier formation in human skin models by chitosan modulated dermal matrices.

Full thickness human skin models (FTMs) contain an epidermal and a dermal equivalent. The latter is composed of a collagen dermal matrix which harbours fibroblasts. Current epidermal barrier properties of FTMs do not fully resemble that of native human skin (NHS), which makes these human skin models less suitable for barrier related studies. To further enhance the resemblance of NHS for epidermal morphogenesis and barrier formation, we modulated the collagen dermal matrix with the biocompatible polymer chitosan. Herein, we report that these collagen-chitosan FTMs (CC-FTMs) possess a well-organized epidermis and maintain both the early and late differentiation programs as in FTMs. Distinctively, the epidermal cell activation is reduced in CC-FTMs to levels observed in NHS. Dermal-epidermal interactions are functional in both FTM types, based on the formation of the basement membrane. Evaluation of the barrier structure by the organization of the extracellular lipid matrix of the stratum corneum revealed an elongated repeat distance of the long periodicity phase. The ceramide composition exhibited a higher resemblance of the NHS, based on the carbon chain-length distribution and subclass profile. The inside-out barrier functionality indicated by the transepidermal water loss is significantly improved in the CC-FTMs. The expression of epidermal barrier lipid processing enzymes is marginally affected, although more restricted to a single granular layer. The novel CC-FTM resembles the NHS more closely, which makes them a promising tool for epidermal barrier related studies.

Inflammatory and antimicrobial responses to methicillin-resistant Staphylococcus aureus in an in vitro wound infection model.

Treatment of patients with burn wound infections may become complicated by the presence of antibiotic resistant bacteria and biofilms. Herein, we demonstrate an in vitro thermal wound infection model using human skin equivalents (HSE) and biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) for the testing of agents to combat such infections. Application of a liquid nitrogen-cooled metal device on HSE produced reproducible wounds characterized by keratinocyte death, detachment of the epidermal layer from the dermis, and re-epithelialization. Thermal wounding was accompanied by up-regulation of markers for keratinocyte activation, inflammation, and antimicrobial responses. Exposure of thermal wounded HSEs to MRSA resulted in significant numbers of adherent MRSA/HSE after 1 hour, and multiplication of these bacteria over 24-48 hours. Exposure to MRSA enhanced expression of inflammatory mediators such as TLR2 (but not TLR3), IL-6 and IL-8, and antimicrobial proteins human ß-defensin-2, -3 and RNAse7 by thermal wounded as compared to control HSEs. Moreover, locally applied mupirocin effectively reduced MRSA counts on (thermal wounded) HSEs by more than 99.9% within 24 hours. Together, these data indicate that this thermal wound infection model is a promising tool to study the initial phase of wound colonization and infection, and to assess local effects of candidate antimicrobial agents.

Effects of serially passaged fibroblasts on dermal and epidermal morphogenesis in human skin equivalents.

Serial passaging has a profound effect on primary cells. Since serially passaged cells show signs of cellular aging, serial passaging is used as an in vitro model of aging. To relate the effect of in vitro aging more to in vivo aging, we generated human skin equivalents (HSEs). We investigated if HSEs generated with late passage fibroblasts show characteristics of aged skin when compared with HSEs generated with early passage fibroblasts. Late passage fibroblasts had enlarged cell bodies and were more often positive for myofibroblast marker α-smooth muscle actin, senescence associated ß-galactosidase and p16 compared with early passage fibroblasts. Skin equivalents generated with late passage fibroblasts had a thinner dermis, which could partly be explained by increased matrix metalloproteinase-1 secretion. In equivalents generated with late passage fibroblasts epidermal expression of keratin 6 was increased, and of keratin 10 slightly decreased. However, epidermal proliferation, epidermal thickness and basement membrane formation were not affected. In conclusion, compared with HSEs generated with early passage fibroblasts, HSEs generated with late passage fibroblasts showed changes in the dermis, but no or minimal changes in the basement membrane and the epidermis.

Marine-derived nutrient improves epidermal and dermal structure and prolongs the life span of reconstructed human skin equivalents.

INTRODUCTION: Imedeen™ is a cosmeceutical that provides nutrients to the skin. One of its active ingredients is the Marine Complex™ (MC). AIM: The aim of this study was to evaluate whether MC affects skin morphogenesis differently in female and male human skin equivalents (HSEs). METHODS: Human skin equivalents were established with cells obtained from female or male donors between 30 and 45 years of age and cultured for seven or 11 weeks in the presence or absence of MC. Using immunohistochemistry, we examined early differentiation by keratin 10 expression, (hyper)proliferation by keratin 17 and Ki67, and basement membrane composition by laminin 332 and collagen type VII. In addition, the expression of collagen type I and the secretion of pro-collagen I were measured. RESULTS: Marine Complex strongly increased the number of Ki67-positive epidermal cells in female HSEs. In the dermis, MC significantly stimulated the amount of secreted pro-collagen I and increased the deposition of laminin 332 and collagen type VII. Furthermore, MC prolonged the viable phase of HSEs by slowing down its natural degradation. After 11 weeks of culturing, the MC-treated HSEs showed higher numbers of viable epidermal cell layers and a thicker dermal extracellular matrix compared with controls. In contrast, these effects were less pronounced in male HSEs. CONCLUSION: The MC nutrient positively stimulated overall HSE tissue formation and prolonged the longevity of both female and male HSEs. The ability of MC to stimulate the deposition of basement membrane and dermal components can be used to combat 2 human skin aging in vivo.

Three-dimensional human skin equivalent as a tool to study Acinetobacter baumannii colonization.

Acinetobacter baumannii can colonize body surfaces of hospitalized patients. From these sites, invasion into the host and spread to other patients and the hospital environment may occur. The eradication of the organism from the patient's skin is an important infection control strategy during epidemic and endemic episodes. In this study, a three-dimensional (3D), air-exposed human epidermal skin equivalent was exploited to study Acinetobacter skin colonization. We characterized the adherence of A. baumannii ATCC 19606(T) and Acinetobacter junii RUH2228(T) to and biofilm formation on the skin equivalent and the responses to these bacteria. Furthermore, we assessed the ability of the disinfectant chlorhexidine to decolonize the skin equivalents. The results revealed that both strains replicated on the stratum corneum for up to 72 h but did not invade the epidermis. A. baumannii, in contrast to A. junii, formed large biofilms on the stratum corneum. Bacterial colonization did not affect keratinocyte activation, proliferation, or differentiation, nor did it induce a strong inflammatory response. Disinfection with chlorhexidine solution resulted in complete eradication of A. baumannii from the skin, without detrimental effects. This 3D model is a promising tool to study skin colonization and to evaluate the effects of novel disinfectant and antimicrobial strategies.

Unraveling barrier properties of three different in-house human skin equivalents.

Human skin equivalents (HSEs) are three-dimensional culture models that are used as a model for native human skin. In this study the barrier properties of two novel HSEs, the fibroblast-derived matrix model (FDM) and the Leiden epidermal model (LEM), were compared with the full-thickness collagen model (FTM) and human skin. Since the main skin barrier is located in the lipid regions of the upper layer of the skin, the stratum corneum (SC), we investigated the epidermal morphology, expression of differentiation markers, SC permeability, lipid composition, and lipid organization of all HSEs and native human skin. Our results demonstrate that the barrier function of the FDM and LEM improved compared with that of the FTM, but all HSEs are more permeable than human skin. Further, the FDM and LEM have a relatively lower free fatty acid content than the FTM and human skin. Several similarities between the FDM, LEM and FTM were observed: (1) the morphology and the expression of the investigated differentiation markers were similar to those observed in native human skin, except for the observed expression of keratin 16 and premature expression of involucrin that were detected in all HSEs, (2) the lipids in the SC of all HSEs were arranged in lipid lamellae, similar to human skin, but show an increase in the number of lipid lamellae in the intercellular regions and (3) the SC lipids of all HSEs show a less densely packed lateral lipid organization compared with human SC. These findings indicate that the HSEs mimic many aspects of native human skin, but differ in their barrier properties.

Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products.

Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.