Environmentally Controlled Microfluidic System Enabling Immune Cell Flow and Activation in an Endothelialised Skin-On-Chip.

Integration of reconstructed human skin (RhS) into organ-on-chip (OoC) platforms addresses current limitations imposed by static culturing. This innovation, however, is not without challenges. Microfluidic devices, while powerful, often encounter usability, robustness, and gas bubble issues that hinder large-scale high-throughput setups.

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function.

Background: Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited.

Methods: Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line (MUTZ-3 DC).

Proof-of-Concept Organ-on-Chip Study: Topical Cinnamaldehyde Exposure of Reconstructed Human Skin with Integrated Neopapillae Cultured under Dynamic Flow.

Pharmaceutical and personal care industries require human representative models for testing to ensure the safety of their products. A major route of penetration into our body after substance exposure is via the skin. Our aim was to generate robust culture conditions for a next generation human skin-on-chip model containing neopapillae and to establish proof-of-concept testing with the sensitizer, cinnamaldehyde.

A Multi-Organ-on-Chip Approach to Investigate How Oral Exposure to Metals Can Cause Systemic Toxicity Leading to Langerhans Cell Activation in Skin.

Investigating systemic toxicity is still a huge challenge. Here, a multi-organ-on-chip approach is presented as a typical case of topical exposure of oral mucosa to metals, which are known to activate the immune system and in turn may result in skin inflammation. Reconstructed human gingiva (RHG) and reconstructed human skin containing MUTZ-3-derived Langerhans cells (MUTZ-LC) in the epidermis (RHS-LC) were incorporated into a HUMIMIC Chip3plus, connected by dynamic flow and cultured for a total period of 72 h.

Carcinogenic effects of prolonged daily low-emission phototherapy in psoriasis.

Background: Low-dose UV treatment has been shown to be effective in mild psoriasis. However, the prolonged use of this treatment modality may raise concerns about its safety. These concerns are mainly focused on potential carcinogenic risks and overuse of this treatment modality.

Patch test-relevant concentrations of metal salts cause localized cytotoxicity, including apoptosis, in skin ex vivo.

Background: Metal alloys containing contact sensitizers (nickel, palladium, titanium) are extensively used in medical devices, in particular dentistry and orthopaedic surgery. The skin patch test is used to test for metal allergy.

Objective: To determine whether metal salts, when applied to freshly excised skin at patch test-relevant concentrations and using a method which mimics skin patch testing, cause in changes in the epidermis and dermis.

Assessment of cytotoxicity and sensitization potential of intradermally injected tattoo inks in reconstructed human skin.

Background: The number of people within the European population having at least one tattoo has increased notably, and with it the number of tattoo-associated clinical complications. Despite this, safety information and testing regarding tattoo inks remain limited.

Objective: To assess cytotoxicity and sensitization potential of 16 tattoo inks after intradermal injection into reconstructed human skin (RHS).

Targeting of the C-Type Lectin Receptor Langerin Using Bifunctional Mannosylated Antigens.

Langerhans cells (LCs) are antigen-presenting cells that reside in the skin. They uniquely express high levels of the C-type lectin receptor Langerin (CD207), which is an attractive target for antigen delivery in immunotherapeutic vaccination strategies against cancer. We here assess a library of 20 synthetic, well-defined mannoside clusters, built up from one, two, and three of six monomannosides, dimannosides, or trimannosides, appended to an oligopeptide backbone, for binding with Langerin using surface plasmon resonance and flow cytometric quantification.

Titanium salts tested in reconstructed human skin with integrated MUTZ-3-derived Langerhans cells show an irritant rather than a sensitizing potential.

Background: The nature of clinically related adverse reactions to titanium is still unknown.

Objective: To determine whether titanium salts have irritant or sensitizing potential in a reconstructed human skin (RHS) model with integrated Langerhans cells (LCs).

Methods: RHS-LCs (ie, reconstructed epidermis) containing primary differentiated keratinocytes and CFSE CD1a -LCs generated from the MUTZ-3 cell line on a primary fibroblast-populated collagen hydrogel (dermis) were topically exposed to titanium(IV) bis(ammonium lactato)dihydroxide (TiALH).

Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity.

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS.

Reconstructed human skin shows epidermal invagination towards integrated neopapillae indicating early hair follicle formation in vitro.

Application of reconstructed human Skin (RhS) is a promising approach for the treatment of extensive wounds and for drug efficacy and safety testing. However, incorporating appendages, such as hair follicles, into RhS still remains a challenge. The hair follicle plays a critical role in thermal regulation, dispersion of sweat and sebum, sensory and tactile functions, skin regeneration, and repigmentation.

Comparison of the skin sensitization potential of 3 red and 2 black tattoo inks using interleukin-18 as a biomarker in a reconstructed human skin model.

Background: During the last decade, the number of people with ≥1 tattoo has increased noticeably within the European population. Despite this, limited safety information is available for tattoo inks.

Objectives: To test the skin sensitization potential of 5 tattoo inks in vitro by using reconstructed human skin (RHS) and the contact sensitization biomarker interleukin (IL)-18.

Allergens of permanent hair dyes induces epidermal damage, skin barrier loss and IL-1 α increase in epidermal in vitro model.

Allergic and irritant skin reactions caused by topical exposure to permanent hair dyes are a common problem. For regulatory and ethnical purposes, it is required to perform chemical safety assessment following the replacement, reduction, and refinement of animal testing (3Rs). Permanent hair dyes are formed by a mixture of ingredients that vary from low to extreme skin sensitizing potency and that inter-react to form unknown by-products.

Assessment of metal sensitizer potency with the reconstructed human epidermis IL-18 assay.

According to the new EU Medical Devices (MDR) legislation coming into effect in 2017, manufactures will have to comply with higher standards of quality and safety for medical devices in order to meet common safety concerns regarding such products. Metal alloys are extensively used in dentistry and medicine (e.g.

MUTZ-3 Langerhans cell maturation and CXCL12 independent migration in reconstructed human gingiva.

Here we describe a reconstructed full thickness human oral mucosa (gingiva) equivalent with integrated Langerhans Cells (GE-LC) and use it to compare LC activation and migration from oral versus skin epithelium. The physiologically representative models consist of differentiated reconstructed epithelium (keratinocytes and Langerhans-like cells derived from the MUTZ-3 cell line) on a fibroblast-populated collagen hydrogel which serves as a lamina propria for gingiva and dermis for skin. Topical exposure of GE-LC and the skin equivalent (SE-LC) to sub-toxic concentrations of the allergens cinnamaldehyde, resorcinol and nickel sulphate, resulted in LC migration out of the epithelia.

Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems.

Immune-competent human skin disease models.

All skin diseases have an underlying immune component. Owing to differences in animal and human immunology, the majority of drugs fail in the preclinical or clinical testing phases. Therefore animal alternative methods that incorporate human immunology into in vitro skin disease models are required to move the field forward.

Gingiva Equivalents Secrete Negligible Amounts of Key Chemokines Involved in Langerhans Cell Migration Compared to Skin Equivalents.

Both oral mucosa and skin have the capacity to maintain immune homeostasis or regulate immune responses upon environmental assault. Whereas much is known about key innate immune events in skin, little is known about oral mucosa. Comparative studies are limited due to the scarce supply of oral mucosa for ex vivo studies.

MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure.

After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure.

International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity.

This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x).

An epidermal equivalent assay for identification and ranking potency of contact sensitizers.

The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens.

CCL5 and CCL20 mediate immigration of Langerhans cells into the epidermis of full thickness human skin equivalents.

Epidermal Langerhans cells (LC) play a key role in initiation and regulation of immune responses. Whereas LC migration out of the epidermis upon environmental assault is extensively studied, the mechanisms involved in the (re)population of the epidermis with LC are poorly understood. Here, we investigated the immigration of LC derived from the human MUTZ-3 cell line (MUTZ-LC) into the epidermis of a full thickness skin equivalent, comprising a fully differentiated epidermis on a fibroblast-populated dermis.

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency.

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers.

Dendritic cell migration assay: a potential prediction model for identification of contact allergens.

This manuscript describes methodology and a prediction model for the MUTZ-LC migration assay. The assay represents the physiological change in Langerhans cell (LC) behavior after exposure to a sensitizing chemical, resulting in LC migration from the epidermis to the dermis. MUTZ-LC are derived from the commercially available MUTZ-3 cell line.