Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II).

To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus, the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87% and the inter-laboratory reproducibility of 85% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404.

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion.

Investigation of percutaneous helminth infection is generally based on animal models or excised skin. As desirable replacement of animal experiments, tissue-engineered skin equivalents have recently been applied in microbial and viral in vitro infection models. In the present study, the applicability of tissue-engineered skin equivalents for the investigation of percutaneous helminth invasion was evaluated. Epidermal and a full-thickness skin equivalents that suit the requirements for helminth invasion studies were developed. Quantitative invasion assays were performed with the skin-invading larvae of the helminths Strongyloides ratti and Schistosoma mansoni. Both skin equivalents provided a physical barrier to larval invasion of the nematode S. ratti, while these larvae could invade and permeate a cell-free collagen scaffold and ex vivo epidermis. In contrast, the epidermal and full-thickness skin equivalents exhibited a human host-specific susceptibility to larvae of trematode S. mansoni, which could well penetrate. Invasion of S. mansoni in cell-free collagen scaffold was lowest for all experimental conditions. Thus, reconstructed epidermis and full-thickness skin equivalents confirmed a high degree of accordance to native tissue. Additionally, not only tailless schistosomula but also cercariae could permeate the skin equivalents, and thus, delayed tail loss hypothesis was supported. The present study indicates that the limitations in predictive infection test systems for human-pathogenic invading helminths can be overcome by tissue-engineered in vitro skin equivalents allowing a substitution of the human skin for analysis of the interaction between parasites and their hosts' tissues. This novel tissue-engineered technology accomplishes the endeavor to save animal lives.

Impedance spectroscopy for the non-destructive evaluation of in vitro epidermal models.

PURPOSE: Reconstructed human epidermis (RHE) is standardly used for the risk assessment of chemical compounds. However, analysis is dependent on invasive methods such as histological processing or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. METHODS: As an alternative, we have developed a non-destructive technology to analyze the integrity of epidermal equivalents based on impedance spectroscopy. RHEs were generated and impedance spectra were recorded. from these spectra, we extrapolated electrical characteristics such as the capacitance and the ohmic resistance. Furthermore, the measurable electrical parameters were used to quantify the effects of mechanical and chemical disruption of the epidermal integrity. RESULTS: A fully matured RHE exhibits typical impedance spectra in a frequency ranging between 1 Hz and 100 kHz, which is comparable to the spectra of freshly isolated human epidermal biopsies. We could show that, during RHE maturation, these characteristics change significantly. Thus, capacitance and ohmic resistance can be employed as a criterion for the quality control of skin equivalents. Additionally, our application of impedance spectroscopy reveals sufficient sensitivity to detect a transient decreased ohmic resistance caused by 2-propanol, which is classified as a non-irritant by MTT assays. CONCLUSION: These results indicate that impedance spectroscopy can be employed as a non-destructive complementary method to assess mild irritative effects, which is currently not possible.

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing.

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.