The aim of this study was to evaluate LabCyte EPI-MODEL, a commercially available reconstructed, cultured human epidermal model, on the basis of a morphological characterization using optical microscopy, electron microscopy and the expression of specific differentiation markers through immunohistochemistry. Histological examination showed a completely stratified epidermis containing all major epidermal layers, including a stratum basal (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum corneum (SC). In addition, specific epidermal differentiation markers and basement membrane constituents were expressed in the appropriate regions, as seen in human skin. Ultrastructurally, it was possible to observe a fully developed basement membrane zone, consisting of a highly developed lamina densa, lamina lucida, and anchoring filaments. Extrusion of lamellar bodies was observed at the interface between the SG and SC. Lipid lamellae, showing a characteristic electron dense and electron lucent pattern, were present. Keratohyalin granules were ubiquitously present in the granular cells at the SG. In conclusion, the LabCyte EPI-MODEL, a reconstructed, cultured human epidermal model, is highly similar to that of native skin. Therefore, this epidermal model provides a promising alternative to animal testing, as a means to assess skin irritation, percutaneous absorption, and other skin-related research.
To establish alternative methods to animal testing and experimentation for hemocompatible materials, we had been developed an apparatus consisting of a modified cone and plate-type rheometer combined with an upright epi-fluorescence microscope. Through this apparatus, we could conduct real-time evaluation of platelet-material interactions, the initial event of thrombus formation, under shear flow conditions using small platelet suspension volumes (7.5∼500 µl per material). The use of human blood from a blood bank allows easy access to large amounts of human blood and does not require medical doctors and volunteer donors to draw the blood. Therefore, to test the hemocompatibility of materials, we used human blood from a blood bank. In order to compare the properties of platelets between fresh and bank blood, the number of adhering platelets, the trigger of the thrombus formation, on two test materials (acrylate resin and polyethylene) was counted under shear flow conditions of 1s-1 and 50s-1. Results showed that there was no difference in the number of adhering platelets to the two material urfaces between the fresh and bank blood under shear flow. However, increase in shear flow decreased the number of adhering platelets. In addition, shear stress changed the rank of the materials. In conclusion, it was suggested that our cone and plate-type rheometer system with human blood from a blood bank is a good alternative to animal experiments for testing and screening the hemocompatibility of materials.
A skin-mimicking artificial membrane consisting of a silicone membrane as a model stratum corneum and laminated dialysis membranes as a model of viable skin was prepared to imitate the layered structure of skin. The permeability of flurbiprofen (FP) through the laminated membranes agreed with that calculated, suggesting that the interfacial resistance between each membrane was negligible. When the FP concentration in the laminated membranes was determined by microdialysis, the observed concentration was similar to that calculated according to Fick's law of diffusion. When bovine serum albumin (BSA) solution was placed between the dialysis membranes to mimic the protein leaching in skin, the FP permeation profiles through the membranes showed a long lag-time without dependence on the depth of the region of the inserted BSA from the surface. However, the FP concentration-time profiles in a region between the membranes showed dependence on the region of the inserted BSA. Since the FP concentration in the region and the permeation rates at steady-state were similar to those without BSA solution, BSA could act as a capacity factor to the delayed reaching to a steady-state. The skin mimicking laminated membranes will be useful to evaluate drug permeation quantitatively and to imitate some events which can happen in inflamed tissues in skin without requiring animals.