Preparation And Evaluation Of Cell Co - Cultured Skin Analogues

Subjective:The subject of this research was to provide new skin models for transdermal drug delivery system (TDDS). Human skin equivalents (HSEs) were prepared preliminary and characterized for it’s preliminary profile. Based on this, we screened the culture time of air-liquid interfance (ALI), relative humidity (RH) of growth envionment and if there any epidermal growth factor (EGF) added in the growth condition indicated by the epidermal morphology, expression of differentiation markers, barrier function, molecular structure of HSEs, then the barrier function was compared with before screening. We would except to provid a new models that similar to human skin (HS) for TDDS as a substitute for experimental animals.Methods:(1) The preliminary prepartion and evaluation of HSEsHSEs were prepared at the following environmental conditions:95% RH, EGF free, cultured for 3weeks after exposed to ALI. HSEs were characterized by macroscopic observation, electron microscopy observation and penetration testing.(2) The optimization of growth condition of HSES① HSEs were harvested at the setted time after ALI culture, macroscopic observation, electron microscopy observation, immunohistochemical method, Fourier Transform Infrared Spectrometer (FTIR), penetration testing were taken to screen the culture time after exposed to air.② HSEs were prepared at different environmental RH that setted before:75% RH or 95% RH. The prepared HSEs were characterized for their penetration profile.③ When rosed to ALI, EGF was added to the culture medium or not, penetration test was used to study the effects of EGF on the barrier properties of HSE.(3) The evaluation of HSEs④ Macroscopic observation of the surface of the skin equivalents, and compared to in vivo HS.② Masson staining was used to investigate the distribution of cells and protein fiber, and compared to HS.③ Scanning electron microscope (SEM) was used to study the surface of HSEs, and compared to HS.④ Transmission electron microscopy (TEM) was used to observe the structure of the layers of HSEs.⑤ Indomethacin, caffeine and hydrocortisone were choosed as model drugs, which represent different oil-water partition coefficient of compounds, to study the barrier function of HSEs, and compared with SD rats, ICR mice, nude mouse and Start-M membrane.⑥ The barrier function of HSEs before and after optimization.Results:(1) HSEs were composed of epidermal and dermal layer, Ultrastructural features was similar to HS, but the surface of HSEs was incomplete. The barrier function for the penetration of indomethacin was similar to Start-M membrane, but impaire when compared with SD rats.(2)The optional culture condition of HSEs was defined as 95% RH, EGF free and cultured for 3 weeks after exposed to ALI.(3) The morphological structure, expresson of early differention marker keratin 10(K10) and the molecular structure of SC lipids and keratin of HSEs were very similar to HS, the expresson of filaggrin was activated. The barrier function was formed. On the whole, HSEs performe comparatively high barrier function for Lipophilic compound, which is close to mice, while comparatively low barrier function for hydrophilic compounds, similar to Start-M membrane.Conclusion:Our in house HSEs were similar to HS to some extent, the barrier function was improved by screening of culture conditionds. There are more efforts needed to improve it’s barrier function with the purpose of HSEs can be a alternate for experimental animals in TDDS.