Martin Luther University Halle-Wittenberg, Department of Pharmaceutical Technology and Biopharmaceutics, D-06120 Halle/Saale, Germany; University of Southern Denmark, Department of Physics, Chemistry and Pharmacy, DK-5230 Odense, Denmark.
Eur J Pharm Sci. 2013 Dec 18;50(5):577-85. doi: 10.1016/j.ejps.2013.06.003. Epub 2013 Jun 11.
Cell cultured skin equivalents present an alternative for dermatological in vitro evaluations of drugs and excipients as they provide the advantage of availability, lower variability and higher assay robustness compared to native skin. For penetration/permeation studies, an adequate stratum corneum barrier similar to that of human stratum corneum is, however, a prerequisite. In this study, the stratum corneum lipid organization in an epidermal cell culture model based on rat epidermal keratinocytes (REK organotypic culture, ROC) was investigated by small-angle X-ray scattering (SAXS) in dependence on ROC cultivation time and in comparison to native human and rat stratum cornea. In addition, the thermal phase behavior was studied by differential scanning calorimetry (DSC) and barrier properties were checked by measurements of the permeability of tritiated water. The development of the barrier of ROC SC obtained at different cultivation times (7, 14 and 21 days at the air-liquid interface) was connected with an increase in structural order of the SC lipids in SAXS measurements: Already cultivation for 14 days at the air-liquid interface resulted overall in a competent SC permeability barrier and SC lipid organization. Cultivation for 21 days resulted in further minor changes in the structural organization of ROC SC. The SAXS patterns of ROC SC had overall large similarities with that of human SC and point to the presence of a long periodicity phase with a repeat distance of about 122Å, e.g. slightly smaller than that determined for human SC in the present study (127Å). Moreover, SAXS results also indicate the presence of covalently bound ceramides, which are crucial for a proper SC barrier, although the corresponding thermal transitions were not clearly detectable by DSC. Due to the competent SC barrier properties and high structural and organizational similarity to that of native human SC, ROC presents a promising alternative for in vitro studies, particularly as it can be obtained under overall rather straightforward cell culture conditions and thus low assay costs.
细胞培养皮肤等效物为药物和赋形剂的皮肤体外评估提供了一种替代方法，因为与天然皮肤相比，它们具有可获得性、变异性更低和分析稳健性更高的优势。然而，对于渗透/渗透研究，需要类似于人角质层的足够的角质层屏障。在这项研究中，通过小角 X 射线散射（SAXS）研究了基于大鼠表皮角质形成细胞（REK 器官型培养物，ROC）的表皮细胞培养模型中的角质层脂质组织，这取决于 ROC 培养时间，并与天然人角质层和大鼠角质层进行了比较。此外，通过差示扫描量热法（DSC）研究了热相行为，并通过测量氚化水的渗透性检查了屏障性能。在不同培养时间（在气液界面培养 7、14 和 21 天）获得的 ROC SC 屏障的发展与 SAXS 测量中 SC 脂质结构有序度的增加有关：在气液界面培养 14 天已经导致整体上具有足够的 SC 渗透性屏障和 SC 脂质组织。培养 21 天会导致 ROC SC 的结构组织进一步发生较小的变化。ROC SC 的 SAXS 图谱总体上与人类 SC 非常相似，并表明存在具有约 122Å重复距离的长周期性相，例如略小于本研究中确定的人类 SC（127Å）。此外，SAXS 结果还表明存在共价结合的神经酰胺，这对于适当的 SC 屏障至关重要，尽管 DSC 未清楚地检测到相应的热转变。由于具有足够的 SC 屏障特性以及与天然人 SC 高度相似的结构和组织，ROC 为体外研究提供了一种有前途的替代方法，特别是因为它可以在相对简单的细胞培养条件下获得，因此分析成本较低。
