Sharma Ruchi, Barakzai Safia Z, Taylor Sarah E, Donadeu F Xavier
The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, UK.
Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, UK.
J Tissue Eng Regen Med. 2016 Aug;10(8):627-36. doi: 10.1002/term.1788. Epub 2013 Jul 30.
Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three-dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK-5 and CK-14, which are associated in vivo with proliferating keratinocyte populations. Explant-derived keratinocytes were seeded into Alvetex™ 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK-5 and CK-14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal-like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd.
尽管马匹皮肤病的患病率很高，但关于马皮肤细胞培养和生物学的文献却很匮乏，部分原因是缺乏合适的体外皮肤模型。本研究的目的是开发一种三维（3D）培养系统，以支持马角质形成细胞的增殖和分化，类似于在天然表皮中观察到的情况。从马皮肤外植体获得细胞单层，并根据细胞角蛋白的表达，包括CK-5和CK-14，将其作为高度纯化的角质形成细胞群体（>95%的细胞）进行连续传代，这些细胞角蛋白在体内与增殖的角质形成细胞群体相关。将外植体来源的角质形成细胞接种到Alvetex™ 3D组织支架中，在促进细胞分化的条件下培养30天。超微结构、免疫组织化学和生化分析表明，支架内的角质形成细胞能够增殖并获得组织极性，包括分化为基底层和基底层以上的层。基底层含有明显的立方体细胞，细胞核大，并对增殖标记物如CK-5和CK-14进行染色。相比之下，基底层以上的层由具有明显多面体形态、丰富细胞质突起和桥粒的细胞组成，这些细胞指示棘层，还有明显扁平的角质化细胞，表达终末分化标记物兜甲蛋白。因此，源自马原发性皮肤外植体的角质形成细胞能够在培养中获得类似表皮的结构。这个新系统可以为皮肤疾病建模、药物测试/毒性研究以及潜在的马再生医学提供一个非常有用的工具。版权所有© 2016约翰威立父子有限公司。
