Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair.
Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair.
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DOI：
10.3791/62326
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发表时间：
2021-02
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影响因子：
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通讯作者：
Holly N. Wilkinson;Alexandria Sarah Kidd;E. R. Roberts;M. Hardman
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文献类型：
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作者：
Holly N. Wilkinson;Alexandria Sarah Kidd;E. R. Roberts;M. Hardman
慢性不愈合伤口主要影响老年人和糖尿病患者，是临床未满足需求的重要领域。不幸的是，目前的慢性伤口治疗是不够的，而可用的临床前模型预测新疗法的临床疗效很差。在这里，我们描述了一种高通量的临床前模型，以评估人类皮肤修复反应的多个方面。在人离体皮肤中产生部分厚度伤口，并在愈合时程中培养。在固定剂中收集皮肤伤口活检用于整体封片染色程序。将固定的样品封闭并在一抗中孵育，通过荧光缀合的二抗实现检测。在计算每次活检中的伤口闭合百分比（上皮再形成）之前，通过共聚焦显微镜对伤口进行复染和成像。应用该方案，我们揭示了在健康供体皮肤中产生的2 mm切除伤口在创伤后4-5天完全再上皮化。相反，糖尿病皮肤伤口的闭合率显著降低，伴随着屏障重建的扰动。将人类皮肤创伤与新的整体包埋染色方法相结合，可以提供一种快速且可重复的方法来量化离体伤口修复。总的来说，该方案提供了一个有价值的人类平台，以评估潜在伤口治疗的有效性，改变临床前测试和验证。
Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.