Wang Yinjuan, Tissot Marion, Rolin Gwenaël, Muret Patrice, Robin Sophie, Berthon Jean-Yves, He Li, Humbert Philippe, Viennet Céline
University of Bourgogne Franche-Comté, INSERM, EFS BFC, UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, Besançon, France.
Clinical Investigation Center, Inserm CICB 1431, University Hospital, Besançon, France.
Cytotechnology. 2018 Aug;70(4):1167-1176. doi: 10.1007/s10616-018-0207-7. Epub 2018 Mar 21.
Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products. Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as melanocytes, are described in literature. They are usually based on two enzymatic steps, Trypsin in combination with Dispase, in order to separate dermis from epidermis and subsequently to provide a suspension of epidermal cells. The objective of this work was to develop and validate an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. TrypLE™ product was tested on very limited size of human skin, equivalent of multiple 3-mm punch biopsies, and was compared to Trypsin/Dispase enzymes. Functionality of extracted cells was evaluated by analysis of viability, morphology and melanin production. In comparison with Trypsin/Dispase incubation method, the main advantages of TrypLE™ incubation method were the easier of separation between dermis and epidermis and the higher population of melanocytes after extraction. Both protocols preserved morphological and biological characteristics of melanocytes. The minimum size of skin sample that allowed the extraction of functional cells was 6 × 3-mm punch biopsies (e.g., 42 mm) whatever the method used. In conclusion, this new procedure based on TrypLE™ incubation would be suitable for establishment of optimal primary melanocytes cultures for clinical applications and research.
培养中的原代黑素细胞是研究表皮色素沉着和黑素生成化合物功效或开发先进治疗用药品的有用模型。细胞提取是建立细胞培养物中不可避免的关键步骤。文献中描述了许多用于提取和培养源自人皮肤的细胞（如黑素细胞）的酶法。它们通常基于两个酶促步骤，即胰蛋白酶与中性蛋白酶联合使用，以分离真皮和表皮，随后提供表皮细胞悬液。这项工作的目的是开发并验证一种简单、有效且适用于较小皮肤样本且避免使用动物试剂的人皮肤黑素细胞提取方法。将重组胰蛋白酶产品用于尺寸非常有限的人皮肤（相当于多个3毫米打孔活检样本）进行测试，并与胰蛋白酶/中性蛋白酶进行比较。通过分析细胞活力、形态和黑色素生成来评估提取细胞的功能。与胰蛋白酶/中性蛋白酶孵育方法相比，重组胰蛋白酶孵育方法的主要优点是真皮与表皮的分离更容易，提取后黑素细胞数量更多。两种方案都保留了黑素细胞的形态和生物学特性。无论使用何种方法，能够提取出功能细胞的皮肤样本最小尺寸为6×3毫米打孔活检样本（例如42平方毫米）。总之，这种基于重组胰蛋白酶孵育的新方法适用于建立用于临床应用和研究的最佳原代黑素细胞培养物。
