Abstract：
Keratinocyte culture is a standard method used to study gene expression, cell differentiation and proliferation. Numerous protocols exist, however their application is frequently unsuitable for small specimens, such as 4-mm punch skin biopsies.
This study compared 3 different methods of keratinocyte culture from paediatric skin biopsies to evaluate which one ensures adequate cell growth for RNA extraction and sequencing.
Thirty-six skin samples were obtained from 4-mm punch skin biopsies from residual human body material from healthy children. They were cultured in vitro according to 3 different methods: enzymatic method, epidermis explant and direct explant method. Keratinocytes were characterized by immunocytochemistry using pan-cytokeratin. RNA extraction was performed with RNeasy Mini kit. Quantity and quality of the extracted RNA was assessed to meet the requirements of library preparation for sequencing.
The direct explant method had largely shown its superiority over the two other methods, with a 100% success rate and an average of 15 days of culture. RNA extraction yielded a mean of 8545.85 ng of RNA per sample with an RQN of 10. Cover-clip immunochemistry staining with pan-cytokeratin had confirmed the absence of fibroblast contamination.
Although the enzymatic method is the most frequently used for keratinocyte culture, it is not suitable small samples required in dermatology. The direct explant method guarantees a high growth rate and the extraction of high quality RNA. Variation in the amount of RNA harvested are related to inter- and intra-individual variations and to the conditions of the experiment.
This study allowed to conclude that the direct explant method is the most efficient and easy method to ensure cell growth when the samples are from 4-mm punch skin biopsies. This technique avoids fibroblasts contamination and obtains a sufficient quantity and quality of RNA to sequence it.
This study compared 3 different methods of keratinocyte culture from paediatric skin biopsies to evaluate which one ensures adequate cell growth for RNA extraction and sequencing.
Thirty-six skin samples were obtained from 4-mm punch skin biopsies from residual human body material from healthy children. They were cultured in vitro according to 3 different methods: enzymatic method, epidermis explant and direct explant method. Keratinocytes were characterized by immunocytochemistry using pan-cytokeratin. RNA extraction was performed with RNeasy Mini kit. Quantity and quality of the extracted RNA was assessed to meet the requirements of library preparation for sequencing.
The direct explant method had largely shown its superiority over the two other methods, with a 100% success rate and an average of 15 days of culture. RNA extraction yielded a mean of 8545.85 ng of RNA per sample with an RQN of 10. Cover-clip immunochemistry staining with pan-cytokeratin had confirmed the absence of fibroblast contamination.
Although the enzymatic method is the most frequently used for keratinocyte culture, it is not suitable small samples required in dermatology. The direct explant method guarantees a high growth rate and the extraction of high quality RNA. Variation in the amount of RNA harvested are related to inter- and intra-individual variations and to the conditions of the experiment.
This study allowed to conclude that the direct explant method is the most efficient and easy method to ensure cell growth when the samples are from 4-mm punch skin biopsies. This technique avoids fibroblasts contamination and obtains a sufficient quantity and quality of RNA to sequence it.
摘要：
角质细胞培养是用于研究基因表达的标准方法，细胞分化和增殖。存在许多协议，然而，它们的应用往往不适合小标本，如4毫米穿孔皮肤活检。
本研究比较了来自儿科皮肤活检的3种不同的角质形成细胞培养方法，以评估哪一种方法确保了RNA提取和测序的足够细胞生长。
从来自健康儿童的残余人体材料的4mm穿孔皮肤活检获得36个皮肤样品。根据3种不同的方法进行体外培养：酶法，表皮外植体法和直接外植体法。使用全细胞角蛋白通过免疫细胞化学表征角质形成细胞。用RNeasyMini试剂盒进行RNA提取。评估提取的RNA的数量和质量以满足测序文库制备的要求。
直接外植体方法在很大程度上显示出其优于其他两种方法，100％的成功率和平均15天的培养。RNA提取产生每个样品8545.85ngRNA的平均值,RQN为10。用泛细胞角蛋白进行的盖夹免疫化学染色已证实没有成纤维细胞污染。
尽管酶法是最常用于角质形成细胞培养的方法，它不适合皮肤病学所需的小样本。直接外植体方法保证了高生长速率和高质量RNA的提取。收获的RNA量的变化与个体间和个体内的变化以及实验条件有关。
这项研究可以得出结论，当样品来自4毫米穿孔皮肤活检时，直接外植体方法是确保细胞生长的最有效和最简单的方法。该技术避免了成纤维细胞污染并获得足够数量和质量的RNA以对其进行测序。
本研究比较了来自儿科皮肤活检的3种不同的角质形成细胞培养方法，以评估哪一种方法确保了RNA提取和测序的足够细胞生长。
从来自健康儿童的残余人体材料的4mm穿孔皮肤活检获得36个皮肤样品。根据3种不同的方法进行体外培养：酶法，表皮外植体法和直接外植体法。使用全细胞角蛋白通过免疫细胞化学表征角质形成细胞。用RNeasyMini试剂盒进行RNA提取。评估提取的RNA的数量和质量以满足测序文库制备的要求。
直接外植体方法在很大程度上显示出其优于其他两种方法，100％的成功率和平均15天的培养。RNA提取产生每个样品8545.85ngRNA的平均值,RQN为10。用泛细胞角蛋白进行的盖夹免疫化学染色已证实没有成纤维细胞污染。
尽管酶法是最常用于角质形成细胞培养的方法，它不适合皮肤病学所需的小样本。直接外植体方法保证了高生长速率和高质量RNA的提取。收获的RNA量的变化与个体间和个体内的变化以及实验条件有关。
这项研究可以得出结论，当样品来自4毫米穿孔皮肤活检时，直接外植体方法是确保细胞生长的最有效和最简单的方法。该技术避免了成纤维细胞污染并获得足够数量和质量的RNA以对其进行测序。
