Isolating and cryopreserving pig skin cells for single-cell RNA sequencing study.
Isolating and cryopreserving pig skin cells for single-cell RNA sequencing study.
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DOI：
10.1371/journal.pone.0263869
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Lei Y
中科院分区：
文献类型：
--
作者：
Han L;Jara CP;Wang O;Shi Y;Wu X;Thibivilliers S;Wóycicki RK;Carlson MA;Velander WH;Araújo EP;Libault M;Zhang C;Lei Y
猪的皮肤结构和生理机能与人类相似。因此，猪模型对于研究皮肤生物学和测试治疗方法非常有价值。单细胞RNA测序（scRNA-seq）技术允许在单细胞分辨率和高通量下定量分析细胞类型，组成，状态，信号传导和受体-配体相互作用组。scRNA-seq已被用于研究小鼠和人类皮肤。然而，用scRNA-seq研究猪皮仍然很少见。scrna测序成功的关键一步是从猪皮肤组织中获得高质量的单细胞。在这里，我们报告了一种分离和低温保存猪皮单细胞用于scrna测序的可靠方法。使用Miltenyi Human Whole skin Dissociator试剂盒和Miltenyi gentleMACS Dissociator，我们发现猪皮肤可以高效地分离成具有高细胞活力的单细胞。此外，获得的单细胞可以使用90% FBS + 10% DMSO进行低温保存，而不会引起额外的细胞死亡、细胞聚集或基因表达谱的变化。使用开发的方案，我们能够识别所有主要的皮肤细胞类型。本研究的方案和结果对皮肤研究科学界具有重要价值。
The pig skin architecture and physiology are similar to those of humans. Thus, the pig model is very valuable for studying skin biology and testing therapeutics. The single-cell RNA sequencing (scRNA-seq) technology allows quantitatively analyzing cell types, compositions, states, signaling, and receptor-ligand interactome at single-cell resolution and at high throughput. scRNA-seq has been used to study mouse and human skins. However, studying pig skin with scRNA-seq is still rare. A critical step for successful scRNA-seq is to obtain high-quality single cells from the pig skin tissue. Here we report a robust method for isolating and cryopreserving pig skin single cells for scRNA-seq. We showed that pig skin could be efficiently dissociated into single cells with high cell viability using the Miltenyi Human Whole Skin Dissociation kit and the Miltenyi gentleMACS Dissociator. Furthermore, the obtained single cells could be cryopreserved using 90% FBS + 10% DMSO without causing additional cell death, cell aggregation, or changes in gene expression profiles. Using the developed protocol, we were able to identify all the major skin cell types. The protocol and results from this study are valuable for the skin research scientific community.