Verrillo Lucia, Mangano Eleonora, Drongitis Denise, Merelli Ivan, Pischedda Francesca, Piccoli Giovanni, Consolandi Clarissa, Bordoni Roberta, Miano Maria Giuseppina
Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, Naples, Italy; University of Campania "Luigi Vanvitelli", Caserta, Italy.
Institute of Biomedical Technologies, National Research Council, Segrate, Milan, Italy.
J Neurosci Methods. 2021 Jan 1;347:108960. doi: 10.1016/j.jneumeth.2020.108960. Epub 2020 Sep 25.
The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas.
Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing.
We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity.
By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis.
Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations.
单细胞RNA测序（scRNASeq）的应用代表了一种独特的方法，可用于识别处于胚胎发育不同阶段的哺乳动物皮质多层结构中的数百至数百万个细胞。应用于神经学研究的scRNASeq技术需要使用新鲜的起始材料，因为标准的冷冻保存方法不能保证源自解剖脑区的皮质原代细胞具有高活力。
在此，我们建立并验证了一种创新策略，用于对从E15.5小鼠胚胎分离的冷冻保存的原代皮质细胞进行scRNASeq研究。为了冷冻皮质原代细胞，我们采用了Neurostore，一种能够保证解冻后胚胎皮质具有高活力和细胞组成的培养基。
我们首次展示了在离线设置下对原代皮质细胞进行scRNASeq实验的可能性，确保了细胞的完整性和多样性。
通过台盼蓝测定和流式细胞术分析，我们发现经Neurostore冷冻保存的皮质细胞显示出约95%的活力。实现了令人满意的RNA回收和cDNA文库构建。对35763个冷冻保存的单细胞进行转录组测序产生了一个可靠的数据集，在三个生物学样本中鉴定出25个细胞簇。通过标记基因表达和免疫荧光分析验证了冷冻保存-复苏过程前后特定神经群体的普遍性。
我们的研究结果支持了以下证据，即冷冻保存的原代皮质细胞可成功用于scRNASeq实验，这在实验程序方面提供了前所未有的灵活性，例如在不同地点进行的样品制备和后续处理步骤。
