10x chromium single cell dissociation trypsin

Factors including tissue necrosis, hypoxia and therapeutic treatments often result in poor viability of cells in solid tumour tissues. Cold preservation is yet to be evaluated for complex tissues such as solid tumours, which possess distinct features in tissue viability. Cell type-specific transcriptional changes can emerge after longer cold preservation periods (> 24 h), particularly affecting immune subpopulations in normal tissues. benchmarked short-term cold preservation of tissue prior to scRNA-Seq, which showed little impact on transcriptome integrity within the first 24 h. Several approaches have been developed to address such issues. The need to process fresh tissue specimens at the time of tissue availability, often as a single specimen, often introduces large experimental costs and confounding batch effects upon studies with large numbers of patients and prevents the selection and triage of cases for analysis based on histopathological analysis. Performing scRNA-seq on clinical samples remains logistically and technically challenging, mainly due to the transport of patient tissue from operation rooms to laboratories for processing, which are often complicated by short notices and core-facility access hours. Advances in high-throughput single-cell RNA sequencing (scRNA-seq) technologies have rapidly developed in recent years, providing a powerful platform to resolve the aetiology of the TME in solid cancers. The tumour microenvironment (TME) is composed of neoplastic cells, mesenchymal and immune cells that interact to shape cancer progression and therapeutic response. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies. We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers.

10x chromium single cell dissociation trypsin