Question: Are there any recommendations for working with post-mortem tissue?
Answer: Post-mortem tissues from autopsies are a valuable source of biological materials widely used in forensic science, biology research, pathology, or clinical research. Below, you will find a schematic diagram of an ideal workflow along with some of our recommendations for working with post-mortem tissue as input to 10x Genomics Single-Cell Assays (Note that we have not specifically tested these approaches, they are meant to be general guidelines. A pilot study is recommended before starting a large scale experiment).
- Minimize post-mortem interval time (PMI) ('rapid autopsy' is preferred).
- During tissue collection, submerge or rapidly perfuse post-mortem tissue with a cold (4˚C) preservation solution (Hypothermosol-FRS, or Miltenyi MACS Tissue Storage Solution) or PBS or media to prevent exposure to air (Step A). See: Can I store samples in a tissue storage solution?
- For an extended period of storage time (hours), place the tissue (after PMI) into a cold preservation solution such as Hypothermosol-FRS, or Miltenyi MACS Tissue Storage Solution or following manufacturer's recommendations (Steps B-C).
- Alternatively, after PMI, tissue can be directly snap-frozen for single nuclei gene expression analysis (Step D) and stored. After thawing the tissue, proceed directly to nuclei isolation for nuclei gene expression analysis (Step E). See: How do you isolate nuclei from snap-frozen tissue for 3’ gene expression profiling?
- A third option is to cryopreserve tissue in a cryopreservation medium (Step F). Tissues can be directly placed and cryopreserved in a cryopreservation medium (similar to a cell suspension). After cryopreservation, the tissue can be thawed, and intact cells are recovered. This method may allow for antibody staining for cell surface protein followed by our feature barcoding assay.
- After the preservation process (Step C or F), proceed to dissociate the tissue into a single cell/nuclei suspension as soon as possible (step G). See: How do I dissociate my tissue of interest?
- Once a single-cell suspension is obtained, it is possible to cryopreserve the cell suspension (step H). See: Can cryopreserved cells be used for gene expression profiling? We do not recommend cryopreserving/freezing a single-nuclei suspension.
Factors influencing the quality of post-mortem tissues
The most important factor that affects the quality of post-mortem tissue is the post-mortem interval or PMI. PMI is defined as the interval between the time of death or the cessation of blood flow and the time that the sampled tissue is stabilized and/or preserved. It is the most crucial factor for obtaining high-quality post mortem tissue; it is known to influence tissue degradation and DNA, RNA, and protein integrity. In general, DNA is relatively stable over long post-mortem periods, but RNA is much more labile in nature and sensitive to degradation in a tissue-specific manner. Other factors like tissue necrosis, hypoxia, and therapeutic treatments often result in poor cell viability in post-mortem tumor tissues.
Gene expression changes in post-mortem tissues
The most important factors influencing RNA stability, organ and cell homeostasis are temperature and time. At temperatures above 15˚C (warm ischemia), RNA stability varies in cell types and tissue types. The changes in transcript types involved genes in stress, immunity, inflammation, apoptosis, transport, cancer, and epigenetic regulations. Also, most of the transcripts level changes happen as early as 30 minutes post-mortem but can also happen after 24 or 48 hours post-mortem depending on tissue type and patient or animal health. Thus after PMI, post-mortem tissue should be transferred as soon as possible into a cold preservation solution (such as Hypothermosol-FRS or Miltenyi MACS Tissue Storage Solution). Cell type-specific transcriptional changes have been shown to emerge after longer cold preservation periods (>24 hours), particularly affecting immune subpopulations in normal tissues. After 24 hours, a decrease in specific cell types (such as T cells in the lung at 72 hrs), a higher percentage of mitochondrial reads, and increased background ambient RNA (in the spleen at 72 hrs) are observed in a tissue-specific manner.
References:
https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1906-x
https://www.nature.com/articles/s41467-017-02772-x
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811979/
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115675
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303275/
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056507
https://www.embopress.org/doi/full/10.15252/embj.2018100811
https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-021-00885-z
The same guidelines apply to post-mortem tissue for Multiome(ATAC+RNA) assay however the nuclei isolation protocol for this assay is different from the nuclei isolation protocols for 3' Single Cell Gene Expression assay.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC + GEX