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What are the best practices for dissociating tissue samples into single cells?

Question: What are the best practices for dissociating tissue samples into single cells?

Answer: The following are some best practices to dissociate tissue into single cells : 

 

1. Use a dissociation protocol that works for your tissue:

Tissue dissociation methods can impact the viability of the resulting cell suspension, so it's important to pick a dissociation protocol suitable for your tissue type. Various resources and publications outline common dissociation techniques for respective tissue types. Additionally, consider whether your cell type of interest has been known to be affected by warm enzymatic dissociation. See: 

2. Unbiased tissue sampling:

Bias in cell-type composition can be introduced during tissue sampling, impacting sequencing results. This consideration is especially important for tissues structurally different like the brain and maybe less important for something more homogenous like the liver. 

It is important to ensure the same region(s) are sampled across replicates or samples that will be compared. If starting with a large piece of tissue and only using a portion of the sample for single-cell sequencing, mix the dissociated tissue/cell suspension/nuclei suspension thoroughly before subsampling to reduce bias in cell types recovered.

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3. Sample QC can provide information on tissue quality and dissociation method :

Some sample metrics can be informative on the quality of the starting tissue and dissociation method. 

  • Assessing the RIN of a tissue sample may be a way to exclude poor-quality samples; we recommend a RIN > 7
  • Cell viability and debris can be indicative of inefficient dissociation or over digestion. 

Issue

Possible Causes

Possible remediation

Large pieces of tissue are still present

Inefficient dissociation
  • Use an appropriate enzyme for dissociation
  • Optimize dissociation method (pipetting, pestle, homogenizer, douncer, pulverization); may need to increase the number of homogenization steps to break up large tissues. 
    • For tissues that are difficult to dissociate into single cells (i.e., Heart), consider nuclei isolation. 
  • Filter out large fragments using a cell strainer

Lots of debris

Inefficient dissociation or overdigestion
  • Filter out debris with a cell strainer
  • Add an extra wash and spin if possible
  • Consider gentler handling of tissue during dissociation
  • Optimize digestion enzymes and time

Low cell viability

The method is too harsh
  • Optimize tissue digestion
  • Consider shortening enzymatic digestion or homogenization steps to preserve cell viability.
  • Consider gentler handling of tissue during dissociation
  • Dead cell removal methods may be appropriate if starting with > X cells 

 

4. Perform a pilot when possible before proceeding with large sample batches:

If planning for a large experiment and handling a new sample type for the first time, it may be useful to run a pilot first to test the experimental workflow and gain confidence before proceeding to scale up. A pilot may also help inform which experimental condition may be more apt. See:

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