Optimizing tissue and cell dissociation for single-cell preparation

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Why optimal cell dissociation is critical

In the early stages of cell dissociation, cells are still intact and alive. This means their transcriptomes can still be altered in response to certain stimuli. Accordingly, exerting excessive chemical or mechanical pressure on cells is a ‘stressor’ that may significantly change the expression levels of genes.

In fact, studies have shown that cells exhibit major transcriptomic changes that are directly related to the method of dissociation. [1], [2] In addition, variables such as duration of dissociation were also found to affect gene expression.

These detected transcriptomes are therefore artifacts of the employed technique and do not represent real cellular states. This makes it challenging to generate accurate conclusions about the different cell populations that exist in a sample or a tissue.

In the case of insufficient dissociation, which may occur if the technique chosen is too gentle for the sample, the number of sequenced cells and transcripts will be lower than expected. On the other hand, excessively harsh dissociation techniques can cause cell death and result in fewer cells.

Finally, the presence of debris (particularly in the case of tissue samples) can interfere with all output parameters. In extreme cases, debris can even lead to a complete failure of the sequencing experiment depending on the platform.

Main factors to consider when choosing a cell dissociation technique

The factors that determine the suitability of cell dissociation techniques are highly variable from one sample to another. Accordingly, there are certain questions you should ask before choosing a handful of techniques to test.

Are you starting from tissue or cells?

This is perhaps the most important factor that determines how gentle or harsh the dissociation technique needs to be. Since tissues are thick, very dense in cells, and highly concentrated in extracellular matrix proteins, they require extra steps and harsher dissociation protocols. Cells, on the other hand, can be subjected to shorter and gentler protocols.

How fragile are the cells?

Some cells are notoriously fragile and die when subject to chemical or mechanical pressure. They must therefore be handled with extreme care. This includes cells such as neurons. In terms of dissociation techniques, fragile cells greatly restrict the viable options. However, there are specific protocols that have been validated on these cell types using gentle dissociation techniques. The goal is to yield a single-cell suspension without compromising the structure, viability, or gene expression of the cells.

How ‘clean’ is your input?

This question is also related to whether you are starting with tissues or cells. Tissues typically produce a lot of debris that can interfere with downstream steps. Consequently, dissociation steps must include ample clean-up of debris. The strength of the chosen dissociation method will typically increase in intensity if debris is expected to be high.

One of the biggest challenges in single-cell sequencing experiments is choosing the appropriate method for tissue or cell dissociation. Having a clean, rich, and dense single-cell suspension without significant cell loss may seem like a feat that is impossible to accomplish. Fortunately, various benchmarking studies have compared the efficiency of different cell dissociation techniques and the effect of each on the quality of downstream sequencing and final results.

Wondering about other parameters impacting your scRNA-seq data? Have a look at our article listing common technical artefacts in sample preparation

Experimental parameters that can be manipulated in a cell/tissue-dependent manner

Dissociation protocols typically include enzymatic dissociation or dissociation with mechanical pressure. Most include some form of the former. Enzymes like serine proteases, collagenase, dispase, and/or hyaluridonase, are the most commonly used for cell dissociation. In some protocols, trypsin is added to speed up the process of dissociation and subsequently reduce the duration.

Practical tip: Run a test experiment on your sample before even thinking about sequencing! Depending on your sample and the questions above, you should already have an idea of the options that are suitable for dissociation. Based on these options, manipulate parameters like the enzymes used and their concentration, the duration of dissociation, and the temperature at which dissociation occurs. The readout could be as simple as looking at the final suspension under a light microscope to check if cells are dissociated (little to no aggregates), look healthy (viability), and to ensure there is little to no debris. When you have evaluated a range of conditions, choose the option that is the least harsh yet efficient at producing single-cell suspensions

Another important factor related to enzyme dissociation is whether it should take place on ice (4ºC) or at 37ºC. Cell dissociation was traditionally performed at 37 ºC but cold enzyme dissociation is gaining popularity since it was shown to lead to fewer transcriptional changes. One study found a major increase in the expression of apoptosis genes during warm enzymatic dissociation. Alarmingly, entire cell populations were absent from annotated clusters in warm dissociation samples, indicating that some cell types may be more sensitive to stress.1 The dissociation technique can therefore determine whether the resulting single-cell data includes all major cell types. On ice, there are very few transcriptional changes, but the cold temperature can also induce cellular stress. Another drawback of cold dissociation is lower efficiency. One possibility to counteract warm dissociation effects on gene expression is to add an inhibitor of the transcriptional machinery, but the best option is to find the shortest possible duration for enzymatic dissociation that is still effective. There is also a trade-off between the duration of dissociation and concentration of the enzymes, as these factors can have different effects on viability and stress and should therefore be tested independently.

A final factor to consider adding to the dissociation protocol is a viability assay. If high levels of cell death are expected, fluorescence-activated cell sorting (FACS) can be used to sort living cells. Another option is to use a viability assay to at least get an approximation of the proportion of living versus dead cells to ensure that viability is high enough. If so, then adding steps for the enrichment of viable cells is not necessary,

There is clearly no one size fits all solution when it comes to cell dissociation. It might seem laborious to optimize what works best for your samples, but once it is done you will have accurate, clean results that are worth the price you pay for sequencing! Remember, garbage in, garbage out!

For detailed protocols that are cell- and tissue-specific, see the following references.[3],[4]


[1] https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02048-6

[2] https://www.nature.com/articles/nmeth.4437

[3] https://link.springer.com/protocol/10.1007%2F978-1-4939-9021-4_5

[4] https://link.springer.com/protocol/10.1007%2F978-1-4939-9240-9_2