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What is Tissue Dissociation? 

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Tissue Dissociation: The First Step Toward Cellular Analysis

Tissue dissociation is a crucial preparatory step in many biological and clinical research workflows, involving the breakdown of complex tissue into a suspension of viable single cells. This process enables researchers to isolate individual cells for downstream applications such as flow cytometry, single cell RNA sequencing (scRNA-seq), cell culture, and more. Dissociation can be achieved through mechanical, enzymatic, or combined approaches, tailored to preserve cell viability and molecular integrity. 

Why is it Important to Optimise Tissue Dissociation in Our Workflow? 

Optimizing tissue dissociation is important because the quality of tissue dissociation significantly impacts the success of downstream analyses. Poorly dissociated samples may lead to low cell yield, reduced viability, loss of fragile cell types, or biased cell population representation. For sensitive applications like single cell sequencing, suboptimal dissociation can introduce artifacts, affect gene expression profiles, or skew results. Therefore, optimizing the tissue dissociation step ensures: 

  • Maximal cell recovery 
  • Maximal viability 
  • Cell membrane integrity 
  • Preservation of cell surface markers mRNA quality and stability 
  • Minimized cellular stress and batch variability 
  • Reproducibility across experiments and sample types 

Careful optimization is essential not only for project success/data quality, but also for cost-effectiveness and efficiency, especially when large numbers of samples are needed. 

Tissue Dissociation Methods 

Tissue dissociation methods generally fall into three categories: 

Mechanical Dissociation:

We first start by collecting the tissue sample under sterile conditions, and keeping the tissue in a cold, isotonic buffer to preserve cell viability. Sterile scissors or scalpels are used to finely chop the tissue into small pieces (1–2 mm³). This increases the surface area and facilitates further dissociation.

Lastly, for mechanical disruption, you can use one or more of the following tools:

  • Pestle and mortar: Gentle grinding to break up tissue.
  • Syringe and needle: Repeatedly pass the tissue through a narrow-gauge needle.
  • Tissue grinders (e.g., Dounce homogenizer): Manually shear cells apart.
  • Automated dissociators: To provide standardized mechanical dissociation.

Physical methods such as cutting, mincing, and grinding to break apart tissue, however fast, may damage cells or result in incomplete dissociation. 

Enzymatic Dissociation:

After mincing, the next step in enzymatic dissociation is the preparation of the enzyme cocktail. Depending on the tissue type, a solution is prepared containing one or more of enzymes, such as:

Papain: Gentle enzyme often used for neural tissues. 

Collagenase: Breaks down collagen in connective tissue.

Trypsin: Cleaves peptide bonds in proteins.

DNase I: Reduces viscosity by digesting free DNA from lysed cells.

Dispase: Separates cells by cleaving extracellular matrix proteins.

Proteolytic enzymes like collagenase, trypsin, and dispase are used to digest the extracellular matrix. These enzymes are often used in specific combinations depending on tissue type and desired cell populations. The minced tissue is incubated in the enzyme solution at 37°C with gentle agitation (e.g., on a shaker or rotator). Duration varies depending on tissue type and enzyme strength.

Similarly to the mechanical method, next step is filtration, passing the digested tissue through a cell strainer to remove undigested clumps, centrifuge the cells at low speed, and wash to remove residual enzymes before resuspending in fresh buffer.

Singleron’s sCelLiVE® Tissue Dissociation Kit is validated for 400+ different tissue types.

Learn more here

Combined Approaches:

Most workflows use a combination of mechanical and enzymatic techniques to balance efficiency with cell integrity. Timing, temperature, and enzyme concentration are carefully calibrated for each tissue type. 

The choice of dissociation method must be tailored to the tissue of interest, taking into account its density, composition, and the sensitivity of target cells. Which brings us to the question:

How to dissociate my tissue of interest?

The method you choose depends heavily on the tissue type, its structural complexity, and the downstream application. For single-cell RNA-seq, prioritize high viability and minimal debris whereas for FACS, you need to ensure cells are free of clumps. For culture, avoid harsh enzymes and maintain sterile conditions. While protocols vary by tissue type, several core principles apply across the board that can remedy common challenges in tissue dissociation:

Optimize Conditions

Temperature

Keep samples cold, unless enzymes require incubation at 37°C.

Time

Over-digestion can damage cells; under-digestion can reduce yield.

Enzyme concentration

Start with published protocols and adjust based on tissue density and cell type.

Minimize Cell Stress

  • Use gentle pipetting to avoid shear stress.
  • Include BSA or FBS in buffers to protect cells.
  • Add RNase inhibitors if RNA integrity is critical.

 Validate and Count

  • Check cell viability using Trypan Blue or automated counters.
  • Assess cell integrity under a microscope before proceeding.

Automated Tissue Dissociation with PythoN Junior and PythoN i from Singleron 

To standardize and streamline the tissue dissociation process, Singleron Biotechnologies has developed PythoN Junior and PythoN i, automated tissue dissociation systems designed for reproducibility, efficiency, and ease of use. 

PythoN Junior is a compact instrument with 2 independent channels. The instrument is suitable for low- to medium-throughput laboratories. It provides consistent dissociation protocols with pre-programmed settings optimized for various tissue types. This eliminates manual variability and ensures higher reproducibility between experiments. 

PythoN i is the 8 independent channels version, supporting simultaneous processing of multiple samples with customizable protocols. It is ideal for clinical research and industrial applications where consistency and speed are paramount. 

Both systems offer the following benefits: 

Standardized protocols 

for reproducible cell yield and viability 

Compatible consumables

including disposable dissociation units and broad-spectrum dissociation buffers 

Time-saving automation 

with reduced hands-on time 

Flexible programming 

to accommodate diverse tissue types and research goals 

Enhanced safety and sterility 

in a closed system 

Well-balanced enzymatic and mechanical dissociation,

to ensure successful tissue dissociation in optimal timing 

The PythoN i comes preloaded with 100+ customizable programs.

Discover PythoN i

PythoN in use 

PythoN® was used to dissociate murine heart, testicle, liver, lung, kidney, and spleen tissues, resulting in high-quality single cell suspensions with high cell viability and cell yield. All the instruments of the PythoN family are also suitable for low-input samples, even needle biopsies.

Performance Data

Python Tissue Dissociation Unit Performance Data

In our latest publication featuring PythoN, our automated tissue dissociation platform delivered high-quality, high-viability single cell suspensions from human colorectal tumor samples, ensuring reliable input for single cell RNA sequencing.  

Read this and more publications by visiting https://singleron.bio/publicationsPythoN® was used to dissociate murine heart, testicle, liver, lung, kidney, and spleen tissues, resulting in high-quality single cell suspensions with high cell viability and cell yield. All the instruments of the PythoN family are also suitable for low-input samples, even needle biopsies

 

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Conclusion

Tissue dissociation is a foundational step in single-cell research, enabling the transformation of complex tissues into viable single-cell suspensions for high-resolution analysis. The success of downstream applications depends heavily on the quality of this process. By carefully selecting and optimizing mechanical, enzymatic, or combined dissociation methods, researchers can preserve cell viability and integrity. Instruments like the PythoN systems further enhance reproducibility and efficiency, making tissue dissociation more accessible and standardized.

Are you planning an experiment soon and would like to know how our solutions can help your tissue dissociation?

Let’s discuss!  

A post by Juan Vicente Lorenzo

Juan earned his master’s degree in biology from the University of Málaga. After a 12-year career in marine biology, he moved to Germany, where he successfully transitioned into the field of molecular biology. With a strong background in routine automation and in vitro diagnostics (IVD), Juan joined Singleron in January 2022.
He has contributed to the development and optimization of several Singleron kits and he has played a key role in starting the local production in Cologne.
Juan is now part of the System Integration team, where he has worked in the optimization of Python Junior, and lately, the development and optimization of Tensor High-Throughput Automation instrument.

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