Tissue dissociation to achieve a single-cell suspension of live, intact cells is required for various techniques, such as flow cytometry, single-cell transcriptomics, and single-cell functional assays. This differs significantly from homogenization techniques that aim to disrupt membranes and release cellular macromolecules for isolation. Efficiently dissociating tissue while preserving cellular integrity is a technically challenging process, often involving a combination of mechanical and enzymatic methods to prepare single-cell suspensions. There are a variety of protocols for tissue dissociation depending on the tissue and downstream application. One popular approach is using orbital shakers or shaking water baths in combination with enzymatic digestion. Non-enzymatic approaches often involve paddle blenders (often called Stomachers), tissue grinders, or bead mills.
The Benchmark Scientific Incu-Shaker 10L has a temperature range of 5°C to 60°C while the refrigerated model (10RL) can be cooled to -15°C, making them suitable for nearly all enzymatic dissociation protocols.
Applications for Tissue Dissociation
Flow cytometry and FACS
Flow cytometry and flow-assisted cell sorting (FACS) are used for cell identification and quantitation, to determine the expression and abundance of proteins of interest, and to sort cells for downstream applications. Most protocols for flow cytometry depend on surface antigen recognition to identify cell types. Thus, these surface antigens must remain intact during tissue digestion. Some enzymes cleave surface receptors and other antigens. Sorting specific cell populations from tissue using FACS requires a high level of viability and cellular integrity as sorting purity depends on antigen integrity, and downstream applications require representative cell biology.
Single-Cell Transcriptomics
For any transcriptomic technique, preparing the sample in a way that minimizes alterations to the transcriptional state is crucial. The most appropriate protocol will depend on the tissue and the cell types of interest. Certain cell types, such as neurons, are often underrepresented in single-cell transcriptomic datasets because it is challenging to extract intact neuronal cells. In cases where effective tissue digestion is difficult or impossible—like in frozen samples or adipose tissue— single-nuclei preparations are preferable [1].
Functional Assays
Viability and cellular integrity are paramount for applications requiring downstream cell function. Additionally, if your functional assay requires the activation of specific surface proteins, like transmembrane receptors, you must ensure that these proteins are unaffected by your dissociation protocol.
Cell Culture and Cell Line Development
Tissues can be dissociated for various cell culture applications, such as isolating primary cells, developing cell lines, performing functional assays in a tissue-specific context, screening novel therapeutics, and preparing co-cultures or organoid cultures. If long-term culture or storage is needed, sterility in the dissociation process—including the reagents, equipment, and personnel—is essential. Even the smallest amount of contamination can ruin mammalian cell culture or even cross-contaminate other cultures. Contamination can also alter biological function even before it is detected.
Enzymatic & Mechanical Approaches
Most dissociation protocols involve some combination of enzymatic and mechanical approaches. Before you select a protocol, you’ll want to consider your application and requirements for viability, cellular integrity, and retention of surface proteins, as we discussed above. Some enzymes can degrade surface proteins, particularly surface receptors. This can be highly problematic for flow cytometry applications where proteins located on the cell surface are used as antigens for cell identification. If you utilize cell suspension for assays that require cellular responses to extracellular cues, you must be selective with your enzymatic choice or opt for non-enzymatic dissociation methods. Enzymatic dissociation can compromise surface receptors, blunting ligand-mediated responses.
Enzymatic Approaches
Collagens, the most abundant proteins in the body, hold cells and tissues together. Collagenases hydrolyze collagens. In addition to collagenases, serine proteases, dispases, and hyaluridonases are common enzymes used in tissue dissociation.
Serine Proteases
Trypsin is the most commonly used serine protease for tissue dissociation. It is generally considered the harshest class of enzymes used for this purpose. Though highly efficient, trypsin and other serine proteases have the greatest impact on cell viability and are incompatible with the integrity of many surface antigens.
Collagenases
Collagenase, derived from C. histolyticum, is the most commonly used class of enzymes used for dissociation of various tissue types, including lung, heart, muscle, bone, liver, kidney, mammary gland, and tumors. There are many different types of collagenases. Collagenases A, B, D, H, and P are most common in tissue dissociation protocols. Among these, Collagenase D is recommended when the functionality and integrity of cell-surface proteins are important.
Dispases
Dispases are generally gentler than serine proteases or collagenases. They cleave fibronectin and collagen IV and do not disrupt cell membranes.
Hyaluronidases
Hyaluronic acid is a significant component of the extracellular matrix that holds tissues together. Hyaluronidases are a type of glycosidase that cleave glycosidic bonds, degrading hyaluronic acid. Hyaluronidases are usually used in combination with other enzymes for more thorough dissociation.
Cold-Active Enzymes
Most enzymes for tissue dissociation work in only a small range of temperatures (around 37°C). However, for transcriptomic approaches, it’s important to consider that prolonged incubation at 37°C will allow transcriptional machinery to remain active and may impact the transcriptomic landscape measured. For this reason, you may consider a cold-active enzyme that works at temperatures below 25°C. These enzymes are much less efficient for tissue dissociation.
Mechanical Approaches
Enzymatic approaches are usually combined with mechanical approaches, including shaking water baths and orbital shakers. Because most enzymes function in a narrow range of temperatures, incubation is usually essential. There are also enzyme-free approaches that are solely mechanical, such as using paddle blenders (often referred to as Stomachers), tissue grinders, or bead mills. These often have the worst cell viability but avoid the limitations of antigen cleavage caused by enzymatic approaches.
Shaking Water Baths
For enzymatic tissue dissociation, a shaking water bath is often preferred over an incubated orbital shaker, mainly because the heat transfer from water is more efficient than air, improving overall temperature consistency.
The Julabo SW Series Reciprocating Water Baths have a temperature range of 20-99.9°C They also have a splash-proof design to reduce the likelihood of sample contamination.
Incubated Orbital Shakers
Incubated orbital shakers may be preferable where sterility is important, as bacteria grow well in 37°C water, increasing the likelihood of sample contamination. Incubated orbital shakers come in smaller benchtop and larger floor models.
Shaking CO2 incubators, like the New Brunswick™ S41i CO2 Incubator Shaker, address concerns with cell physiology or viability that may arise from prolonged exposure to ambient air.
Paddle Blenders/Stomachers
Paddle blenders, often called Stomachers, can be used with or without enzymes for tissue dispersion. For more on what to consider when selecting a Paddle Blender, see our prior blog post.
The Stomacher® 80 Biomaster paddle blender is great for small tissue samples, though it can process samples from 250µl to 80 mL.
Tissue Grinders
There have been several studies comparing the viability and integrity of cells following tissue dissociation using mechanical and enzymatic approaches. One such study compared cell viability following the dissociation of mouse and human colon biopsy samples using a tissue grinder compared to an enzymatic approach and reported that efficiency was similar and the mechanical approach was preferred for downstream flow cytometry as surface antigens remain intact [3]. There are multiple types of tissue grinders, including the mortar & pestle style BioSpec TissueGrinder.
Bead Mill Homogenizers
Bead mill homogenizers utilize tubes filled with beads to rapidly dissociate tissue samples. To dissociate tissues rather than homogenizing them, it is important to use a bead mill homogenizer which can attain a very low minimum speed, as many such homogenizers have relatively high minimum speeds that still efficiently lyse cells. Bead tubes can be purchased pre-filled, or beads can be purchased separately. The size and material of the bead can impact the cell viability and integrity and are suitable for different tissue hardness. Using larger beads will help preserve cell viability, but may lead to incomplete tissue dissociation.
The Bullet Blender 5E Pro is a great option for tissue dissociation as the speed can be reduced to near-zero. A lower speed is required to maintain cell viability.
Protocol Considerations
Optimizing a tissue digestion protocol requires careful consideration of multiple factors. Key variables include the duration of digestion, the enzyme type and concentration, the digestion buffer composition and volume, the incubation temperature, and even the orbit speed and type of container used. Each of these elements can significantly impact the outcome.
Different tissue types demand tailored approaches. For example, delicate tissues, like those rich in immune cells, require gentler protocols than denser tissues, like solid tumors or those rich in connective tissue. Solid tumors often necessitate more aggressive enzymes and higher buffer volumes, while delicate cell populations—such as immune cells—are more sensitive to harsh conditions. Balancing digestion efficiency with cell viability is crucial, especially for downstream applications like flow cytometry that rely on live cells with intact surface antigens for accurate analysis.
For instance, in our studies with murine lung tissue, using Collagenase D digestion for two hours in a 37°C orbital shaker resulted in significantly improved digestion efficiency compared to one hour under the same conditions. However, the extended duration compromised cell viability, leading to a substantial loss of cells critical for downstream analysis. By increasing the digestion buffer volume from 1 mL to 4 mL per 100 mg of tissue, we achieved a marked improvement in cell viability—surpassing results obtained with a shorter digestion time. This adjustment allowed us to recover more viable cells while maintaining effective digestion.
References:
[1] Ding, J., Adiconis, X., Simmons, S.K. et al. Systematic comparison of single-cell and single-nucleus RNA-sequencing methods. Nat Biotechnol 38, 737–746 (2020). https://doi.org/10.1038/s41587-020-0465-8
[2] Denisenko, E., Guo, B.B., Jones, M. et al. Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biol 21, 130 (2020). https://doi.org/10.1186/s13059-020-02048-6
[3] Soteriou, D., Kubánková, M., Schweitzer, C. et al. Rapid single-cell physical phenotyping of mechanically dissociated tissue biopsies. Nat. Biomed. Eng 7, 1392–1403 (2023). https://doi.org/10.1038/s41551-023-01015-3
