Immune Intergrity: Retain biological fidelity in single-cell spleen prep

Cellsonics' SimpleFlow™ system introduces an innovative approach to spleen dissociation by utilizing acoustic energy, eliminating the tradeoff between harsh enzymatic digestion and labor-intensive manual dissociation. The data show this method better retains cell surface markers and cellular diversity, yielding viable single-cell suspensions that closely reflect the composition of native tissue.

In this study, we compare three tissue dissociation methods—acoustic (SimpleFlow), mechanical, and enzymatic—using murine spleen samples. Our findings demonstrate that SimpleFlow's acoustic dissociation produces a higher live cell yield than traditional mechanical dissociation while better preserving key markers such as CD45, CD4, and CD19. Improved retention of cell surface markers in a well-characterized tissue like the spleen highlights SimpleFlow as a superior dissociation method, offering enhanced cell viability and marker integrity for downstream applications.

Marker Retention and Immune Profiling

In two experiments conducted on different days, every marker tested exhibited reduced mean fluorescence intensity (MFI) following enzymatic digestion, indicating that enzyme treatment cleaves these surface markers.

Figure 1. MFI of a wide panel of immune markers for spleens dissociated using acoustic energy on the SimpleFlow compared to lowered MFI for spleens dissociated using enzymes. Every single marker represented had reduced fluorescence for the enzymatic dissociation.

A selection of the above markers is highlighted in Figure 2. Here it is demonstrated that the epitope expression of CD45, CD138, CD100, CD229, CD44, and CD45RA, preserved by mechanical dissociation of the spleen, is similarly preserved by acoustic dissociation on the SimpleFlow. In contrast, the MFI of these cell surface markers is reduced in the enzyme dissociation.

Figure 2a. CD45 MFI is significantly higher in the SimpleFlow acoustic dissociation compared to dissociation with an enzymatic kit. Nearly all cells resulting from a spleen dissociation should be CD45+, so the apparent loss of epitopes is likely a result of the harsher dissociation conditions.

Figure 2b. CD138, which should be found on all CD19+ B cells, has reduced expression in spleen dissociated enzymatically. This difference is significant according to a two-tailed unpaired t-test.

Figure 2c. CD100 fluorescent signal is nearly twice as high for the SimpleFlow acoustic dissociation compared to the enzymatic dissociation.

Figure 2d. CD229 expression is significantly higher in spleens dissociated on the SimpleFlow than those dissociated using enzymes.

Figure 2e. CD44 expression on live cells was highest for the acoustically dissociated spleen samples compared to mechanical and enzymatic dissociations, which both had reduced MFI.

Figure 2f. CD45RA was significantly higher for the SimpleFlow acoustic spleen dissociations, with higher MFI and percentage positivity compared to the enzyme and mechanical methods.

Additionally, enzymatic dissociation significantly skewed the CD4/CD8 T cell ratio, a key immune stability indicator (1). SimpleFlow dissociations maintained ratios within the expected healthy range of mechanically dissociated murine spleen (CD4:CD8 = 1.5–2.5) (2,3)^[i], whereas enzyme dissociations exhibited elevated ratios (CD4:CD8 = 3.18–4.6). This discrepancy could pose challenges for researchers studying HIV and other immune dysfunctions, as described by McBride et al. In HIV, immune dysfunction is characterized by a CD4/CD8 ratio below 1, which could be masked by enzyme-induced bias, leading to an overestimation of the true ratio.

Figure 3. CD4:CD8 ratio is a key indicator of immune health and function. The enzyme dissociation (orange) had values well above the healthy expected range of 1.5-2.5 CD4 T cells for every CD8 T Cell, which if taken at face value will distort accurate immunological analysis.

Live Cell Yield and Viability Results

Propidium iodide flow cytometry revealed that the SimpleFlow dissociation resulted in 65% cell viability, comparable to enzymatic dissociation (70% viability), whereas mechanical dissociation yielded a significantly lower viability of ~45%, suggesting that cell damage may occur when forcing spleen tissue through a mesh strainer.

Figure 4. Spleens dissociated on the SimpleFlow had statistically equivalent live cell yield and viability to spleens dissociated enzymatically. The mechanical dissociation has the lowest live cell yield and viability.  

SimpleFlow dissociation generated ~10.9 million live cells per ~100 mg spleen. This is slightly lower than the ~21.1 million live cells obtained via enzymatic dissociation. However, this yield remains sufficient for most applications, as flow cytometry requires 10⁵–10⁶ live cells per sample (4), and 10X Genomics’ scRNA-seq assay requires 10³–10⁴ live cells per sample (5). The SimpleFlow more than tripled the live cells yielded by a mechanical method.

Methods

Figure 5. Workflow time is comparable for the SimpleFlow and the mechanical method. Both take half the time required for enzymatic dissociation.

Spleens were extracted from nine 4- to 6-week-old CD1 mice and processed using three dissociation methods:

1. SimpleFlow Acoustic Dissociation: Three spleens were minced 100 times within the Cellsonics cartridge, acoustically dissociated at 4 °C for six minutes, and passed through a 70-micron cell strainer.
2. Enzymatic Dissociation: Three spleens were incubated at 37 °C for 28 minutes with an enzyme mix, followed by periodic mixing and passage through a 70-micron cell strainer.
3. Mechanical Dissociation: Three spleens were gently pressed through a 70-micron cell strainer without enzymatic digestion.

For all groups, red blood cells were lysed using a hypotonic ACK solution. Samples were stained with antibodies, fixed, and analyzed via flow cytometry on the BD Symphony. Data were processed using FlowJo v10.10.0, and statistical comparisons were performed using unpaired two-tailed Student’s t-tests in GraphPad Prism 10.

Conclusion

SimpleFlow acoustic dissociation provides a consistent, automated approach with higher repeatability than manual methods. It achieves a comparable live cell yield while preserving cell surface markers damaged by enzymatic dissociation. The gentle acoustic dissociation of SimpleFlow offers an optimal balance between both strategies—combining the speed and automation of enzymatic digestion with the preservation of cellular diversity and lineage-specific markers seen in enzyme-free dissociation.

[i] Mice from Jackson Laboratory were of a different strain and used an alternate mechanical dissociation comprised of mincing and manipulation with forceps.

References

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2. McBride JA, Striker R. Imbalance in the game of T cells: What can the CD4/CD8 T-cell ratio tell us about HIV and health? PLoS Pathog. 2017 Nov 2;13(11):e1006624.
   
   
3. MPD: Jaxpheno6: project protocol [Internet]. [cited 2025 Feb 28]. Available from: https://phenome.jax.org/projects/Jaxpheno6/protocol?method=immune+cell+quantification#Procedure
   
   
4. Critical-Aspects-of-Staining-Cells.pdf [Internet]. [cited 2025 Feb 6]. Available from: https://med.virginia.edu/flow-cytometry-facility/wp-content/uploads/sites/170/2015/10/Critical-Aspects-of-Staining-Cells.pdf
   
   
5. 10X Genomics [Internet]. [cited 2025 Feb 6]. What is the minimum number of cells that can be profiled? Available from: https://kb.10xgenomics.com/hc/en-us/articles/115001800523-What-is-the-minimum-number-of-cells-that-can-be-profiled