Splenocytes in Immunological Research: Across Species and Isolation, Freezing, Thawing Steps

Keywords: Splenocytes(SPLs); Cynomolgus Monkey Splenocytes; Rhesus Monkey Splenocytes; Dog Splenocytes; Canine Splenocytes; Rat Splenocytes; Mouse Splenocytes; Mice Splenocytes; Rabbit Splenocytes; Splenocytes Isolation; Freezing Splenocytes; Thawing Splenocytes

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Splenocytes (SPLs) are a heterogeneous population of immune cells isolated from the spleen, a vital organ involved in filtering blood, mounting immune responses, and maintaining homeostasis. These cells include lymphocytes (T cells, B cells), macrophages, dendritic cells, and natural killer (NK) cells, all of which play critical roles in adaptive and innate immunity. Splenocytes are widely used in preclinical and translational research to study immune function, vaccine development, autoimmune diseases, and cancer immunotherapy.

The Role and Composition of Splenocytes (SPLs)

Within the spleen, splenocytes reside in distinct regions: the red pulp, which filters blood and recycles old red blood cells, and the white pulp, where immune responses are initiated. The white pulp is organized into T cell zones and B cell follicles, facilitating interactions between antigen-presenting cells and lymphocytes. This unique architecture makes splenocytes (SPLs) a powerful tool in immunological assays such as ELISPOT, flow cytometry, and mixed lymphocyte reactions.

Species-Specific Splenocytes (SPLs)

-Cynomolgus Monkey Splenocytes: Cynomolgus monkey splenocytes are used in preclinical research because they more closely mimic human immune responses, making cynomolgus monkey splenocytes ideal for translational studies.

-Rhesus Monkey Splenocytes: Rhesus monkey splenocytes are similarly valued for their close immunologic similarities to humans. Studies using rhesus monkey splenocytes help bridge the gap between animal models and clinical applications.

-Dog Splenocytes / Canine Splenocytes: In veterinary research, dog splenocytes (or canine splenocytes) are used to study immune disorders in canines and to draw comparisons with human immune responses.

-Rabbit Splenocytes: Rabbit splenocytes are important in studies on antibody production and vaccine development. Rabbit splenocytes help reveal details that may differ from mouse splenocytes or rat splenocytes.

-Mouse Splenocytes / Mice Splenocytes: Mouse splenocytes are one of the most frequently studied splenocytes (SPLs) in immunology. Protocols for isolation splenocytes from mice are well established, and both mouse splenocytes and mice splenocytes are used for flow cytometry, ELISPOT, and other functional assays. 

-Rat Splenocytes: Rat splenocytes provide a complementary model for immunotoxicology and vaccine studies. Rat splenocytes are often compared with mouse splenocytes to understand interspecies differences.

Splenocytes Isolation

Splenocytes Isolation is the process of extracting immune cells from the spleen to be used in various research applications, such as flow cytometry, cytokine production assays, or mixed lymphocyte reactions.

The spleen was removed under aseptic conditions, ground in isolation solution and transferred to a sterile tube containing RPMI 1640 medium. The leukocyte layer was carefully collected after centrifugation. The cells were washed with RPMI 1640 medium, centrifuged and the supernatant discarded. This washing step was repeated 1-2 times before the isolated cells were used for further experiments.

Freezing splenocytes

Freezing Splenocytes is a crucial step for preserving cells for future use. Cryopreservation allows researchers to store splenocytes for extended periods without compromising their viability or functionality.

The supernatant of the well centrifuged cell suspension from the isolation step is discarded and the cell concentration was diluted in freezing medium. Add an aliquot to each freezing tube and transfer to a freezing container, ensuring that the cells remain in suspension. Quickly freeze the freezing containers in a -80°C freezer. Transfer to -150°C freezer container (or liquid nitrogen tank) for long-term storage.

Thawing Splenocytes

Thawing Splenocytes must be done carefully to ensure high cell viability and functionality after cryopreservation. The thawing process is typically fast to minimize the damaging effects of DMSO and ice crystal formation.

The cryotubes are transferred to a 37°C water bath and thawed until only fine ice crystals remained in the tubes. Add 0.5-1mL of cell culture medium to the frozen tube, resuspend and transfer the suspension to a 15mL tube filled with cell culture medium. Centrifuge, remove supernatant and tap the tube to loosen the cell arrangement. Add 1mL of cell culture medium, blow and resuspend with a pipette, add medium to a volume of 15 ml. centrifuge, de-supernatize, add 1mL of cell culture medium, adding medium according to expected cell concentration. Place the cells in a CO2 incubator and incubate for 1h with a slight gap in the lid. At the end of the incubation, resuspend and leave for 1min to allow the aggregated cell debris to precipitate. The cell suspension without precipitation was carefully transferred to a new 15mL tube. Cells were counted to determine cell activity.

Conclusion

Splenocytes, with their diverse composition and pivotal role in immune responses, are invaluable tools in immunological research. Whether derived from rodents, primates, or other species like rabbits and dogs, splenocytes facilitate critical studies in immune function, disease mechanisms, vaccine development, and cancer immunotherapy. The processes of isolation, freezing, and thawing are essential for preserving their integrity and viability, enabling researchers to explore cellular dynamics across various models. As our understanding of immunology deepens, the use of splenocytes will continue to be instrumental in bridging the gap between preclinical studies and clinical applications, ensuring advancements in both human and veterinary medicine.