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Updated: Jul 18, 2026
Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety
Published on: September 15, 2023
Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study
Wayne G Shreffler1, Cynthia M Visness, Melissa Burger
1Mount Sinai School of Medicine, Division of Pediatric Allergy & Immunology, New York, NY, USA. wayne.shreffler@mssm.edu <wayne.shreffler@mssm.edu>
Insights
Cryopreservation significantly alters cytokine responses in peripheral blood mononuclear cells, impacting immunologic measurements. Standardizing cryopreservation methods is crucial for multicenter studies to ensure reliable results.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cryopreservation of peripheral blood mononuclear cells (PBMCs) is vital for multicenter immunologic studies.
- The precise impact of cryopreservation on cytokine responses remains incompletely understood.
- This study addresses knowledge gaps in designing immunologic studies for a childhood asthma cohort.
Purpose of the Study:
- To evaluate the effects of two cryopreservation methods on PBMC cytokine responses.
- To assess cryopreservation's impact on both cord and peripheral blood mononuclear cells.
- To determine the suitability of cryopreserved cells for multicenter immunologic research.
Main Methods:
- Paired PBMC samples were processed fresh, or cryopreserved using Nalgene containers (NC) or a controlled-rate freezer (CRF).
- Cytokine secretion (IL-10, IL-2, TNF-alpha, IL-13, IFN-gamma) was measured following stimulation.
- Reproducibility was assessed by processing identical samples across multiple laboratories.
Main Results:
- Cryopreservation inhibited PHA-induced IL-10 and Der f 1-induced IL-2 secretion.
- Cryopreservation augmented PHA-induced IL-2 and spontaneous TNF-alpha secretion.
- Cord blood mononuclear cell cytokine profiles were significantly affected by NC cryopreservation, with limited correlation between fresh and cryopreserved samples.
Conclusions:
- Cryopreservation alters mononuclear cell cytokine response profiles, particularly IL-10 secretion and antigen-induced responses.
- Standardization of mononuclear cell responses is achievable for multicenter studies.
- Assessing reproducibility and the impact of cryopreservation techniques on specific immunologic outcomes is essential.
Background:
Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells.
Results:
Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-alpha. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-gamma, TNF-alpha) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site.
Conclusion:
Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes.
