Cryopreservation of primary B cells minimally influences their signaling responses

Abstract

Phospho flow is a powerful approach to detect cell signaling aberrations, identify biomarkers and assess pharmacodynamics, and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here, B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions, fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes.

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors

“…of immune cells to freezing compared to other cell types, also observed in other settings 21 , and the lack of a dissociation step in CLL scRNA-Seq (see Methods). Cryopreservation, however, can increase the proportion of damaged cells 22 and may not successfully recover all the malignant and other non-malignant cells in the tumor.…”

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

“…In particular, in CLL, we successfully recovered the expected cell types from a cryopreserved sample, containing viable cells. This reflects the increased resilience of immune cells to freezing compared to other cell types, also observed in other settings(Hermansen et al, 2018), and the lack of a dissociation step in CLL scRNA-Seq ( Methods ). Cryopreservation, however, can increase the proportion of damaged cells(Guillaumet-Adkins et al, 2017) and may not successfully recover all the malignant and other non-malignant cells in the tumor.…”

B cell signalling pathways—New targets for precision medicine in chronic lymphocytic leukaemia

“…94 The method gives highly reproducible results, and cryopreservation does not affect signalling responses in B lymphocytes. 95 By performing high-throughput phospho flow experiments in a 96-well plate format, changes in phosphorylation level of 35 proteins in response to short (20 minutes) treatment with the BTK inhibitor ibrutinib and the PI3K inhibitor idelalisib was systematically analysed in CLL cells. 89 Results showed that the targeted agents primarily affect proteins in the BCR signalosome and in the PI3K pathway.…”

“…Application of phospho flow has provided valuable information about baseline and drug‐induced cell signalling in several haematological malignancies, including CLL, 39,43,88,89 acute myeloid leukaemia (AML) 90‐92 and non‐Hodgkin lymphomas, 93 as well as in normal haematologic subsets present among the malignant cells 94 . The method gives highly reproducible results, and cryopreservation does not affect signalling responses in B lymphocytes 95 . By performing high‐throughput phospho flow experiments in a 96‐well plate format, changes in phosphorylation level of 35 proteins in response to short (20 minutes) treatment with the BTK inhibitor ibrutinib and the PI3K inhibitor idelalisib was systematically analysed in CLL cells 89 .…”