Time dependent changes in the bioenergetics of peripheral blood mononuclear cells: processing time, collection tubes and cryopreservation effects.
Author(s): Werner BA, McCarty PJ, Lane AL, Singh I, Karim MA, Rose S, Frye RE
Publication: Am J Transl Res, 2022, Vol. 14, Page 1628-1639
PubMed ID: 35422946 PubMed Review Paper? No
Purpose of Paper
This paper compared the viability and bioenergetics of fresh and cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from EDTA, sodium citrate (NaC) and cell preparation tubes (CPT) after different processing delays. Potential effects of storing blood in the different collection tubes on wet or dry ice for up to 24 h; storing PBMCs with plasma, without plasma and with various media; and cryopreservation in an isopropanol bath or using the Mr Frosty device were also investigated.
Conclusion of Paper
Storage of blood tubes on dry ice resulted in cracking of the NaC tubes and hemolysis of the EDTA blood specimens, but all NaC and EDTA tubes and 3 of 4 CPT tubes remained intact following storage of blood collection tubes on wet ice for 24 h. Storage of blood, resulted in lower viability of PBMCs from CPT, but had a lesser impact on PBMC from EDTA or NaC tubes where a loss of 0.5%/h was observed. Viability of PBMCs was not affected by removal of plasma, or replacement of the plasma with any of the tested media (DMEM, wash buffer, Seahorse media). Storage of blood prior to PBMC isolation altered ATP linked respiration (ALR), proton leak respiration (PLR) and maximal respiratory capacity (MRC).
Cryopreservation by the standard method (Mr Frosty) resulted in decreased viability, ALR, MRC and reserve capacity (RC) and elevated PLR. PBMCs that were stored for 24 h before cryopreservation displayed changes in ALR, PLR, MRC, and RC, but depended on whether PBMCs were stored with or without plasma as well as the duration of cryostorage. Using an isopropanol bath during cryopreservation led to lower viability, ALR, MRC and RC. During cryostorage of PBMCs in liquid nitrogen changes in viability, ALT, PLR, MRC and RC were observed but depended on the duration of storage and whether PBMCs were stored on wet ice prior to cryopreservation.
Studies
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Study Purpose
This study compared the viability and bioenergetics of fresh and cryopreserved PBMCs isolated from different blood collection tube types (EDTA, NaC and CPT) after different processing delays. Potential effects of storing blood in the different collection tubes on wet or dry ice for up to 24 h; storing PBMCs with plasma, without plasma, and with various media; and of cryopreservation in an isopropanol bath instead of sing the Mr Frosty device were also investigated. Blood was collected from an unspecified number of patients (no details provided) into EDTA, sodium citrate (NaC), and Mononuclear Cell Preparation Tubes (CPT). Tubes were immediately placed on wet ice. Unless otherwise specified, specimens were centrifuged at 1500 g for 10 min at 4°C within 30 min of collection. Plasma was replaced with wash buffer and layered with Histopaque-1077. PBMCs were separated by centrifugation at 400 g for 30 min, washed in wash buffer, and counted using a hemocytometer. PBMCs were suspended in culture media, frozen at -80°C in a Mr Frosty Cryofreeze container overnight and then transfer to liquid nitrogen. Mitochondrial respiration was evaluated using the Seahorse assay. To assess the effects of delayed processing on PBMC viability, blood was stored on wet ice or dry ice for 0 and 24 h before centrifugation. To assess effects of different conditions during post-centrifugation storage on PBMC viability, tubes were stored after initial centrifugation for 24 h with plasma (CPT and NaC tubes), after plasma was removed (CPT and NaC tubes), after plasma was replaced with DMEM and not mixed (NaC tubes only), after plasma was replaced with DMEM and mixed (NaC tubes only), after plasma was replaced with wash buffer (NaC tubes only), after plasma was replaced with Seahorse media and not mixed (NaC tubes only), and after plasma was replaced with Seahorse media and mixed (NaC tubes only). Potential effects of a 1, 2, 3, 4, 5, 6, 7, 8 and 12 h processing delay on mitochondrial respiration were evaluated in NaC and EDTA specimens by storing blood on wet ice . Potential effects of a 5, 6, 7, and 8 h processing delay on mitochondrial respiration were evaluated in CPT specimens by storing blood on wet ice. Potential effects of cryopreservation method on PBMC viability were investigated using NaC and EDTA specimens collected from 4 patients by comparing cryopreserved PBMCs obtained without a processing delay, cryopreserved PBMCs that were stored for 24 h on wet ice with or without plasma prior to cryopreservation, and PBMCs cryopreserved in a -80°C isopropanol bath.
Summary of Findings:
Storage of blood tubes on dry ice resulted in cracking of the NaC tubes and hemolysis of the EDTA blood specimens, but all NaC and EDTA tubes and 3 of 4 CPT tubes remained intact following storage of blood collection tubes on wet ice for 24 h. Viability was significantly reduced when blood was stored on wet ice in CPT for 24 h, but viability was not significantly lower after storage of blood in EDTA or NaC tubes after 24 h of storage. Viability of PBMCs was not affected by removal of plasma, or replacement of the plasma with any of the tested media. In a follow up study with more time-points, viability decreased linearly by 0.5%/h (P<0.05). In this same experiment, PBMC from blood stored in EDTA and NaC tubes had increasing ALR with storage duration for the first 12 h and then decreasing ALR (P<0.01). During blood storage proton leak respiration (PLR) of PBMC decreased and then increased in blood stored in NaC tubes but did not change when blood was stored in EDTA tubes. The maximal respiratory capacity (MRC and reserve capacity increased and then decreased in PBMC from blood stored in NaC (P<0.01 and P=0.001, respectively) and EDTA tubes (P<0.001 and P<0.001, respectively) with peaks observed at 4 h for specimens from NaC tubes and 7 h for specimens from EDTA tubes.
Cryopreservation by the standard method (using the Mr Frosty device at -80°C) resulted in a 9.6% loss in viability (P<0.05), a decrease in ALR (P<0.001), an increase in PLR (P<0.05), a decrease in MRC (P<0.0001), and a decrease in RC (P=0.001) compared to fresh PBMCs. Compared to fresh PBMC, a 24 h delay to cryopreservation resulted in decreased ALR (P<0.001), increased PLR (P<0.05) which declined with subsequent cryostorage, decreased MRC (P<0.0001), and decreased RC (P<0.05). Storing PBMC without plasma for 24 h before cryopreservation increased viability by 14.3% (P<0.05), increased ALR (P<0.001) and PLR (P=0.01) but had no effect on MRC. Compared to fresh PBMC, PBMCs stored after centrifugation had reduced ALR (P<0.001) and increased PLR (P<0.0001) but did not affect MRC. Cryopreservation of PBMCs using a -80°C isopropanol bath led to a 14.3% decrease in viability (P<0.05) and lower ALR, MRC and RC (statistics not provided) than fresh PBMCs. During liquid nitrogen storage of cryopreserved PBMCs viability remained unchanged during the first month but then declined, ALR declined only in specimens stored for 24 h prior to cryopreservation, PLR increased during storage in freshly cryopreserved PBMC but decreased and then increased when PBMC were stored for 24 h before cryopreservation, MRC and RC declined and then increased during storage regardless of storage prior to cryopreservation in both types of PBMC.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Small molecule Clinical chemistry/auto analyzer Cell count/volume Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution NaC tube
EDTA tube
CPT
Biospecimen Acquisition Anticoagulant Sodium citrate
EDTA
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 0 h
24 h
1 h
2 h
3 h
4 h
5 h
6 h
7 h
8 h
12 h
Storage Storage conditions With plasma
Without plasma
With DMEM (mixed)
With DMEM not mixed
With PBMC wash buffer
With Seahorse media (mixed)
With Seahorse media not mixed
Storage Storage temperature Wet ice
Dry ice
Biospecimen Preservation Cooling or freezing method/ rate Mr Frosty
Isopropanol bath
Biospecimen Preservation Type of fixation/preservation Snap frozen
None (fresh)