Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function

Germann, Anja; Oh, Younjae; Schmidt, Tomm; Schön, Uwe; Zimmermann, Heiko; Briesen, Hagen von

doi:10.1016/j.cryobiol.2013.06.012

Cited by 75 publications

(61 citation statements)

References 52 publications

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“…2A), confirming the effect of the process on cellular viability. The median (range) of nonviable PBMCs from healthy children was low (4.6% [1% to 19%]) after cryopreservation and comparable to that reported previously (5,23), demonstrating the efficiency of the freezing protocol used here ( Fig. 2A).…”

Section: Resultssupporting

confidence: 58%

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Perdomo‐Celis

Salgado

Castañeda

et al. 2016

Clin Vaccine Immunol

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bCryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosisinducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-␥]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r ‫؍‬ 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P < 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3 ؉ CD8 ؉ amine-positive cells was found before cryopreservation (P ‫؍‬ 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-␥-producing CD3؉ cells after stimulation compared with healthy controls and convalescent-phase patients (P < 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho ‫؍‬ ؊0.56; P ‫؍‬ 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. Cryopreservation is the maintenance of cells and biological tissues at low temperatures and is based on the use of various media or solutions that form hydrogen bonds with water molecules, preventing cellular damage. The low temperatures allow the cells to enter a quiescent state in which cellular functions are suspended without affecting their intrinsic characteristics (1). Peripheral blood mononuclear cells (PBMCs) are frequently cryopreserved for use in transplants or immunological studies (2, 3). However, the cryopreservation process may affect viability, phenotype, and cellular functionality due to factors such as inadequate temperatures, the freezing protocol used, the expertise of the personnel, and freezing time (4, 5). The disease of the individual from whom the PBMCs come also affects cryopreservation. For example, PBMCs from subjects infected with human immunodeficiency virus (HIV) presented reduced viability after cryopreservation, possibly due to the increased numbers of apoptotic cells circulating during the course of the disease (6). Similar findings have been found in the acute phases of diseases, such as visceral leishmaniasis (7). Particularly for HIV, great efforts have been undertaken to optimize the evaluation and comparability of immune tests...

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Section: Resultssupporting

confidence: 58%

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Perdomo‐Celis

Salgado

Castañeda

et al. 2016

Clin Vaccine Immunol

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“…9,[15][16][17] Although, as expected, fluctuating temperatures experienced by the cells in transit resulted in a lower yield of viable cells, antigen-specific B MEM frequencies could still be reliably measured. Compared with T cells, which have been shown to display reduced antigen-specific responses after experiencing fluctuating storage temperatures, 18 B MEM may be more resilient under these conditions. Although previous studies have shown that low cell viability following cryopreservation can compromise lymphocyte proliferation in response to mitogens including PWM, 33 the present study shows that after allowing for lower viable cell numbers following storage and shipment, freezing of PBMC did not affect the numbers of viable PBMCs that were harvested after a 6-d culture compared with fresh samples.…”

Section: Discussionmentioning

confidence: 99%

Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot

Trück

Mitchell²,

Thompson

et al. 2014

Human Vaccines & Immunotherapeutics

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“…29 The maintenance of T cell functionality during cryopreservation is therefore very important to produce reliable results. 10,26,30–34 …”

Section: Introductionmentioning

confidence: 99%

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

Angel

et al. 2016

Biopreservation and Biobanking

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Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented.

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Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function

Cited by 75 publications

References 52 publications

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Effect of cryopreservation of peripheral blood mononuclear cells (PBMCs) on the variability of an antigen-specific memory B cell ELISpot

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality

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