2020
Cryopreservation Affected the Levels of Immune Responses of PBMCs and Antigen-Presenting Cells
Martikainen, MV
Elsevier BV
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© 2020 Elsevier Ltd.
CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.tiv.2020.104918
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Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells
Maria-Viola Martikainen, Marjut Roponen
PII: S0887-2333(20)30468-9
DOI: https://doi.org/10.1016/j.tiv.2020.104918
Reference: TIV 104918
To appear in: Toxicology in Vitro Received date: 6 April 2020 Revised date: 9 June 2020 Accepted date: 12 June 2020
Please cite this article as: M.-V. Martikainen and M. Roponen, Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells, Toxicology in Vitro(2020),https://doi.org/10.1016/j.tiv.2020.104918
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© 2020 Published by Elsevier.
Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells
Maria-Viola Martikainen* maria.martikainen@uef.fi, Marjut Roponen
Department of Environmental and Biological Sciences, University of Eastern Finland, Kuopio, Finland
*Corresponding author at: Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland.
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ABSTRACT
The effect of cryopreservation on antigen-presenting cells (APCs) is understudied. It is important to understand the effects of cryopreservation on these cells as they play a major role in immune responses, and they could be utilized in different clinical applications.
In this study, we compared fresh and cryopreserved PBMCs in regards of their general immune responsiveness and, furthermore, the effect of cryopreservation on the circulating APCs among PBMCs. We stimulated fresh and cryopreserved PBMCs (N=6) with LPS or Poly(I:C). Cytokine production of PBMCs and expression of functional markers CD80 and ILT4 on major types of APCs, dendritic cells (DCs) and monocytes, were analysed. We also analysed whether cryopreservation affects different subtypes of DCs (plasmacytoid and myeloid DCs) differently.
Cryopreserved PBMCs produced less cytokines than fresh cells in response to stimulation, but the response profiles were comparable. Cryopreservation had also an effect on the relative proportions of APCs. Stimuli-induced responses were somewhat parallel but weaker than those observed in fresh cells.
This study suggests that the use of cryopreserved cells is more suitable in studies that assess general responses to stimuli instead of measuring exact levels of reactions. Thus, the interpretation and comparison of the results of different studies should not be done without considering the differences in cryopreservation techniques and their effects on PBMCs and, more specifically, on APCs.
Keywords: Cryopreservation, Cytokine, Dendritic cell, Immune cell, Monocyte, PBMC
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1. INTRODUCTION
The use of cryopreserved peripheral blood mononuclear cells (PBMCs) has become a standard procedure in several fields of science. For example, in cohort studies the use of cryopreserved cells is usually more practical due to logistical and scheduling reasons.
Several factors may hinder the interpretation of the results obtained using fresh cells i.e.
alterations in data acquired over the whole study period and, in multicenter studies, the differences in instrumentation and technical personnel. In addition, analysis of cells within a reasonable timeframe may not be feasible. Cryopreservation of PBMCs and analysis performed in one center in one batch is widely used response to minimize potential confounders, such as inter-assay and inter-laboratory variability.
Cryopreservation also enables additional analyses as new hypotheses are discovered or methods are developed.
Furthermore, cryopreserved cells can be used in cellular and immune therapies.
These applications are typically time- and cost-intensive and require a large number of cells with minimal batch-to-batch variations. Cryopreservation of large batch of cells at one time point could be the answer to minimize the constraints of these applications.
Additionally, when investigating new biomarkers for early detection and prognosis of various diseases it is important that immunological assays can be performed after the conclusion of the studies when all the endpoints have already been identified. [John et al. 2003]
Although cryopreservation of PBMCs is well-established practice offering several advantages, the effect of cryopreservation on PBMCs is understudied. Some studies have assessed the effect of cryopreservation on cell numbers or relative proportions, whereas other studies have concentrated on functional properties [Kreher et al. 2003,
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Lauer et al. 2017, Lemieux et al. 2016, Reimann et al. 2000, Weinberg et al. 2009].
Furthermore, it is somewhat conflicting whether cryopreserved blood is comparable to fresh blood and is the use of cryopreserved cells practical in clinical studies. Only a few studies have investigated the effects of cryopreservation on antigen-presenting cells (APCs). Studies concentrating on immunological responses of dendritic cells (DCs) are hard to find [Gerrits et al. 2007, John et al. 2003, Zhou et al. 2016]. It would be exceedingly beneficial to understand the effects of cryopreservation on these cells as they play a major role in immune responses, and they could be utilized in different clinical applications.
Of course, not only cryopreservation, but also sample shipment [Kofanova et al.
2014], storage temperature [Smith et al. 2007, Yang et al. 2016], handling [Baboo et al.
2019, Nazarpour et al. 2012] and several other things such as composition of freezing media can have effects on PBMCs.
In this study, we assessed whether cryopreservation alters cytokine secretion of PBMCs. We also analysed whether cryopreservation affects the relative proportion, phenotype or functional properties of antigen-presenting cells, namely myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and monocytes. Freshly isolated and cryopreserved PBMCs were stimulated with different stimulants. Expression of immunological markers cluster of differentiation 80 (CD80) and immunoglobulin-like transcript 4 (ILT4) on circulating mDCs, pDCs and monocytes were analysed by flow cytometry.
We assessed the expression of these two immune receptors, as they are expressed in circulating antigen-presenting cells and represent stimulatory and inhibitory responses.
Cytokine production of PBMCs was analysed by multiplexed ELISA method.
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2. MATERIALS AND METHODS 2.1 Experimental set-up
PBMCs from six (6) healthy adult volunteers were collected for this experiment.
Informed consent from all participants was obtained. Simplified overview of experimental set-up is described in Figure 1. To assess the effects of cryopreservation on studied cells and soluble mediators, we used paired samples; half of the PBMCs from each donor were processed immediately (fresh cells), while the other half were cryopreserved for later processing (cryopreserved cells). Due to the freezing and thawing of the cells, fresh and thawed PBMCs of the same donor could not be tested side-by-side.
Fresh and cryopreserved PBMCs were stimulated with different stimulants (Polyinosinic: polycytidylic acid (POLY(I:C)), and lipopolysaccharide (LPS)) for 18 hours in at 37° C in 5% CO2.
Circulating myeloid DCs (mDCs), plasmacytoid DCs (pDCs) and monocytes were identified by flow cytometry and analysed for the expression of functional markers CD80 and ILT4. Cytokine production (IFN-γ, IL-1β, IL-10, IL-12/IL-23p40, TNFα) of PBMCs was analysed by multiplexed ELISA method.
2.2 Isolation of PBMCs
Blood samples were collected from peripheral vein into EDTA-tubes (10 ml, Vacutainer, K2EDTA, BD). Samples were then were gently mixed by inverting tubes 8- 10 times and placing them onto level stirrer for 15-30 minutes.
PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB) density gradient protocol. For that, blood samples were diluted 1:1 with washing
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medium (RPMI 1640 (Gibco) + 1% L-glutamine (Invitrogen) + 1%
antibiotic/antimycotic (Gibco)). Diluted blood samples were carefully pipetted on top of Ficoll solution to avoid mixing. Blood-Ficoll-tubes were centrifuged for 400g, 30 min, RT without brake. After centrifuging, cell layers were visible. PBMC layer was carefully collected and washed once with washing medium and twice with RPMI 1640 medium (350g, 10min, RT). After washing, cell pellets were resuspended to concentration of 20x106 cells/ml with culture medium (RPMI 1640 + 10 % FBS Good (PAA laboratories) +1% glutamine +1 % antibiotic/antimycotic).
2.3 Cryopreservation and thawing
PBMCs for cryopreservation were diluted 1:1 with ice-cold freezing media (15%
DMSO in heat-inactivated FBS Good) slowly adding one drop at the time, whilst stirring tubes at the same time, working on ice. The final concentration of DMSO was 7.5%. Cell suspensions were aliquoted into cryovials at 1 ml/vial (10x106cells/vial).
Cell vials were stored in -80°C overnight for gradual temperature reduction using Mr.
Frosty devices (Nalgene Cryo 1°C “Mr. Frosty” Freezing Container, Thermo Scientific). Cells were then transferred into liquid nitrogen tanks for 2 weeks.
Cells were stimulated immediately after isolation (fresh cells) or cryopreserved, thawed and then stimulated (cryopreserved cells). Cryopreserved cells were thawed by warming cell vials in hands until the frozen cell media barely detached from the vial.
Cell pellet was poured into a 50 ml Falcon tube and culture medium (RT) was gradually added on top of the cells using a pipette, stirring the tube gently at the same time. Cells were washed with washing medium (300 x g, 10 min, RT) and resuspended with human AB culture medium. Viability of frozen PBMCs was determined by trypan blue
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exclusion. The mean cell viability of frozen cells was 96.9% (SD ±1.2). Cells were processed for stimulation approximately 45 min after thawing.
2.4 Stimulation
For stimulations, cells from each donor were suspended in Human AB culture medium (RPMI 1640 + 10% Human AB serum (Innovative Research) +1% glutamine +1%
antibiotic/antimycotic) to the final concentration of 1x106 cells/ml. Cells (2x106 per well) were stimulated with medium alone (unstimulated control), with POLY(I:C) (50 µg/ml, Sigma Aldrich) or with LPS (0.1 µg/ml, Sigma Aldrich) for 18 hours at 37° C in 5% CO2 on Ultra-Low attachment surface-plates (Corning, Costar). The concentrations were chosen based on preliminary dose-response experiments (data not shown). After stimulation, media were collected for cytokine analysis and cell pellets were collected for immunophenotyping. Plates were inspected after removal of the cells using light microscopy to check if there were noticeable differences in cell adherence after stimulations. Differences between the plates were not observed.
2.5 Immunophenotyping
For the immunophenotyping of blood DCs and monocytes, cell suspensions were stained with fluorochrome- labelled antibodies.
The following steps were performed on ice and protected from light. Before staining, cells were washed with FACS buffer (5% FBS Good, 0.02% NaN3 in PBS) and centrifuged (300g, 5 min, +4°C). The supernatant was discarded, and the cells were suspended in human AB FACS buffer (10% Human AB serum, 0.02% NaN3 in PBS).
Cell suspensions were incubated with fluorochrome-labelled antibody cocktails for 20
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min at +4°C (fluorochrome-labelled antibodies in supplementary table T1). After incubation, cells were washed with FACS buffer (300g, 5 min, +4°C) twice and resuspended in FACS buffer. Samples were analysed by flow cytometry immediately after staining. Immunophenotyping was performed by FACSCantoII cytometer and FACSDiva software v. 8.0.1 (BD Biosciences). A mean of 144 500 (range 42 400 –242 700) PBMCs were acquired in order to obtain sufficient numbers of DCs for accurate enumeration. OneComp eBeads (eBiosciences) were used for single-color compensation controls. Instrument calibration was evaluated by using BD cytometer setup & tracking beads for CST (BD Biosciences). The compensation matrix was calculated with FACSDiva software from unstained and single-color control beads.
Samples were further analysed using FlowJo 10.2 software (Treestar, Ashland, OR, USA) on Windows 10 workstation. Erythrocytes and debris were gated out according to the size (forward scatter, FSC) and cytoplasmic granularity (sideward scatter, SSC) (supplementary figure 1). Doublets were excluded using forward scatter area (FSC-A) and forward scatter height (FSC-H). Remaining dead cells were excluded using Fixable Viability Dye (eBioscience). Live monocytes were identified according to their CD14 expression and FSC-SSC characteristics. The main peripheral blood DC subsets were identified as live CD14-CD19-BDCA2+ pDCs and CD14-CD19- BDCA1+CD11c+ mDC1s. Expression of functional markers ILT4 and CD80 on DCs and monocytes was also analysed. Gating adjustments were based on fluorescence minus one controls.
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2.6 Cytokine measurements
Cytokines were analysed using Meso Scale Discovery (MSD) Sector Imager™ 2400A with Discovery Workbench® 3.0.18 software. Samples were analysed with MSD Biomarker Group 1, custom U- PLEX kit (for IFN-γ, IL-1β, IL-10, IL-12/IL-23p40, TNF-α), (MSD, Rockville, MD, USA) according to manufacturer's instructions, using reagents provided with the kit. The detection limit (DL) was defined for each cytokine separately. Samples with concentrations below the DL were given value corresponding to DL of the respective cytokine assay. Distributions of cytokines and detection ranges are shown in Supplementary table T2.
2.7 Statistical analyses
Data from immunophenotyping (DC and monocyte variables) were expressed as percentages of cells and percentages of cells positive for specific markers. Data from cytokine measurements were expressed as concentrations of cytokines (pg/ml).
In this study, two different statistical comparisons were made; one comparing cryopreserved cells to fresh cells and one comparing stimulated samples with control samples (later referred as response profiles). All pair-wise comparisons were analysed with non-parametric Mann Whitney-test. Statistical analyses were performed using SPSS Statistics 23-software (IBM Corporation, USA). Values of P < 0.05 were considered statistically significant.
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3. RESULTS
3.1 Cryopreservation had more pronounced effects on monocytes than in DCs Unstimulated cell cultures. Generally, the relative proportions of studied cells and cells expressing CD80 and ILT4 were comparable between fresh and cryopreserved cells (Figure 2). Few differences were seen as cryopreservation increased the relative proportions of monocytes and pDCs and decreased the percentages of ILT4+ monocytes as compared to fresh cultures.
Stimulated cell cultures. In LPS-stimulated cultures of cryopreserved
cells, the percentages of ILT4+ monocytes and CD80+mDCs were lower than in fresh cells. The percentages of Poly(I:C) – stimulated pDCs were higher in cryopreserved cells than in fresh cells.
Responses to stimulation. The response profiles of fresh and frozen APCs
were mainly comparable. In monocytes, LPS stimulation significantly decreased the percentages of monocytes in frozen but not in fresh cells and increased the percentages of CD80+monocytes in fresh but not in frozen cells. When studying dendritic cells, only two differences in responses were observed: Stimulation increased the percentages of CD80+ mDCs and pDCs among fresh but not cryopreserved cells.
3.2 Cryopreservation did not affect the cytokine response profiles of PBMCs
Baseline (control) production of cytokines was higher in cryopreserved cells than in fresh cells (Figure 3). On the contrary, cryopreserved PBMCs produced less cytokines in response to LPS and Poly(I:C) stimulation than fresh cells. Only exception was IL- 1β, in which the responses between fresh and frozen cells were comparable. Cytokine
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response profiles of fresh and cryopreserved PBMCs were similar, that is, studied stimulants induced statistically significant responses both in fresh and frozen cells.
4. DISCUSSION
Studies are conflicted whether cryopreserved cells are comparable to fresh cells and whether the use of cryopreserved cells is feasible in research. In this study, we aimed at the overview of the effects of cryopreservation on the percentages and general characteristics of circulating DCs and monocytes and cytokine production of PBMCs.
The strength of our study is that we studied the effects of thawing and freezing not only on general immune responses but also on cell-specific markers in the PBMC samples of the same subjects, collected at the same time, and isolated from the freshly collected blood. We demonstrated that while the levels of responses differed in some extent between fresh and cryopreserved cells, the overall trends and significances were generally similar.
4.1 Cryopreservation could affect the cell percentages via cell loss, altered maturation or disturbed cell communication
Cryopreservation had some effects on the levels of studied immune cells. The differences in the relative proportions could be the consequence of, for example, death and loss of other cells (e.g. lymphocytes) during sample processing. Studies have shown that the effect of cryopreservation on the numbers/percentages of cells depends on cell type [Verschoor et al. 2018]. For example, a decrease in the number of T cells has been reported in cryopreserved PBMCs [Sattui et al. 2012, Wang et al. 2016], whereas number of viable monocytes was not affected by freezing [Hori et al. 2004]. Studies suggest that some cell types may be more resilient to cryopreservation than others as the
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percentages of those cells are elevated after freezing and thawing [Ivison et al. 2018, Kotsakis et al. 2012]. Furthermore, cryopreservation can affect the maturation of precursor cells, such as monocytes, and thus alter proportions of cell populations such as macrophages or DCs [Hori et al. 2004, John et al. 2003].
Studies assessing the effect of freezing on surface receptors have shown that surface markers in PBMCs are generally decreased after cryopreservation [Campbell et al. 2009, Gerrits et al. 2007, Wang et al. 2016]. This loss of functional cells could lead to differences in cell function, as study of Kotsakis et al. 2012 showed that myeloid- derived suppressor cells lost suppressor functions after cryopreservation, whereas Hermansen et al. 2018 has reported that there were no differences in signalling patterns between frozen and fresh B lymphocytes. In our study, cryopreservation did not affect surface marker expression of pDCs. In control samples cryopreservation decreased the relative proportions of ILT4+monocytes and in stimulated samples decreased the relative proportions of ILT4+ monocytes and CD80+ mDCs. Could it be that the cells of mononuclear phagocyte system are more affected by freezing? This is of interest as DCs can be derived from monocytes [Gross et al. 2017], and changes in maturation capabilities could hinder the production of monocyte derived DCs. The impairing effects of freezing on the maturation should be further studied by assessing broader range of receptors related to the maturation status, especially CD83 but also CD40, CD86, and CCR7.
In general, the baseline cytokine levels were higher, whereas stimulated levels were lower in frozen cells than in fresh cells. Studies assessing cytokine secretion have presented conflicting results. In study of Axelsson et al. 2008, baseline secretion of several cytokines was increased after cryopreservation, whereas in John et al. 2003
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cryopreserved DCs produced lower levels of IL-12 p40 and IL-12 p70 compared to fresh DCs. Higher baseline levels after cryopreservation may be due to the processes of freezing and handling, e.g. the components in freezing media could affect the cytokine secretion. However, lower cytokine levels after cryopreservation could be due to loss of effector cells. It could be that this loss of certain cells i.e. specific subset of T cells, affects intercellular communication, and thus modifies the expression of functional markers or cytokine secretion of studied cells. Intercellular and intracellular communication is co-regulated by surface receptors and soluble cytokines. If a part of this communication is lost or altered, immune responses to different antigens could be impaired or reduced. Overall, previous studies and our results suggest that the effects of cryopreservation on cells and cell function are dependent of cell and receptor type, cytokine itself, and the ligand cells are stimulated with.
4.2 Cryopreservation did not alter reactions towards stimulants in general
Although cryopreservation altered the magnitude of the cytokine production as well as relative proportions of cells, studied stimulants induced somewhat parallel reactions in fresh and cryopreserved cells. As discussed in the previous chapters, it seems that cells are less sensitive to stimulation after cryopreservation. When studying e.g. clinical biomarkers, differences in the levels could cause problems in the assessment of clinical threshold values. In the epidemiological research, the main focus is not in the levels but in the differences between groups. Changes in the levels induced by cryopreservation may be especially important when examining weak stimuli and instances when small shifts are expected e.g. in the comparison of diseased and healthy patients or study
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subjects. Of course, researchers should take the effects of freezing on account, especially when interpreting and comparing the results with other studies.
Some studies claim that only fresh samples should be used in clinical settings [Costantini et al. 2003], whereas some studies have successfully used cryopreserved dendritic cells in immune therapies [Feuerstein et al. 2000, Hori et al. 2004, Westdorp et al. 2019]. DCs for vaccines could be prepared either by producing them from monocytes by a several days culture period [Gross et al. 2017] or by freshly isolating them from blood [Westdorp et al. 2019]. As our results suggest that cryopreservation could affect maturation of DCs, it would be important to further investigate how freezing affects the antigen-presenting capability and maturation of these cells, and whether they should be cryopreserved before or after antigen loading. It is also important to study whether freezing affects different types of DCs differently as in our study pDCs seems to be more resistant to cryopreservation than mDCs and monocytes We suggest that different cryopreservation methods need to be tested and, of course, when assessing the effects of cryopreservation, immunological indicators or analyses that are central to each study should be taken into account.
In this study we used freezing medium that contained DMSO and FBS, as they are still widely used in several fields of science. We want to remind that the composition of the freezing medium should be always considered and chosen accordingly to the purpose, especially if the cells are used in immunotherapy or other clinical applications.
When interpreting these results, one should note that this study used FBS in cryomedia instead of human alternatives. DMSO, serum and xeno-free cryomedia should be used when suitable [Schulz et al. 2012]. The presented protocol is in use in our laboratory and it shows a very high survival rate of cells and is suitable for measurements shown in
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this paper. One limitation of our study is that we did not assess cell survival following stimulation experiments. However, data from flow cytometry suggested that the percentage of cells recovered from fresh cells was significantly higher than in cryopreserved cells, but only in stimulated samples (data not shown). We do not know whether the cells died during stimulation, or were they lost during staining procedure and thus recommend that cell counts and cell survival should be followed when cryopreserved cells are studied. These results could indicate that frozen and thawed cells are more sensitive to stress caused by stimulation or processing as control samples did not differ significantly in regard of cell survival. This study assessed the effects of freezing, thawing and a short-term storage as we believe that the majority of cellular changes arise from freezing itself, and less from the cryo-storage time. Furthermore, we focused on responsiveness of the cells to stimuli, and not on other functional properties, such as antigen uptake, processing and presentation. We recommend that the effects of freezing, handling and storage on the studied markers and functional properties should be assessed before cryopreservation, especially in extensive and often expensive cohort studies.
Based on the results of this study, it can be concluded that the use of cryopreserved cells may be more suitable in studies that assess general reactions to stimuli instead of studies that rely on exact levels of reactions. We suggest, however, that the interpretation and comparison of the results of different studies should not be done without considering the differences in cryopreservation techniques and their possible effects on the function of PBMCs.
Acknowledgements
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The authors thank the volunteer blood donors. This work was supported by the Päivikki and Sakari Sohlberg Foundation, Juho Vainio Foundation, Yrjö Jahnsson Foundation, and Doctoral School of University of Eastern Finland.
Conflicts of interest
Authors do not have any actual or potential conflicts of interest including any financial, personal or other relationships that could inappropriately influence, or be perceived to influence, their work.
Statement of contribution
Both authors approved the submitted version. Martikainen was responsible for the immunological analysis, statistical analysis and the interpretation of results, completion of the background literature search, and drafting and revising the manuscript. Roponen obtained funds, designed the study, had responsibility for data collection and management of the study, and contributed to the interpretation of results and to the manuscript.
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Figure 1. Overview of experimental set-up.
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CD80+
0 10 20 30 40 50
% of pDCs
r
pDCs
Control Poly(I:C)
ILT4+
0 20 40 60 80
% of pDCs
Control Poly(I:C) CD80+
0 20 40 60 80 100
% of mDCs
r
a
mDCs
Control LPS
ILT4+
0 10 20 30 40 50
% of mDCs
Control LPS
CD80+
0 10 20 30 40 50
% of Monocytes
r
Monocytes
Control LPS
ILT4+
0 20 40 60 80
% of Monocytes ra
ar
Control LPS
% among PBMCs
0 20 40 60
% of PBMCs
r a
Control LPS
% among PBMCs
0 1 2 3 4
% of PBMCs
Control LPS
% among PBMCs
0.0 0.5 1.0 1.5
% of PBMCs
a
ar
r
Control Poly(I:C)
Cryopreserved cells Fresh cells
Figure 2. The effect of cryopreservation on percentages and response profiles of antigen- presenting cells (N=6). Figures show boxplots with 5-95% whiskers, horizontal line indicates the median. Grey boxplots represent fresh cells, white represent cryopreserved cells. Significances
Journal Pre-proof
were calculated using Mann Whitney U test. a= P-value < 0.05, cryopreserved cells compared to fresh cells. r= P-value <0.05, stimulated cells compared to control (response profile).
Journal Pre-proof
IL-1ß
0 1000 2000 3000 4000
pg/ml
a
r r r r
Control Poly(I:C) LPS
IL-10
0 50 100 150 200
pg/ml
a
ar ar
r
r
Control Poly(I:C) LPS
IL-12/IL-23p40
0 500 1000 1500 2000 2500
pg/ml
a
ar ar
r
r
Control Poly(I:C) LPS
TNF-a
0 1000 2000 3000 4000
pg/ml a
ar
r r ar
Control Poly(I:C) LPS
IFN-y
0 200 400
600 Fresh cells
Cryopreserved cells
pg/ml
a
ar
ar r
r
Control Poly(I:C) LPS
Figure 3. The effect of cryopreservation on the production of cytokines (N=6). Figures show boxplots with 5-95% whiskers, horizontal line indicates the median. Grey boxplots represent fresh cells, white represent cryopreserved cells. Significances were calculated using Mann Whitney U test. a= P-value < 0.05, cryopreserved cells compared to fresh cells. r= P-value <0.05, stimulation compared to control (response profile).
