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Circulating Angiogenic Cells can be Derived from
Cryopreserved Peripheral Blood Mononuclear Cells
Tanja Sofrenovic
1,2
, Kimberly McEwan
1,3
, Suzanne Crowe
1
, Jenelle Marier
1,2
, Robbie Davies
4
,
Erik J. Suuronen
1,2
*, Drew Kuraitis
1,2
*
1 Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Canada, 2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
Canada, 3 Department of Mechanical Engineering, University of Ottawa, Ottawa, Canada, 4 Department of Statistics, University of Ottawa Heart Institute, Ottawa, Canada
Abstract
Background:
Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem
and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for
storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving
peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population,
circulating angiogenic cells (CACs).
Methods:
PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or
cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for
1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of
flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine
secretion, and in vivo angiogenic potential.
Results:
The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after
cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent
CD34
+
VEGFR2
+
CD133
+
population remained unaffected. The derived CACs, while exhibiting changes in inflammatory
cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their
ability to restore perfusion in ischemic muscle.
Conclusion:
Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the
CD34
+
VEGFR2
+
CD133
+
progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic
potential of CACs remain unaffected by cryopreservation.
Citation: Sofrenovic T, McEwan K, Crowe S, Marier J, Davies R, et al. (2012) Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood
Mononuclear Cells. PLoS ONE 7(10): e48067. doi:10.1371/journal.pone.0048067
Editor: Zoran Ivanovic, French Blood Institute, France
Received April 17, 2012; Accepted September 20, 2012; Published October 25, 2012
Copyright: ß 2012 Sofrenovic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grant-in-Aid T6793 from the Heart and Stroke Foundation of Ontario (to ES). TS was supported by Master’s studentship
award from the Heart and Stroke Foundation of Ontario, DK was supported by a Canadian Graduate Scholarship from the Canadian Institutes of Health Research.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: dkuraitis@ottawaheart.ca (DK); esuuronen@ottawaheart.ca (ES)
Introduction
Although a unifying definition regarding their characterization
does not exist [1,2], endothelial progenitor cells (EPCs) often
identified as CD34
+
VEGFR2
+
CD133
+
cells have the ability to
augment postnatal vasculogenesis [3–6]. The therapeutic revas-
cularization that results from EPC transplantation is believed to be
mediated by two main mechanisms: differentiation into new blood
vessels [7,8] and paracrine signaling to augment endogenous vessel
growth via the production of pro-vasculogenic cytokines [9,10]. In
humans, patients with acute myocardial infarction who received
an intracoronary infusion of bone marrow derived progenitors
(sorted for markers CD34/CD45 and CD133) or peripheral
blood-derived progenitors (plated for 3 days and positive for
endothelial markers such as CD31, KDR (VEGFR2), von
Willebrand factor and CD105 as well as uptake of low density
lipoprotein and lectin binding) saw a beneficial effect in post-
infarction remodeling processes, such as a global increase in
ejection fraction and a decrease in infarct size [11,12]. The
number of EPCs in the blood has been shown to be a predictor of
cardiovascular health: low levels of circulating EPCs have been
associated with increased risk of major cardiovascular events and
vascular function [13].
EPCs can be generated from the culture of peripheral blood
mononuclear cells (PBMCs) isolated from blood by density
gradient centrifugation. PBMCs are cultured for 4–7 days in
endothelial-promoting media on fibronectin and the subsequently
generated therapeutic population is referred to as ‘circulating
angiogenic cells’ (CACs), or early EPCs [2,14].
As cardiovascular disease is the number one leading cause of
death in the Western world [15], there is a potential for CAC
therapy to improve the quality of life for patients of this disease by
aiding in the restoration of blood flow to the heart. However,
EPCs and CACs are not available off-the-shelf and their frequency
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in circulating PBMCs is rather low, at about 0.0001% to 0.01%
for EPCs [16] and 2% for CACs [17]. Furthermore, diabetes and
cardiovascular disease decrease EPC numbers and function
[18,19], making it difficult to obtain therapeutically-relevant and
potent cells for application in therapy. Cryopreservation offers a
method to maintain cells as they are generated, until they are
required for therapy. More importantly, cryopreservation may
allow a patient to store his or her own autologous cells until
needed, thereby avoiding the risks and potential of graft-versus-
host disease [20].
Cryopreservation has been applied for some time in the medical
field, ranging from freezing of blood and bone marrow cells for
transplantation, to embryo preservation for in vitro fertilization and
long term gamete storage for cancer patients. This process
preserves cells by dramatically reducing biological metabolism at
low temperatures; however, cryopreservation also causes damage
to some cell types, as well as potentially changing their function
[21,22]. One study demonstrated that cryopreservation of T-cell
subsets caused an increase in the expression of CXCR4 and
CD69, while expression of L-selectin (CD62L) was decreased [23].
The consequence of cryopreservation on CACs, and their
generation from PBMCs, remains to be thoroughly investigated.
The aims of this study were to investigate the outcome that
cryopreservation has on the phenotype and function of: 1) freshly-
isolated PBMCs; and 2) in vitro culture-generated CACs derived
from fresh and cryopreserved PBMCs. In our study, we focused on
the CACs (sometimes referred to elsewhere as early EPCs), which
represent a highly heterogeneous population, thought to mostly
exert their therapeutic effects through paracrine mechanisms.
Results
A summary of the cell populations and experiments is presented
in Figure 1.
Viability of PBMCs but not of Generated CACs is Reduced
after Cryopreservation
Fresh and cryopreserved cells were incubated with 7AAD
-
exclusion stain and the number of viable cells was quantified via
flow cytometry. Fresh samples of PBMCs and CACs showed about
99.760.1% and 95.360.1% viability, respectively. Following
cryopreservation, PBMCs sustained a non-significant reduction
in viability on day 1 (93.161.5%) with a significant loss observed
on day 28 (viability of 85.064.3%; p = 0.0078; Figure 2 A).
However, the viability of CACs remained relatively stable over
time post-cryopreservation at 88.761.4% on day 1 and
94.363.8% on day 28 (p
B
= 1; Figure 2 A). Morphology of the
thawed cells was preserved compared to their fresh counterpart
sample as observed under a light microscope at 106 magnification
(Figure 2 B).
Cryopreservation Affects PBMC Phenotype
The phenotype of the fresh and frozen cells was analyzed by
staining the cells for surface markers: CD31, CD34, KDR
(VEGFR2), CD133 and L-selectin and their appropriate isotype
matched IgGs. The IgGs were used qualitatively [24] as there
were no significant differences observed between the different
sample time points (Figure 3 A–C). Surface markers CD34, KDR
and CD133 were selected as they are most commonly used to
describe a potent subset of CACs sometimes referred to as EPCs.
Other surface markers investigated were CD31, an endothelial cell
marker and L-selectin, an important adhesion protein for PBMCs
and CACs. Expression of the endothelial marker CD31 remained
stable at day 1 but by day 28 it was significantly increased
compared to the earlier time points (Table 1). CD34 and
VEGFR2 expressing cells followed a similar trend with a
significant rise of these populations after cryopreservation com-
pared to their fresh counterparts. Furthermore, the number of
CD133 expression cells increased after cryopreservation while the
number of L-selectin positive cells was reduced in the cryopre-
served PBMC samples when compared to the fresh PBMC
Figure 1. Summary of the methods. In brief, PBMCs were isolated and either analyzed (fresh), plated to generate CACs, or cryopreserved for 1 or
28 days after which the cells were thawed and analyzed or plated to generate CACs from cryopreserved PBMCs.
doi:10.1371/journal.pone.0048067.g001
Effects of Cryopreservation on PBMCs and CACs
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samples (Table 1). Double, triple and even quadruple staining of
cells positive for the markers described above was also investigated
to look at certain subpopulations of PBMCs. Their summary is
shown in Table 1.
Cryopreservation Affects CAC Phenotype
The number of CACs generated was not significantly different
between fresh and cryopreserved samples. CAC analysis followed
the same procedures as the PBMCs and investigated the number
of cells expressing the five markers: CD31, CD34, CD133,
VEGFR2 and L-selectin. Overall, EPC identifying markers
(CD34, VEGFR2 and CD133) did not show any significant
differences after cryopreservation compared to the fresh samples
(Table 2). The number of CD31 positive cells was significantly
decreased after 1 day of cryopreservation; however, a significant
decrease was not observed for the 28-day frozen cells. L-selectin
was significantly up-regulated in cells after 28 days of cryopres-
ervation compared to the fresh samples. The double, triple and
quadruple staining of cells for the selected markers described was
also investigated to look at various subpopulations of CACs. Their
summary is shown in Table 2.
Cryopreservation does Affect Lectin Binding and LDL
Uptake of CACs
Lectin binding and LDL uptake are characteristic functions of
EPCs and other circulating cells, such as leukocytes. We decided to
investigate whether these functions are altered in the cells by the
cryopreservation process. There were no significant differences in
the uptake of LDL and binding of lectin when day 1 and day 28
cryopreserved PBMCs were compared to the fresh PBMCs
(Figure 4 A, B). However, a significant increase in LDL uptake
(by 2.4-fold) was observed in day 28 CACs generated after
cryopreservation, compared to fresh CACs (Figure 4 C, p = 0.004).
There was also a significant increase in lectin binding for CACs
Figure 2. Viability of PBMCs, but not of generated CACs, is reduced after cryopreservation. 7AAD
–
exclusion staining was used to
enumerate the number of viable cells for fresh, early and late cryopreserved PBMCs and CACs. Prior to flow cytometry, the cells were incubated with
7AAD for 5 minutes. The proportion of viable cells is represented as a percentage 6 SE (n = 7) (A). Representative images of fresh and cryopreserved
PBMCs and CACs were taken by light microscopy at 106 magnification; scale bar = 50
mm. (B). *p,0.05 compared to the fresh sample.
doi:10.1371/journal.pone.0048067.g002
Effects of Cryopreservation on PBMCs and CACs
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after cryopreservation with an increase of ,1.1-fold compared to
the fresh samples (Figure 4 D, p = 0.005).
Functional Capacities of PBMCs and CACs after
Cryopreservation
Adhesion and migration are important functional properties of
most cells. Since changes in some of the proteins involved in these
processes (VEGFR2, L-selectin) were observed after cryopreser-
vation, assays measuring the cells
`
adhesive and migratory abilities
were conducted. Overall, cryopreserved PBMCs and CACs
demonstrated no difference in adhesive capabilities on fibronectin
coated plates compared to the fresh samples (Figure 5 A, C).
Cryopreserved PBMCs were also unaffected in their ability to
migrate using a VEGF chemokine (Figure 5 B), whereas CACs
after 1 day of cryopreservation exhibited reduced migration
(p = 0.04), compared to fresh CACs (p = 0.001). Migration of day
28 cryopreserved CACs was not significantly affected (p = 0.12;
Figure 5 D).
Cytokine Secretion of CACs after Cryopreservation
The supernatant from CAC cultures was collected and a
cytokine array was performed to evaluate the secretion of select
cytokines by the cells. The secretion of pro-inflammatory cytokines
interleukin-1a (IL-1a), IL-1b and tumor necrosis factor (TNF-a)
and anti-inflammatory cytokine IL-10 was observed to be higher
in frozen cells compared to their fresh counterparts. In most cases,
day 1 cryopreserved cells seemed to have the highest secretion of
these inflammatory cytokines compared to both fresh and day 28
samples (Figure 6 A–D). Secretion of angiopoietin-1, a protein that
plays a role in vascular development, was significantly reduced in
day 28 cells (Figure 6 E). Granulocyte colony-stimulating factor
(GCSF), which stimulates the BM to produce cells, was increased
in day 1 cryopreserved cells; however, this increase was not seen in
the day 28 cells (Figure 6 F). Furthermore, intracellular adhesion
molecule 1 (ICAM-1), important in cell-to-cell adhesion, was
increased in cryopreserved cells, whereas tissue inhibitor of
metalloproteinases 2 (TIMP-2), which plays a role in extracellular
matrix degradation and suppression of EC proliferation [25], was
Figure 3. Isotype matched IgGs for each surface marker. Controls were conducted for background staining of fluorescent conjugates for
markers CD31, VEGFR2, L-selectin, CD133 and CD34 on PBMCs (A) and CACs (B). No significant difference was observed in positively stained cells
during fresh, day 1 and day 28 time points. The average IgG background staining is presented as fold-change ± SE compared to fresh samples (n = 7).
Representative histograms of the various markers (blue) and corresponding IgG (red) analysis from one donor’s fresh, day 1 and day 28 CACs (C).
doi:10.1371/journal.pone.0048067.g003
Effects of Cryopreservation on PBMCs and CACs
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reduced in the cryopreserved cells (Figure 6 G–H). The secretion
of pro-vasculogenic cytokine basic fibroblast growth factor (bFGF),
pro-myogenic cytokine insulin-like growth factor 1 (IGF-1) and
chemotactic cytokines stromal cell derived factor 1 (SDF-1) and
VEGF were all observed to be unaffected by cryopreservation
(Figure 6 I–L).
Cryopreservation does not Affect in vivo Angiogenic
Potential of CACs
Fresh or cryopreserved-derived human CACs were injected into
the skeletal muscle of immunocompromised mice with hindlimb
ischemia induced by femoral artery ligation. Laser Doppler
perfusion analysis was conducted to measure blood flow over a
2-week post-treatment period, after which the animals were
sacrificed. Animals receiving cryopreserved cell treatments dis-
played restoration in perfusion comparable to those receiving
transplantation of fresh cells. No significant differences were
observed in perfusion between treatments at days 4, 7, 10 and 14
post-op (Figure 7 A). Furthermore, there were no differences in the
number of arterioles (assessed by smooth muscle actin (SMA)
staining) between hindlimbs treated with cryopreserved cells or
fresh cells (Figure 7 B). Staining the tissue with anti-human
mitochondria antibodies yielded no detection of the transplanted
human cells in the hindlimbs at the time of sacrifice, suggesting
that transplanted cells did not persist in the tissue 2 weeks after
delivery.
Discussion
Cryopreservation is an appealing method for storing an
individual’s own autologous stem and progenitor cells until needed
for therapy; however, the effects of freezing cells for storage have
not been fully characterized. In particular, there is limited data
regarding cryopreserved PBMC-generated CACs, which have
been a major focus of regenerative research. Our data demon-
strates that cryopreservation does not adversely affect the viability
of PBMCs and CACs but it does cause phenotypic changes in
PBMCs and to a lesser degree in some subpopulations of CACs.
However, the potent CD34
+
VEGFR2
+
CD133
+
population re-
mained unaffected in the CACs. Also the CACs demonstrated no
changes in the secretion of important regenerative and chemo-
tactic cytokines thought to be involved in the CAC’s regenerative
ability via paracrine mechanisms. Furthermore, the angiogenic
potential of CACs was examined in vivo, and no differences were
seen between the 3 groups in their ability to restore perfusion to
ischemic hindlimbs; demonstrating that cryopreservation did not
affect the therapeutic potency of the cells. Nevertheless, some
differences between the fresh and the cryopreserved CACs were
observed, such as changes in inflammatory and other cytokine
Table 1. PBMC phenotype is affected after cryopreservation.
PBMC Fresh (%) Day 1 (%) Day 28 (%)
CD31
+
67.3862.17 70.4264.12 76.8862.10
a,b
CD31
2
CD34
+
10.1260.47 7.6960.62
a
8.1160.80
a,b
CD31
2
CD34
+
VEGFR2
+
0.6160.14 1.5560.17 2.1660.15
CD31
2
VEGFR2
+
1.6460.39 3.8860.42
a
4.9160.34
a
CD31
+
CD34
2
VEGFR2
+
3.3660.57 8.0460.73
a
5.1462.28
a
CD31
+
CD34
2
L-Selectin
+
VEGFR2
+
3.5060.24 5.1261.10 6.3160.70
a
CD31
+
CD34
+
29.4760.48 40.1366.13 38.862.42
CD31
+
CD34
+
VEGFR2
+
3.7160.30 15.4265.82 13.3061.64
CD31
+
L-Selectin
+
60.8963.11 60.7661.04 62.5461.73
CD31
+
VEGFR2
+
7.3060.42 22.0264.67 22.0261.0
CD34
+
35.8961.36 48.1462.72
a
43.9362.59
a
CD34
+
L-Selectin
2
4.7260.82 5.4260.89 4.7261.02
CD34
+
L-Selectin
-
VEGFR2
+
0.6160.22 3.6660.61
a
2.5860.76
a
CD34
+
L-Selectin
+
27.4461.58 24.9160.61 23.3961.92
a
CD34
+
L-Selectin
+
VEGFR2
+
2.2460.58 6.9460.57
a
4.6362.15
CD34
+
CD133
+
0.1460.053 0.536.077
a
0.4460.17
CD34
+
CD133
+
VEGFR2
+
0.01560.0082 0.1560.043
a
0.1260.049
CD34
+
VEGFR2
+
2.3360.56 11.6662.99
a
7.6362.81
L-Selectin
+
84.3761.52 74.7962.43
a
70.7662.82
a
L-Selectin
2
VEGFR2
+
0.7560.33 4.0660.25
a
3.5760.61
a
L-Selectin
+
VEGFR2
+
3.6660.56 6.4060.79 5.0861.61
CD133
+
0.2760.067 2.6560.97
a
1.9760.23
a
CD133
+
VEGFR2
+
0.002760.0027 0.07260.013
a
0.08460.031
a
VEGFR2
+
12.4762.85 27.5766.19
a
27.0765.44
a
a
p,0.05 vs. ‘fresh’ time point.
b
p,0.05 vs. ‘day 1’ time point.
The values represent the percentage of cells within the total cell population (sample of 200,000 cells) that express the given phenotype.
doi:10.1371/journal.pone.0048067.t001
Effects of Cryopreservation on PBMCs and CACs
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secretion and the numbers of cells expressing certain non-
progenitor surface markers (L-selectin and CD31).
The maintenance of PBMC viability following cryopreservation
observed in the present work is in accordance with other studies
that also did not find considerable cell death after cryopreservation
of PBMCs [26,27]; however, it is believed that the cell viability
achieved after cryopreservation is largely dependent on the
expertise/technique of the laboratory that performs the cryopres-
ervation [26,27]. In particular, one report showed cell viability
post-cryopreservation ranging from 1% to more than 90% [27].
The effect of cryopreservation on CAC viability has not been
studied in great detail [28,29], especially when considering
PBMC-derived CACs. One investigation using 7-AAD, examined
the viability of CACs from cryopreserved umbilical cord blood and
found a significant level of apoptosis [29]. In contrast, our results
illustrated no significant decline in CAC viability after 1 day and
28 days time points of cryopreservation. It is possible that this is
attributable to differences between the cells under study or that
after being cultured for 4 days the CAC viability differences are
negated. The importance of cell viability after cryopreservation is
imperative as CACs make up a small fraction of the total PBMCs.
The ability to generate a large number of viable cells would allow
for a better stem cell therapy outcome.
The cells’ phenotype was analyzed by staining for cell surface
markers CD31, L-selectin, CD34, CD133 and VEGFR2 using
flow cytometry. In our investigation, CACs generated from
cryopreserved PBMCs showed no difference in expression of
surface markers CD34, VEGFR2 and CD133; however a
difference in CD31 and L-selectin expression was observed.
CD34, CD133 and VEGFR2 are markers used to characterize
CACs and the potent subpopulation of EPCs, although they are
not exclusive to these populations and can be expressed by other
hematopoietic lineages [30]. As previously mentioned,
CD34
+
VEGFR2
+
CD133
+
cells have been shown to be a potent
subpopulation of cells involved in vascular regeneration [3–6].
The fact that this population and their vasculogenic derivatives
(ex. CD34
+
VEGFR2
+
cells) are unaffected by the cryopreservation
in CACs, and are even increased in cryopreserved PBMCs,
indicates that perhaps this population is able to better tolerate the
harsh process of freezing without significant losses, compared to
other populations.
CD31 is a marker of endothelial cells and L-selectin is an
adhesion molecule found on CACs and other PBMCs. The
expression of CD31 by day 28 cryopreserved PBMCs was
increased; whereas the frequency of L-selectin
+
PBMCs decreased,
from about 85% to 70%, after 28 days of cryopreservation. This
result is in accordance with previous studies that have also found
down-regulation of L-selectin in PBMCs following cryopreserva-
tion [31], which may be a cellular response to stress [32].
Regarding progenitor markers CD34, CD133 and VEGFR2, we
Table 2. CAC phenotype is affected after cryopreservation.
CAC Fresh (%) Day 1 (%) Day 28 (%)
CD31
+
72.8162.94 61.9761.09
a
58.1065.12
CD31
2
CD34
+
8.3861.25 12.5160.58
a
12.9860.78
a
CD31
2
CD34
+
VEGFR2
+
0.7960.23 2.7060.32
a
1.7660.53
CD31
2
VEGFR2
+
1.8060.50 7.3560.96
a
4.6261.20
CD31
+
CD34
2
VEGFR2
+
3.8360.66 8.8360.32
a
6.8060.83
a
CD31
+
CD34
2
L-Selectin
+
VEGFR2
+
3.0560.51 6.8860.26
a
4.6560.72
CD31
+
CD34
+
31.4561.68 29.2061.20 19.3963.24
a
CD31
+
CD34
+
VEGFR2
+
3.8160.63 8.4260.89
a
5.2760.82
b
CD31
+
L-Selectin
+
64.2662.69 58.4561.36 45.7966.69
a
CD31
+
VEGFR2
+
6.7561.08 16.0461.14
a
11.1961.63
CD34
+
42.5361.89 42.7362.17 38.5163.26
CD34
+
L-Selectin
2
4.5360.82 2.6960.37 2.1860.61
CD34
+
L-Selectin
2
VEGFR2
+
0.9060.19 1.9560.29
a
0.7860.24
b
CD34
+
L-Selectin
+
31.2961.61 28.0361.06 23.7863.27
CD34
+
L-Selectin
+
VEGFR2
+
3.4460.54 9.3261.16
a
6.3161.29
CD34
+
CD133
+
0.2460.11 0.7160.25 4.3562.43
CD34
+
CD133
+
VEGFR2
+
0.07060.042 0.2360.064 0.4460.22
CD34
+
VEGFR2
+
3.5760.63 10.0761.28 5.8860.99
L-Selectin
+
82.9962.65 87.2661.47 92.0461.98
a
L-Selectin
2
VEGFR2
+
1.0060.31 2.8860.36
a
2.8360.72
a
L-Selectin
+
VEGFR2
+
3.9760.67 10.3460.67
a
6.3261.30
b
CD133
+
2.5461.15 4.9361.02 3.6061.89
CD133
+
VEGFR2
+
0.05060.030 0.1260.022 2.3961.40
VEGFR2
+
14.6763.15 26.2662.19 28.4466.47
a
p,0.05 vs. ‘fresh’ time point.
b
p,0.05 vs. ‘day 1’ time point.
The values represent the percentage of cells within the total cell population (sample of 200,000 cells) that express the given phenotype.
doi:10.1371/journal.pone.0048067.t002
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observed an increased expression following cryopreservation of
PBMCs. Ketheesan et al. showed that the proportion of CD34
+
cells in frozen cord blood did not change after freezing [33], while
Lanza et al. demonstrated that 90-day cryopreserved PBMCs
derived from patients with non-Hodgkin’s lymphoma had an
increase in CD34
+
cells compared to fresh samples, attributed to a
decrease in mature myeloid cells [34]. Many hypotheses exist to
explain the changes in phenotypes after cryopreservation: 1) more
cells could indeed shift towards expression of particular markers
during the cryopreservation process; 2) due to cell death of other
subpopulations, there is a relative increase of more cells expressing
markers CD34, CD133 and VEGFR2 used to identify EPCs; or 3)
an increase in false positives as perceived by the flow cytometry
analyses. Given that the observed non-specific binding IgG
controls did not differ among time points, it is plausible that the
observed changes are due to one of the first two hypotheses;
however, it is not yet clear whether one or both are predominant
reasons for changes in stored cell phenotypes.
Although studies have shown that L-selectin decreases in
immunomagnetically-purified CD34
+
CACs immediately after
thawing, the cells do recover their expression of L-selectin after a
short period of culture [35]. This may explain why, in our study,
we observed a decrease in PBMC L-selectin expression, which
were analyzed immediately, while the CACs were generated by a
4-day culture protocol before analysis, and demonstrated no
change in L-selectin on day 1 cryopreserved cells and even an
increased expression by day 28 cryopreserved cells. Surprisingly,
the levels of CD31 were decreased in cryopreserved CACs in
contrast to cryopreserved PBMCs where the expression of CD31
was unchanged or increased.
We also assessed the function of the populations’ capacity for
lectin-binding and LDL uptake, which are further characteristics
used to assess CAC populations [1,2]. As expected, a higher
percentage of cells in the CAC sample showed lectin binding and
DiI-LDL uptake, compared to PBMCs. In PBMCs there was no
significant difference between the number of fresh and cryopre-
served cells staining positive for either one of these factors.
However, for CACs a significant increase was seen for lectin
binding and LDL uptake in 28 day cryopreserved cells. This is in
contrast to an investigation by Mieno et al. that showed that LDL
uptake and lectin-binding of EPCs is unaffected by cryopreserva-
tion [28].
The cells’ function was further assessed by their adhesion and
VEGF-mediated migration capabilities. The adhesion of PBMCs
was unaffected by cryopreservation, which is somewhat contra-
dictory to the observed decrease in L-selectin after cryopreserva-
tion. Binding of L-selectin inside the cell results in the activation of
b1 and b2 integrins which promotes adhesion of leukocytes to
fibronectin [36]. It is possible that while the loss of L-selectin in
PBMCs is statistically significant, it is not, however, physiologically
Figure 4. Cryopreservation affects LDL uptake and lectin binding of CACs. The numbers of positive PBMCs that took up fluorescent LDL (A)
or that bound fluorescent lectin (B) were quantified using flow cytometry. The same procedure was conducted for CACs (C, D). Data is reported as
fold-change from donor-matched fresh samples ± SE (n = 7). *p,0.05 compared to the fresh sample.
doi:10.1371/journal.pone.0048067.g004
Effects of Cryopreservation on PBMCs and CACs
PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e48067
relevant, as compensatory mechanisms may be in action (e.g. other
selectins may assume similar functions [37]). The migration
potential of PBMCs using VEGF for chemotaxis was also
unaffected by cryopreservation. The fact that there was no change
in VEGF-mediated migration even though there was increased
expression of one of its receptors (VEGFR2) was not further
investigated. One theory could be attributed to the newly
expressed receptor proteins being non-functional, and therefore
being unable to contribute to the cells’ migration.
CAC adhesion and migration capacities were mostly unaffected
by cryopreservation. There is a significant decrease in CAC
migration between fresh and 1-day frozen samples, but no
significant difference between fresh and 28-day frozen samples.
The loss of migratory function in the 1-day samples may be
attributed to shock that the cells underwent during the short
succession between freezing/thawing procedures; whereas the
maintenance of migratory potential in the 28-day cells is in
Figure 5. Cryopreservation does not affect adhesion and migration of PBMCs and CACs. PBMCs were pre-stained with DAPI for
visualization by electron microscope for analysis of their adhesive (A) and migration (B) capabilities. The same procedure was performed for CACs (C,
D). Five pictures were taken per well and the number of DAPI
+
cells (blue) were counted per FOV and representative pictures of each assay are
shown. Data is reported as fold-change from donor-matched fresh samples ± SE (n = 7), scale bar = 80
mm. *p,0.05 compared to the fresh sample.
doi:10.1371/journal.pone.0048067.g005
Effects of Cryopreservation on PBMCs and CACs
PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e48067
accordance with other studies showing that migration is not
affected by cryopreservation [28,38].
Secretion of cytokines is an important aspect of the CAC’s
ability to repair tissue after an ischemic injury [10]. Using an array
we looked at a variety of cytokines released by cultured fresh and
cryopreserved CACs. Inflammatory cytokines were increased a
few fold in the cryopreserved cells; and this increase was greatest in
day 1 cryopreserved cells, even compared to day 28 cells. Why the
day 1 cells increase their secretion of inflammatory cytokines
remains unknown. As the cryopreservation preparation proce-
dures are the same, the difference between day 1 and day 28 might
be explained by the short period of time between the freezing/
thawing procedures. While changes in inflammatory cytokine
production in cryopreserved CACs have not been reported,
PBMCs have been previously shown in some studies to have a
different cytokine secretion pattern post-cryopreservation [39,40].
In one study, 12 months frozen PBMCs were found to
spontaneously show an increase of inflammatory cytokines IL-6,
IL-10, IL-13 and IFN-c [39]. In our study, we found that the
secretion of several other cytokines was also changed after
cryopreservation. ICAM-1, an adhesion factor and, in its soluble
form, an indicator of endothelial dysfunction [41], was found to
have an increased secretion in cells after cryopreservation.
However, this might be attributed to the increase in IL-1a, IL-b
and TNF-a which have been found to up-regulate ICAM-1 in ECs
[42]. Similarly, the observed angiopoietin-1 and TIMP-2 decrease
in our cryopreserved cells may also be attributed to the effect of
the increased IL-1b and TNF-a secretion by the cells [43,44].
While differences in the secretion of certain cytokines was
observed in cryopreserved cells, some of the most important pro-
regenerative and chemotactic cytokines such as IGF-1, bFGF,
SDF-1a and VEGF remained unchanged between the fresh and
Figure 6. Cytokine secretion of CACs after cryopreservation. Fresh and cryopreserved PBMCs were cultured for 4 days to generate CACs. The
media was collected and analyzed using a cytokine array (n = 526). Results of some of the cytokines are shown as a fold-change in light intensity 6
SE; (A–L). *p,0.05 compared to the fresh sample, unless otherwise indicated.
doi:10.1371/journal.pone.0048067.g006
Effects of Cryopreservation on PBMCs and CACs
PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e48067
cryopreserved CACs. Although some changes between fresh and
cryopreserved cells were found, when their angiogenic potential
was investigated in vivo, no differences in the recovery of perfusion
or arteriole density were observed between the animals treated
with cryopreserved cells vs. fresh cells, underscoring the conser-
vation of therapeutic function after cryopreservation.
Figure 7. Cryopreservation does not affect the
in vivo
therapeutic function of CACs in a hindlimb ischemia model. Five million fresh,
d1 or d28 cryopreserved-derived cells were injected into the skeletal muscle of mice that had undergone femoral artery ligation to induce hindlimb
ischemia. Laser Doppler perfusion analysis was conducted over a period of 2 weeks (A). The tissue was harvested at 2 weeks and stained for SMA. The
number of arterioles (per FOV 6 SE) was determined (B), with representative images shown at 206 magnification (n = 526); scale bar = 50
mm.
doi:10.1371/journal.pone.0048067.g007
Effects of Cryopreservation on PBMCs and CACs
PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e48067
The extraction and cryopreservation of PBMCs is a well-
established protocol, already in use for treatment of patients
suffering with bone marrow cancers and other blood diseases.
While the amount of serum used during our freezing step is 6%
and other protocols have used storage solutions containing up to
90% serum [45,46], we did not observe a great loss of viable cells.
This is an important finding, considering that a study investigating
cryopreservation of MNCs reported that viability of ,70% is
associated with compromised proliferative responses to antigens
and mitogens, and are not suitable for cytokine production studies,
flow cytometric analyses, or immunomagnetic cell separation
[47,48].
Overall, our data indicate that the function of therapeutic CAC
populations is generally preserved after short- and longer-term
cryopreservation, with the cells retaining their adhesion and
migratory capabilities. Despite the more drastic phenotypic
changes in PBMCs following cryopreservation, these cells can still
generate CACs with no changes in the population of the potent
vasculogenic CD34
+
VEGFR2
+
CD133
+
cells compared to their
fresh counterparts. Furthermore, the secretion of important
regenerative and chemotactic cytokines is unchanged in the
cryopreserved CACs along with their angiogenic potential in vivo.
However, the cryopreserved CACs do show a significant increase
in secretion of inflammatory factors, which may affect the
expression of additional cytokines such as angiopoietin-1, GCSF,
TIMP-2 and ICAM-1. Our findings also have clinical implication,
as more research is showing that PBMCs and CACs have a
positive impact on neovascularization in ischemic and cardiovas-
cular diseases. One important factor for successful cell therapy is
generating enough therapeutic cells to achieve sufficient repair of
the damaged tissue. Progenitor cells from the peripheral blood are
much easier to obtain than the traditional bone marrow harvest,
which is more painful and requires anesthesia. The PBMCs may
be collected over a given period and stored each time until enough
cells are collected and the patient is primed to receive the cell
therapy, at which time the PBMCs may be cultured to generate
the therapeutic CACs.
Conclusions
In conclusion, the CACs generated from the cryopreserved
PBMCs show no significant difference in viability, in in vitro
functions such as adhesion and migration, or in in vivo angiogenic
potential; however, an increase in LDL uptake and lectin binding
was observed. Also while there are some phenotypic changes, the
therapeutically potent CD34
+
VEGFR2
+
CD133
+
population re-
mains unchanged. Additionally, the secretion of important
regenerative and chemotactic cytokines involved in tissue repair
through paracrine mechanisms is unaffected in CACs after
cryopreservation. The ability to store these therapeutic cells for
a long period of time without large cell losses and disruption of
function will offer more patients an opportunity to experience
successful cell therapy.
Materials and Methods
A summary of the cell populations, methods and time points are
presented in Figure 1.
Ethics Statement
The study was approved by the Human Research Ethics Board
of the University of Ottawa Heart Institute, and informed consent
was obtained from all donors.
Cell Isolation
Total PBMCs were isolated from 100 ml of fresh blood samples
of seven healthy human donors both male and female (20–35 years
of age) by Histopaque 1077 (Sigma-Aldrich, Oakville, Canada)
density-gradient centrifugation as described previously [49]. The
cells within the buffy coat were then either analyzed (baseline
PBMCs), cultured to generate CACs, or cryopreserved.
Cryopreservation
PBMCs retrieved from the buffy coat were reconstituted in a
1 ml solution of Isocove’s Modified Dulbecco’s Medium (IMDM;
Stem Cell Technologies, Vancouver, Canada) containing 5%
dimethyl sulfoxide (DMSO; Sigma-Aldrich) and 6% donor cell-
matched serum and placed into a cryovial (Corning Incorporated,
Corning, USA). The DMSO was mixed with the IMDM prior to
addition of blood serum and cells in order to prevent clump
formation. The cells were cooled to 280uC over 3 hours before
being transferred to liquid nitrogen for storage.
For rapid thawing and to prevent toxicity to cells, cryovial tubes
were taken from the freezer and immediately placed in a 37uC
water bath until ice crystals disappeared, after which the cells were
diluted 56 with IMDM and centrifuged at 1400 rpm and
immediately resuspended in fresh Endothelial Basal Media
(EBM-2; Clonetics, Guelph, Canada).
CAC Culture
CACs were generated by seeding fresh PBMCs, or PBMCs
thawed at days 1 or 28 post-freezing, on fibronectin (Sigma-
Aldrich)-coated plates in EBM supplemented with EGM-2-MV-
SingleQuots (Clonetics) containing 5% fetal bovine serum, human
VEGF, human insulin-like growth factor 1, human epidermal
growth factor and antibiotics for 4 days. After 4 days, the media
was removed and the cells were lifted using gentle pipetting with
PBS and prepared for subsequent assays.
Flow Cytometry Staining
Cells were counted using a Vi-Cell analyzer (Beckman Coulter,
Mississauga, Canada). PBMCs or CACs (3610
5
) in 200 ml of EBM
were stained for 30 minutes at 4uC with pre-conjugated antibodies
against the following antigens: CD31-FITC (Beckman Coulter),
CD34-PECy7 (BD Biosciences, Mississauga, Canada), KDR-PE
(R&D Systems, Minneapolis, USA), CD133-APC (Miltenyl Biotec,
Auburn, USA) and L-selectin-ECD (Beckman). Samples were also
stained with appropriate IgG isotype-matched controls. Immedi-
ately prior to analysis, the viability stain 7-actinomycin D (7-AAD)
(Invitrogen, Burlington, Canada) was added to samples to a final
concentration of 8.3
mg/ml. Characterization also included
incubating 3610
5
cells with 2 mg/ml of 1,19-dioctadecyl-
3,3,39,39-tetramethylindocarbocyanine–labeled acetylated low
density lipoprotein (DiI-LDL) (Invitrogen) for one hour at 37uC
after which the cells were pelleted, fixed for 10 minutes with
fixation buffer (BD Biosciences) and further incubated for an hour
at 37uC with 10
mg/ml of fluorescein isothiocyanate (FITC)
labelled lectin from Ulex europaeus agglutinin-1 (Sigma-Aldrich).
Cells were analyzed and quantified with a BD FACSaria cell sorter
(BD Biosciences) until the number of events (cells) reached
200,000.
Functional Assays
Cells were suspended in 50 mg/ml of 49,6-diamidino-2-pheny-
lindole (DAPI; Sigma-Aldrich) in PBS for 30 minutes at 37uC,
after which the cells were pelleted and counted. Migration.
DAPI-stained PBMCs or CACs (2610
4
) were suspended in 100 ml
Effects of Cryopreservation on PBMCs and CACs
PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e48067
of VEGF-free EBM and placed in the top compartment of a 24-
well Boyden chamber (Corning Inc.) with the lower chamber
coated with fibronectin and containing 350
ml of 0.05 mg/ml
VEGF (Cedarlane, Burlington, Canada) in EBM. The cells were
incubated for 24 hours in 5% CO
2
after which they were fixed
with 4% paraformaldehyde and washed with PBS. Adhesion.
DAPI-stained cells (2610
4
) were plated in 1 ml of EBM on
fibronectin coated 24-well plates and incubated for 1 hour in 5%
CO
2
after which they were fixed with 4% paraformaldehyde. The
number of DAPI stained cells for both assays were counted in 5
random fields-of-view for each well at 206 magnification using
Olympus B660 fluorescent microscope (Olympus Canada Inc.,
Markham, Canada). Assays were performed in duplicate.
Cytokine Array
Three million PBMCs were plated on fibronectin-coated 6-well
plates in 2 ml of EBM media. After culturing for 4 days to
generate CACs, the media was collected. Cytokine arrays
(RayBiotech, Norcross, USA) were incubated with 100
mlof
media per sample according to manufacturer’s protocol. Following
the cytokine array procedures, the membranes were exposed and
the light produced at each spot (proportional to the amount of
cytokine bound) was quantified using AlphaEaseFC.
Hindlimb ischemia mouse model. Nude BALB/C mice
(7–8 weeks old) underwent ligation of proximal femoral arteries
under 2% isoflurane, as previously described [14]. Subsequently,
the ischemic hindlimb was injected downstream of the ligation site
with 2 equivolumetric injections (50
ml total, suspended in PBS)
containing a total of 5610
6
CACs that were derived from: i) fresh
PBMCs; ii) day 1 cryopreserved PBMCs; or, iii) day 28
cryopreserved PBMCs, prepared as described above. Injections
were performed with a 27 gauge needle. Prior to injection, cell
viability was confirmed to be .85%.
Laser Doppler
Hindlimb perfusion was measured using laser Doppler analysis
pre-operatively, post-operatively, and at days 4, 7, 10 and 14, as
described previously [14]. Briefly, while mice were anaesthetized
with 2% isoflurane, single point measurements were recorded
(moorLD12; Moor Instruments, Axminster, UK) in both hind-
limbs and used to evaluate perfusion. The data is expressed as a
ratio of ischemic to non-ischemic hindlimb blood flow.
Immunohistochemistry
Two weeks post-CAC treatment, the mice were sacrificed and
the ischemic hindlimb tissue was collected, frozen and sectioned.
After fixing tissue sections with acetone for 10 minutes, the sections
were washed for 365 minutes with PBS, blocked for an hour using
10% fetal bovine serum in PBS and stained with anti-SMA
antibody (1:100; abcam, Cambridge, USA). Following 3 washes (5
minutes each) and secondary antibody application (1:600 anti-
rabbit Alexa 488; Invitrogen,) the slides were mounted with DAPI
and analyzed at 206 magnification with an Olympus BX50
microscope using Image Pro Plus. The number of arterioles was
counted by 2 blind observers and expressed as the average number
of SMA
+
vessels per field of view (FOV). Additionally, to visualize
the injected CACs, hindlimb sections were stained with anti-
human mitochondria antibody (1:100; Chemicon, Temecula,
USA), following the manufacturers instruction.
Statistical Analysis
Results obtained for each phenotype and each cell type were
analyzed for differences between days using a one-way Anova
adjusted for donor. Sub-analyses were performed when a
Bonferonni significant result was obtained using a paired t-test.
Results were Bonferonni adjusted for the number of tests
performed for each of viability, phenotype and function separately,
to achieve an alpha of 0.05; p-values given for sub-analyses are not
corrected. Statistical analyses were performed using R (R
development core team, Vienna, Austria).
Acknowledgments
The authors would like to thank Branka Vulesevic for her assistance with
animal procedures.
Author Contributions
Conceived and designed the experiments: TS ES DK. Performed the
experiments: TS KM SC JM. Analyzed the data: TS KM RD DK.
Contributed reagents/materials/analysis tools: TS KM SC JM RD. Wrote
the paper: TS ES DK.
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