Extracted Text
lyophilzation of BM.pdf
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894 889
Preliminary study on the freeze-drying of human bone
marrow-derived mesenchymal stem cells
*
Shao-zhi ZHANG
1
, Huan QIAN
2
, Zhen WANG
2
, Ju-li FAN
1
, Qian ZHOU
2
,
Guang-ming CHEN
1
, Rui LI
2
, Shan FU
2
, Jie SUN
†‡2
(
1
Refrigeration and Cryogenic Engineering Institute, Zhejiang University, Hangzhou 310027, China)
(
2
Bone Marrow Transplantation Center, the First Affiliate Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China)
†
E-mail: jsun1492@gmail.com
Received May 19, 2010; Revision accepted Aug. 1, 2010; Crosschecked Oct. 13, 2010
Abstract: Long-term preservation and easy transportation of human bone marrow-derived mesenchymal stem cells
(hBM-MSCs) will facilitate their application in medical treatment and bioengineering. A pilot study on the freeze-drying
of hBM-MSCs was carried out. hBM-MSCs were loaded with trehalose. The glass transition temperature of the
freeze-drying suspension was measured to provide information for the cooling and primary drying experiment. After
freeze-drying, various rehydration processes were tested. The highest recovery rate of hBM-MSCs was (69.33±
13.08)%. Possible methods to improve freeze-drying outcomes are discussed. In conclusion, the present study has
laid a foundation for the freeze-drying hBM-MSCs.
Key words: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs), Freeze-drying, Trehalose, Rehydration
doi:10.1631/jzus.B1000184 Document code: A CLC number: Q27
1 Introduction
Human bone marrow-derived mesenchymal
stem cells (hBM-MSCs) are multipotent stem cells
that can proliferate, support the immune system, and
differentiate into multiple lineages. They are ideal
stem cells for tissue engineering and clinical treat-
ment (Nakamizo et al., 2005; Winer et al., 2009).
Resuscitated hBM-MSCs could be used for estab-
lishing an abundant hBM-MSC reservoir for further
experiment and treatment of various clinical diseases
(Xiang et al., 2007). However, the difficulties faced in
the transportation and prolonged storage of hBM-
MSCs have hindered their use in clinical studies and
industrial application. The traditional method for
hBM-MSCs storage is cryopreservation. Under
standard cryopreservation procedures, cell recovery
rates were from 87.67% to 94.76% (Luo et al., 2006;
Carvalho et al., 2008). The recovery rate was high and
the proliferating ability could be maintained. How-
ever, cryopreservation always requires expensive and
clumsy equipments, and usually the supply and
management of liquid nitrogen, which limits its usage.
Gordon et al. (2001) conducted a primary study on the
air-drying of hBM-MSCs, and the rehydrated cells
maintained high viability and proliferation capacity.
Nevertheless, considering the damage of air-drying,
including shrinkage of membrane, elevation in in-
tracellular salt concentration, changes in biophysical
properties and physiological processes, the air-drying
of hBM-MSCs is still not the preferential method
(Potts et al., 2005).
Freeze-drying has been used in the preparation
of pharmaceuticals and vaccines as one of the most
important processes for the preservation of heat-sen-
sitive biological materials. Compared to other tech-
niques, freeze-drying has some well-known advantages,
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
www.zju.edu.cn/jzus; www.springerlink.com
E-mail: jzus@zju.edu.cn
Rapid Communication:
‡
Corresponding author
*
Project (Nos. 30600256 and 50606032) supported by the National
Natural Science Foundation of China
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2010
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894
890
including sample stability at room temperature, de-
fined porous product structure, easy reconstitution by
the addition of water or aqueous solution, and easy
transportation (Xiao et al., 2004). Freeze-drying of
cells used to be limited to prokaryotes. Over recent
years, a few types of mammalian cells have been
successfully freeze-dried, such as human erythrocytes
and human platelets (Crowe et al., 2001; Wolkers et
al., 2001; 2002; Han et al., 2005; Li et al., 2005;
Török et al., 2005). It has been proven that
freeze-dried platelets can be stored at room tempera-
ture for several months while their physiological
viabilities are remained
(Wolkers et al., 2002). Yang
et al. (2005) tried different lyoprotectants to lyophi-
lize hBM-MSCs and found 30% (w/v) polyvi-
nylpyrrolidone 40 (PVP40)+20% (w/v) trehalose to
be better. However, the recovery rate of rehydrated
cells was 16.4%, rather lower than that of cryopre-
servation. Vacuum-drying was also tried and good
results were obtained (Jamil et al., 2005). However,
the final water content was 0.3 g H
2O/g dry weight,
which may influence long-term stability of the prod-
ucts. Obtaining a better recovery rate after freeze-
drying still requires further study.
The success of freeze-drying mammalian cells
was largely dependent on the usage of trehalose.
Anhydrobiotic organisms can tolerate the lack of
water because of their ability to synthesize large
quantities of trehalose (Alpert, 2006). The major
challenge of using trehalose for cell preservation is to
increase its intracellular concentration. Many meth-
ods have been proposed to load trehalose into the cell
across the membrane which is naturally impermeable
to this chemical, such as ultrasound, electroperme-
abilization, transgenes, and
microinjection (Chen et
al., 2001; Eroglu et al., 2002; Shirakashi et al., 2002;
Zhang et al., 2009). Fluid-phase endocytosis is easier
to perform compared to other methods and has been
employed in the successful loading of trehalose into
human platelets (Wolkers et al., 2001). Oliver et al.
(2004) have investigated the factors that influenced
the loading of trehalose into hBM-MSCs by fluid-
phase endocytosis. They found that the uptake process
was inhibited below 20 °C, and the quantity of uptake
was proportional to the length of incubation and de-
pendent on extracellular trehalose concentration. In
this study, the method of fluid-phase endocytosis was
also employed to load trehalose into hBM-MSCs.
To reduce the injuries of both desiccation and
hypothermia during the freeze-drying process, PVP40
was added as a protectant. It can inhibit sucrose
crystallization and stabilize the glassy structure of
sugar
(Zeng et al., 2001). As to the rehydration
process, phosphate buffered saline (PBS) was often
used as rehydration solution (Xiao et al., 2004; Li et
al., 2005). Both colloidal osmotic pressure and crys-
talloid pressure of the rehydration solution would
affect the recovery of cells, while the colloidal os-
motic pressure was more influential on cell survival
than crystalloid pressure during rehydration (Han et
al., 2004). Based on this finding, rehydration solution
containing trehalose and PVP40 will be tried in this
study, different formulas were tried in order to obtain
the best colloidal osmotic pressure and crystalloid
pressure, and the recovery rates were detected.
2 Materials and methods
2.1 Culture of hBM-MSCs
Human bone marrows were collected from three
healthy adult donors from the First Affiliated Hospital,
School of Medicine, Zhejiang University, China.
Mesenchymal stem cells were obtained by density
gradient centrifugation. The cells were incubated in
Dulbecco’s modified Eagle’s medium-low glucose
(DMEM-LG, Gibco, USA) with 10% (w/v) fetal
bovine serum (FBS) at 37 °C in 5% CO
2 atmosphere.
After 48-h incubation, nonadherent cells were dis-
carded, and adherent cells were washed twice with
PBS. Fresh culture medium was added every 3 d. In
14 d, the cells grew to 70%–90% confluence. Then
the cells were harvested with 0.25% (w/v) trypsin/
1 mmol/L ethylenediamine tetraacetic acid (EDTA)
(Life Technologies, Gaithersburg, MD, USA) and
diluted 1:3 (v/v) for passage. hBM-MSCs of the 3rd
passage were used for experiment.
2.2 Trehalose loading and preparation of freeze-
drying suspension
In order to load trehalose into hBM-MSCs, the
cells were incubated with trehalose (Sigma-Aldrich)
solution at 37 °C in 5% CO
2 atmosphere for 24 h. The
concentrations of trehalose solution tested included
10, 20, 50, 80, 100, and 200 mmol/L. Anthrone-
sulfuric acid reaction method (anthrone and sulfuric
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894 891
acid bought from Sigma-Aldrich) was applied
to determine intracellular trehalose concentration
(Umbreit et al., 1972). The absorbance of the solu-
tion was measured at 620 nm on a VIS-723 spec-
trophotometer (Precision & Scientific Instrument Co.,
Ltd., Shanghai, China) and compared with a standard
curve. Assuming that 14
000 fl of the cell volume is
taken up by the cytosol (Oliver et al., 2004), the
concentration of intracellular trehalose was calcu-
lated as follows:
/
,
AXM
C
vNV
=
where C (mmol/L) is the concentration of intracellu-
lar trehalose, A (ml) is the volume of sample solution,
X (μg/ml) is the trehalose concentration of sample
solution (derived by the standard curve), M (g/mol) is
molecular weight of trehalose, ν (ml) is the volume of
the solution used to measure the intracellular treha-
lose; N (L
−1
) is the concentration of cells, and V (fl) is
the mean cytosol volume.
An appropriate concentration was chosen
(20–200 mmol/L) and further experiments were car-
ried out to investigate the impact of incubation dura-
tion of 6, 12, and 24 h. To estimate the possible effect
of trehalose loading, the growth curve of post-loading
hBM-MSCs was compared with non-loading hBM-
MSCs. Cell growth was evaluated by counting cell
numbers with a counting chamber (n=3). Freeze-
drying suspension was composed of 30% PVP40,
100 mmol/L trehalose, and cell suspension at a vol-
ume ratio of 2:1:2. The cell concentration in the cell
suspension was 10
7
cells/ml.
2.3 Differential scanning calorimetry (DSC) analysis
The glass transition temperature of the freeze-
drying system was measured with DSC-Q100 (TA
Instruments, New Castle, DE, USA). An empty
sealed aluminum pan was used as reference. Nitrogen
was used as carrier gas. Both the cooling rate and the
warming rate were 10 °C/min. The Universal Analy-
sis software was used to analyze the DSC curves.
2.4 Residual water content
Residual water content after freeze-drying was
measured with the thermogravimetry analysis (WCT-2
A-DTA, Beijing Optical Instrument Factory, China).
2.5 Freeze-drying
The freeze-drying process was carried out on a
self-made freeze-dryer as shown in Fig. 1 (Weng et
al., 2004). A total of 1 ml freeze-drying system was
filled into a sterilized glass bottle covered with a
semipermeable film to prevent contamination. The
glass bottle’s inner diameter was 1.6 cm (Fig. 6). The
bottle was put on the shelf of the freeze-dryer, which
had been precooled to −60 °C, so that quick freezing
could be realized
(Li et al., 2003; Wang and Zhang,
2007). After cooling for 2 h, the primary drying began.
The shelf temperature was set at −32 °C, and the
vacuum was controlled under 10 Pa. The drying
process was conducted similarly to what we did on
human platelets (Zhou et al., 2007). The primary
drying process lasted for 16 h. Then the shelf was
heated up to 20 °C at a rate of 0.2 °C/min and held for
6 h. After freeze-drying, the bottle was sealed and
kept at room temperature for 2 h.
2.6 Rehydration
Three kinds of solution were used: (1) PBS
buffer (pH 6.8), made of 100 mmol/L NaCl, 9.4 mmol/L
Na
2HPO4, and 0.6 mmol/L KH2PO4; (2) rehydration
solution A, DMEM-LG containing 20% (w/v) FBS;
(3) rehydration solution B, composed of 30% PVP40,
100 mmol/L trehalose, and PBS buffer at a volume
ratio of 2:1:2. Four different procedures of rehydra-
tion were tested: (1) 5 ml rehydration solution A was
Fig. 1 Self-made freeze-dryer
An auto-cascade refrigeration system was employed. The
shelf could be cooled down to −60 °C and the condenser
temperature could reach −80 °C. The pressure in the drying
chamber could reach 2 Pa (Weng et al., 2004)
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894
892
gradually added into the dried sample; (2) 2 ml PBS
was added into the dried sample and held for 5 min,
then another 6 ml PBS was added, and the suspension
was centrifuged at 900×g for 1 min, then the pre-
cipitated sample was resuspended in 5 ml rehydration
solution A; (3) the same with (2) except that the dried
sample was first equilibrated in 2 ml rehydration
solution B for 5 min; and, (4) the dried samples was
equilibrated in 0.5 ml rehydration solution B for 5 min,
and then added with 4.5 ml rehydration solution A.
2.7 Cell viability evaluation
Cell number and viability were determined by
trypan blue (ScienCell, USA) staining. The recovery
rate (RR) was defined as the percentage: RR=N
1/N2×
ER×100%, where N
1 is the number of cells after re-
hydration, N
2 is the number of cells before freeze-
drying, and ER is trypan blue excluding rate after
rehydration. The recovery rate evaluation was dupli-
cated thrice for each rehydration procedure. The re-
covered cells were cultured in a 25-cm
2
flask with
DMEM-LG medium at 37 °C and 5% CO
2.
2.8 Statistical analysis
The experimental data were statistically ana-
lyzed using two-sample t-test. A level of P≤0.05 was
accepted as being statistically significant.
3 Results
3.1 Trehalose uptake by hBM-MSCs
Intracellular trehalose concentration of hBM-
MSCs increased with increasing extracellular tre-
halose concentration up to 100 mmol/L. While the
extracellular trehalose concentration increased from
100 mmol/L to 200 mmol/L, the magnitude of the
increasing of intracellular trehalose concentration was
very limited (Fig. 2).
Intracellular trehalose concentration increased
linearly (r=0.99) with incubation time when incu-
bated with 100 mmol/L trehalose solution. After 24 h
of incubation, the intracellular trehalose concentration
reached (14.57±0.74) mmol/L (Fig. 3). This value
was a little lower than former reports as 19 mmol/L
(Oliver et al., 2004). It may be caused by different cell
conditions. Twenty-four hours and 100 mmol/L were
chosen as the best conditions for trehalose loading.
No significant difference (P>0.05) was observed
between the two growth curves of post-loading and
non-loading cells (Fig. 4).
20 50 80 100 200
C
e (mmol/L)
16
12
8
4
0
C
i (mmol/L)
Fig. 2 Trehalose uptake by hBM-MSCs as a function of
extracellular trehalose concentration
Loading experiments were conducted at 37 °C in the
presence of 20–200 mmol/L trehalose solutions for 24 h.
Data were derived in triplicates. Intracellular trehalose
concentration (C
i) increased with increasing extracellular
trehalose concentration (C
e)
6 12 24
t
i (h)
20
16
12
8
4
0
C
i (mmol/L)
Fig. 3 Uptake of trehalose by hBM-MSCs as a function
of incubation time
hBM-MSCs were incubated in 100 mmol/L trehalose so-
lution for 6, 12, and 24 h. Data were derived in triplicates
and the error bars represent standard deviations. Intracel-
lular trehalose concentration (C
i) increased linearly (r=
0.99) with incubation time (t
i)
1 2 3 4 5 6 7 8
t (d)
4
3
2
1
0
N (×10
4
)
Non-loading
Loading
Fig. 4 Growth curves of trehalose loading and non-
loading hBM-MSCs at passage 3
No significant difference was found between two growth
curves (P>0.05). N: cell number; t: incubation time
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894 893
3.2 Glass transition temperature
The mid-point of the transition was considered
to be the glass transition temperature T
g. The T g of
the freeze-drying suspension was determined to be
−26.6 °C (Fig. 5). During the primary drying process,
the sample temperature should be lower than this
temperature; thus, the shelf temperature was set at
−32 °C.
3.3 Residual water content
After freeze-drying, a dried and sterile sample
was obtained (Fig. 6). The residual water content of
the sample was determined to be 2.9%.
3.4 Cell recovery after rehydration
Freeze-dried samples were rehydrated with four
procedures mentioned above. The recovery rates after
12 h planting for procedures 1, 2, 3, and 4 were
(61.75±15.64)%, (41.17±7.56)%, (48.56±18.10)%,
and (69.33±13.08)%, respectively. Procedures 1 and
4 showed higher viability than procedures 2 and 3
(P<0.05), but there was no statistical differences
between procedures 1 and 4 (P>0.05). The mor-
phologies of recovered cells immediately after rehy-
dration and after 12-h incubation are shown in Fig. 7.
The recovery rate was higher than former reports by
Yang et al. (2005). However, recovered hBM-MSCs
had impaired adhere and proliferation abilities and
died within a week.
Compared to procedures 1 and 4, procedures 2
and 3 tended to have lower concentrations of ex-
tracellular trehalose and more physical disturbance,
which could lead to more susceptibility to physical
and chemical stresses during rehydration. It seemed
that the rehydration solution containing trehalose was
beneficial for cell survival, which might be due to its
higher colloidal osmotic pressure than that of other
solutions without trehalose.
4 Conclusions
In this study, hBM-MSCs undergoing freeze-
drying had a recovery rate ranging from (41.17±
7.56)% to (69.33±13.08)% at 12 h after recovering.
However, the proliferation ability of rehydrated hBM-
MSCs was not shown. Nevertheless, the present study
has laid a foundation for the freeze-drying of hBM-
MSCs. The attractive prospects of freeze-drying
hBM-MSCs prompt us to put more effort into de-
signing better protocols in future studies.
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Preliminary study on the freeze-drying of human bone
marrow-derived mesenchymal stem cells
*
Shao-zhi ZHANG
1
, Huan QIAN
2
, Zhen WANG
2
, Ju-li FAN
1
, Qian ZHOU
2
,
Guang-ming CHEN
1
, Rui LI
2
, Shan FU
2
, Jie SUN
†‡2
(
1
Refrigeration and Cryogenic Engineering Institute, Zhejiang University, Hangzhou 310027, China)
(
2
Bone Marrow Transplantation Center, the First Affiliate Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China)
†
E-mail: jsun1492@gmail.com
Received May 19, 2010; Revision accepted Aug. 1, 2010; Crosschecked Oct. 13, 2010
Abstract: Long-term preservation and easy transportation of human bone marrow-derived mesenchymal stem cells
(hBM-MSCs) will facilitate their application in medical treatment and bioengineering. A pilot study on the freeze-drying
of hBM-MSCs was carried out. hBM-MSCs were loaded with trehalose. The glass transition temperature of the
freeze-drying suspension was measured to provide information for the cooling and primary drying experiment. After
freeze-drying, various rehydration processes were tested. The highest recovery rate of hBM-MSCs was (69.33±
13.08)%. Possible methods to improve freeze-drying outcomes are discussed. In conclusion, the present study has
laid a foundation for the freeze-drying hBM-MSCs.
Key words: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs), Freeze-drying, Trehalose, Rehydration
doi:10.1631/jzus.B1000184 Document code: A CLC number: Q27
1 Introduction
Human bone marrow-derived mesenchymal
stem cells (hBM-MSCs) are multipotent stem cells
that can proliferate, support the immune system, and
differentiate into multiple lineages. They are ideal
stem cells for tissue engineering and clinical treat-
ment (Nakamizo et al., 2005; Winer et al., 2009).
Resuscitated hBM-MSCs could be used for estab-
lishing an abundant hBM-MSC reservoir for further
experiment and treatment of various clinical diseases
(Xiang et al., 2007). However, the difficulties faced in
the transportation and prolonged storage of hBM-
MSCs have hindered their use in clinical studies and
industrial application. The traditional method for
hBM-MSCs storage is cryopreservation. Under
standard cryopreservation procedures, cell recovery
rates were from 87.67% to 94.76% (Luo et al., 2006;
Carvalho et al., 2008). The recovery rate was high and
the proliferating ability could be maintained. How-
ever, cryopreservation always requires expensive and
clumsy equipments, and usually the supply and
management of liquid nitrogen, which limits its usage.
Gordon et al. (2001) conducted a primary study on the
air-drying of hBM-MSCs, and the rehydrated cells
maintained high viability and proliferation capacity.
Nevertheless, considering the damage of air-drying,
including shrinkage of membrane, elevation in in-
tracellular salt concentration, changes in biophysical
properties and physiological processes, the air-drying
of hBM-MSCs is still not the preferential method
(Potts et al., 2005).
Freeze-drying has been used in the preparation
of pharmaceuticals and vaccines as one of the most
important processes for the preservation of heat-sen-
sitive biological materials. Compared to other tech-
niques, freeze-drying has some well-known advantages,
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)
ISSN 1673-1581 (Print); ISSN 1862-1783 (Online)
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E-mail: jzus@zju.edu.cn
Rapid Communication:
‡
Corresponding author
*
Project (Nos. 30600256 and 50606032) supported by the National
Natural Science Foundation of China
© Zhejiang University and Springer-Verlag Berlin Heidelberg 2010
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894
890
including sample stability at room temperature, de-
fined porous product structure, easy reconstitution by
the addition of water or aqueous solution, and easy
transportation (Xiao et al., 2004). Freeze-drying of
cells used to be limited to prokaryotes. Over recent
years, a few types of mammalian cells have been
successfully freeze-dried, such as human erythrocytes
and human platelets (Crowe et al., 2001; Wolkers et
al., 2001; 2002; Han et al., 2005; Li et al., 2005;
Török et al., 2005). It has been proven that
freeze-dried platelets can be stored at room tempera-
ture for several months while their physiological
viabilities are remained
(Wolkers et al., 2002). Yang
et al. (2005) tried different lyoprotectants to lyophi-
lize hBM-MSCs and found 30% (w/v) polyvi-
nylpyrrolidone 40 (PVP40)+20% (w/v) trehalose to
be better. However, the recovery rate of rehydrated
cells was 16.4%, rather lower than that of cryopre-
servation. Vacuum-drying was also tried and good
results were obtained (Jamil et al., 2005). However,
the final water content was 0.3 g H
2O/g dry weight,
which may influence long-term stability of the prod-
ucts. Obtaining a better recovery rate after freeze-
drying still requires further study.
The success of freeze-drying mammalian cells
was largely dependent on the usage of trehalose.
Anhydrobiotic organisms can tolerate the lack of
water because of their ability to synthesize large
quantities of trehalose (Alpert, 2006). The major
challenge of using trehalose for cell preservation is to
increase its intracellular concentration. Many meth-
ods have been proposed to load trehalose into the cell
across the membrane which is naturally impermeable
to this chemical, such as ultrasound, electroperme-
abilization, transgenes, and
microinjection (Chen et
al., 2001; Eroglu et al., 2002; Shirakashi et al., 2002;
Zhang et al., 2009). Fluid-phase endocytosis is easier
to perform compared to other methods and has been
employed in the successful loading of trehalose into
human platelets (Wolkers et al., 2001). Oliver et al.
(2004) have investigated the factors that influenced
the loading of trehalose into hBM-MSCs by fluid-
phase endocytosis. They found that the uptake process
was inhibited below 20 °C, and the quantity of uptake
was proportional to the length of incubation and de-
pendent on extracellular trehalose concentration. In
this study, the method of fluid-phase endocytosis was
also employed to load trehalose into hBM-MSCs.
To reduce the injuries of both desiccation and
hypothermia during the freeze-drying process, PVP40
was added as a protectant. It can inhibit sucrose
crystallization and stabilize the glassy structure of
sugar
(Zeng et al., 2001). As to the rehydration
process, phosphate buffered saline (PBS) was often
used as rehydration solution (Xiao et al., 2004; Li et
al., 2005). Both colloidal osmotic pressure and crys-
talloid pressure of the rehydration solution would
affect the recovery of cells, while the colloidal os-
motic pressure was more influential on cell survival
than crystalloid pressure during rehydration (Han et
al., 2004). Based on this finding, rehydration solution
containing trehalose and PVP40 will be tried in this
study, different formulas were tried in order to obtain
the best colloidal osmotic pressure and crystalloid
pressure, and the recovery rates were detected.
2 Materials and methods
2.1 Culture of hBM-MSCs
Human bone marrows were collected from three
healthy adult donors from the First Affiliated Hospital,
School of Medicine, Zhejiang University, China.
Mesenchymal stem cells were obtained by density
gradient centrifugation. The cells were incubated in
Dulbecco’s modified Eagle’s medium-low glucose
(DMEM-LG, Gibco, USA) with 10% (w/v) fetal
bovine serum (FBS) at 37 °C in 5% CO
2 atmosphere.
After 48-h incubation, nonadherent cells were dis-
carded, and adherent cells were washed twice with
PBS. Fresh culture medium was added every 3 d. In
14 d, the cells grew to 70%–90% confluence. Then
the cells were harvested with 0.25% (w/v) trypsin/
1 mmol/L ethylenediamine tetraacetic acid (EDTA)
(Life Technologies, Gaithersburg, MD, USA) and
diluted 1:3 (v/v) for passage. hBM-MSCs of the 3rd
passage were used for experiment.
2.2 Trehalose loading and preparation of freeze-
drying suspension
In order to load trehalose into hBM-MSCs, the
cells were incubated with trehalose (Sigma-Aldrich)
solution at 37 °C in 5% CO
2 atmosphere for 24 h. The
concentrations of trehalose solution tested included
10, 20, 50, 80, 100, and 200 mmol/L. Anthrone-
sulfuric acid reaction method (anthrone and sulfuric
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894 891
acid bought from Sigma-Aldrich) was applied
to determine intracellular trehalose concentration
(Umbreit et al., 1972). The absorbance of the solu-
tion was measured at 620 nm on a VIS-723 spec-
trophotometer (Precision & Scientific Instrument Co.,
Ltd., Shanghai, China) and compared with a standard
curve. Assuming that 14
000 fl of the cell volume is
taken up by the cytosol (Oliver et al., 2004), the
concentration of intracellular trehalose was calcu-
lated as follows:
/
,
AXM
C
vNV
=
where C (mmol/L) is the concentration of intracellu-
lar trehalose, A (ml) is the volume of sample solution,
X (μg/ml) is the trehalose concentration of sample
solution (derived by the standard curve), M (g/mol) is
molecular weight of trehalose, ν (ml) is the volume of
the solution used to measure the intracellular treha-
lose; N (L
−1
) is the concentration of cells, and V (fl) is
the mean cytosol volume.
An appropriate concentration was chosen
(20–200 mmol/L) and further experiments were car-
ried out to investigate the impact of incubation dura-
tion of 6, 12, and 24 h. To estimate the possible effect
of trehalose loading, the growth curve of post-loading
hBM-MSCs was compared with non-loading hBM-
MSCs. Cell growth was evaluated by counting cell
numbers with a counting chamber (n=3). Freeze-
drying suspension was composed of 30% PVP40,
100 mmol/L trehalose, and cell suspension at a vol-
ume ratio of 2:1:2. The cell concentration in the cell
suspension was 10
7
cells/ml.
2.3 Differential scanning calorimetry (DSC) analysis
The glass transition temperature of the freeze-
drying system was measured with DSC-Q100 (TA
Instruments, New Castle, DE, USA). An empty
sealed aluminum pan was used as reference. Nitrogen
was used as carrier gas. Both the cooling rate and the
warming rate were 10 °C/min. The Universal Analy-
sis software was used to analyze the DSC curves.
2.4 Residual water content
Residual water content after freeze-drying was
measured with the thermogravimetry analysis (WCT-2
A-DTA, Beijing Optical Instrument Factory, China).
2.5 Freeze-drying
The freeze-drying process was carried out on a
self-made freeze-dryer as shown in Fig. 1 (Weng et
al., 2004). A total of 1 ml freeze-drying system was
filled into a sterilized glass bottle covered with a
semipermeable film to prevent contamination. The
glass bottle’s inner diameter was 1.6 cm (Fig. 6). The
bottle was put on the shelf of the freeze-dryer, which
had been precooled to −60 °C, so that quick freezing
could be realized
(Li et al., 2003; Wang and Zhang,
2007). After cooling for 2 h, the primary drying began.
The shelf temperature was set at −32 °C, and the
vacuum was controlled under 10 Pa. The drying
process was conducted similarly to what we did on
human platelets (Zhou et al., 2007). The primary
drying process lasted for 16 h. Then the shelf was
heated up to 20 °C at a rate of 0.2 °C/min and held for
6 h. After freeze-drying, the bottle was sealed and
kept at room temperature for 2 h.
2.6 Rehydration
Three kinds of solution were used: (1) PBS
buffer (pH 6.8), made of 100 mmol/L NaCl, 9.4 mmol/L
Na
2HPO4, and 0.6 mmol/L KH2PO4; (2) rehydration
solution A, DMEM-LG containing 20% (w/v) FBS;
(3) rehydration solution B, composed of 30% PVP40,
100 mmol/L trehalose, and PBS buffer at a volume
ratio of 2:1:2. Four different procedures of rehydra-
tion were tested: (1) 5 ml rehydration solution A was
Fig. 1 Self-made freeze-dryer
An auto-cascade refrigeration system was employed. The
shelf could be cooled down to −60 °C and the condenser
temperature could reach −80 °C. The pressure in the drying
chamber could reach 2 Pa (Weng et al., 2004)
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894
892
gradually added into the dried sample; (2) 2 ml PBS
was added into the dried sample and held for 5 min,
then another 6 ml PBS was added, and the suspension
was centrifuged at 900×g for 1 min, then the pre-
cipitated sample was resuspended in 5 ml rehydration
solution A; (3) the same with (2) except that the dried
sample was first equilibrated in 2 ml rehydration
solution B for 5 min; and, (4) the dried samples was
equilibrated in 0.5 ml rehydration solution B for 5 min,
and then added with 4.5 ml rehydration solution A.
2.7 Cell viability evaluation
Cell number and viability were determined by
trypan blue (ScienCell, USA) staining. The recovery
rate (RR) was defined as the percentage: RR=N
1/N2×
ER×100%, where N
1 is the number of cells after re-
hydration, N
2 is the number of cells before freeze-
drying, and ER is trypan blue excluding rate after
rehydration. The recovery rate evaluation was dupli-
cated thrice for each rehydration procedure. The re-
covered cells were cultured in a 25-cm
2
flask with
DMEM-LG medium at 37 °C and 5% CO
2.
2.8 Statistical analysis
The experimental data were statistically ana-
lyzed using two-sample t-test. A level of P≤0.05 was
accepted as being statistically significant.
3 Results
3.1 Trehalose uptake by hBM-MSCs
Intracellular trehalose concentration of hBM-
MSCs increased with increasing extracellular tre-
halose concentration up to 100 mmol/L. While the
extracellular trehalose concentration increased from
100 mmol/L to 200 mmol/L, the magnitude of the
increasing of intracellular trehalose concentration was
very limited (Fig. 2).
Intracellular trehalose concentration increased
linearly (r=0.99) with incubation time when incu-
bated with 100 mmol/L trehalose solution. After 24 h
of incubation, the intracellular trehalose concentration
reached (14.57±0.74) mmol/L (Fig. 3). This value
was a little lower than former reports as 19 mmol/L
(Oliver et al., 2004). It may be caused by different cell
conditions. Twenty-four hours and 100 mmol/L were
chosen as the best conditions for trehalose loading.
No significant difference (P>0.05) was observed
between the two growth curves of post-loading and
non-loading cells (Fig. 4).
20 50 80 100 200
C
e (mmol/L)
16
12
8
4
0
C
i (mmol/L)
Fig. 2 Trehalose uptake by hBM-MSCs as a function of
extracellular trehalose concentration
Loading experiments were conducted at 37 °C in the
presence of 20–200 mmol/L trehalose solutions for 24 h.
Data were derived in triplicates. Intracellular trehalose
concentration (C
i) increased with increasing extracellular
trehalose concentration (C
e)
6 12 24
t
i (h)
20
16
12
8
4
0
C
i (mmol/L)
Fig. 3 Uptake of trehalose by hBM-MSCs as a function
of incubation time
hBM-MSCs were incubated in 100 mmol/L trehalose so-
lution for 6, 12, and 24 h. Data were derived in triplicates
and the error bars represent standard deviations. Intracel-
lular trehalose concentration (C
i) increased linearly (r=
0.99) with incubation time (t
i)
1 2 3 4 5 6 7 8
t (d)
4
3
2
1
0
N (×10
4
)
Non-loading
Loading
Fig. 4 Growth curves of trehalose loading and non-
loading hBM-MSCs at passage 3
No significant difference was found between two growth
curves (P>0.05). N: cell number; t: incubation time
Zhang et al. / J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2010 11(11):889-894 893
3.2 Glass transition temperature
The mid-point of the transition was considered
to be the glass transition temperature T
g. The T g of
the freeze-drying suspension was determined to be
−26.6 °C (Fig. 5). During the primary drying process,
the sample temperature should be lower than this
temperature; thus, the shelf temperature was set at
−32 °C.
3.3 Residual water content
After freeze-drying, a dried and sterile sample
was obtained (Fig. 6). The residual water content of
the sample was determined to be 2.9%.
3.4 Cell recovery after rehydration
Freeze-dried samples were rehydrated with four
procedures mentioned above. The recovery rates after
12 h planting for procedures 1, 2, 3, and 4 were
(61.75±15.64)%, (41.17±7.56)%, (48.56±18.10)%,
and (69.33±13.08)%, respectively. Procedures 1 and
4 showed higher viability than procedures 2 and 3
(P<0.05), but there was no statistical differences
between procedures 1 and 4 (P>0.05). The mor-
phologies of recovered cells immediately after rehy-
dration and after 12-h incubation are shown in Fig. 7.
The recovery rate was higher than former reports by
Yang et al. (2005). However, recovered hBM-MSCs
had impaired adhere and proliferation abilities and
died within a week.
Compared to procedures 1 and 4, procedures 2
and 3 tended to have lower concentrations of ex-
tracellular trehalose and more physical disturbance,
which could lead to more susceptibility to physical
and chemical stresses during rehydration. It seemed
that the rehydration solution containing trehalose was
beneficial for cell survival, which might be due to its
higher colloidal osmotic pressure than that of other
solutions without trehalose.
4 Conclusions
In this study, hBM-MSCs undergoing freeze-
drying had a recovery rate ranging from (41.17±
7.56)% to (69.33±13.08)% at 12 h after recovering.
However, the proliferation ability of rehydrated hBM-
MSCs was not shown. Nevertheless, the present study
has laid a foundation for the freeze-drying of hBM-
MSCs. The attractive prospects of freeze-drying
hBM-MSCs prompt us to put more effort into de-
signing better protocols in future studies.
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