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Co-culture of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood using human autoserum.

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ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
Published online 20 September 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI:10.1002/apj.507
Special theme research article
Co-culture of hematopoietic stem cells and mesenchymal
stem cells derived from umbilical cord blood using human
autoserum
Kedong Song,1† Yiqun Yin,1† Chao Lv,1† Tianqing Liu,1 * Hugo M. Macedo,2 Meiyun Fang,3 Fangxin Shi,4 Xuehu Ma1
and Zhanfeng Cui5
1
Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, Dalian 116023, China
Biological Systems Engineering Laboratory, Department of Chemical Engineering, Department of Chemical Engineering, South Kensington Campus,
London SW7 2AZ, UK
3
Department of Hematology, First Affiliated Hospital, Dalian Medical University, Dalian 116011, China
4
Department of Obstetrics and Gynecology, First Affiliated Hospital, Dalian Medical University, Dalian 116011, China
5
Oxford Centre for Tissue Engineering and Bioprocessing, Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, UK
2
Received 31 October 2009; Revised 12 July 2010; Accepted 19 July 2010
ABSTRACT: The feasibility of co-culturing hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stem cells
(MSCs) derived from human umbilical cord blood (UCB) using cytodex-3 microcarriers was investigated in this paper
in order to assess this as a possibility for future clinical therapies. Considering the safety requirements of clinical
applications, we have tried to use human autologous serum (HAS), collected from UCB, to replace the widely used
fetal bovine serum (FBS). Moreover, both UCB-hematopoietic stem cells (HSCs) and UCB-MSCs could be harvested
simultaneously after their ex vivo culture. In addition, the two different types of stem cells could be easily separated by
sedimentation after the co-culture due to the distinct weight differences between cytodex-3 microcarriers (containing
adherent MSCs) and the suspended HSCs. To optimize the culture conditions, we have assessed the effect of the
concentration of HAS (2.8, 5.6, 8.3 and 11.1%) on the expansion of UCB-MSCs and UCB-HSCs. The results have
indicated that the expansion of UCB-HSCs supplied with 5.6% autoserum was at least (1.88 ± 0.33)-fold, better than
in the other groups, while it had no to little effect on the expansion of UCB-MSCs. We hence conclude that the optimal
concentration of HAS for the co-culture conditions herein reported is 5.6%. Finally, the co-culture system we have
developed and herein report is feasible to provide expanded UCB-HSPCs and UCB-MSCs for clinical applications,
especially the former.  2010 Curtin University of Technology and John Wiley & Sons, Ltd.
KEYWORDS: HAS; UCB; HSCs; MSCs; co-culture
INTRODUCTION
Hematopoietic stem cells (HSCs) can be widely used in
the fields of gene therapy, tumor defecation, bone marrow (BM) transplantation, etc. Particularly, hematopoietic stem cell transplantation, which is widely applied
in hematopoietic functional reconstruction for cancer
patients who have undergone radioactive therapy, has
become a routine therapeutic method.[1] However, the
limited cell numbers from one unit of cord blood
(CB) have up to now been confining the HSC clinical
potential.[2,3] An effective way to solve this problem is
by promoting the large-scale ex vivo amplification of
*Correspondence to: Tianqing Liu, Dalian R&D Center for Stem
Cell and Tissue Engineering, Dalian University of Technology,
Dalian 116023, China. E-mail: liutq@dlut.edu.cn.
†
These authors contributed equally to this work.
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
Curtin University is a trademark of Curtin University of Technology
HSCs. However, although such method could indeed
significantly improve the life quality of patients, most
of them would inevitably suffer the pain of severe neutropenia and/or thrombocytopenia. It is thus necessary
to find out a new therapeutic scheme to reduce the
occurrence of such complications appearing after transplantation.
It has been proven that mesenchymal stem cells
(MSCs) have the ability to support hematogenesis
in vitro. Reconstruction experiments conducted in
NOD/SCID mice have also confirmed that transplanting
HSCs together with MSCs can accelerate the restoration of hematopoietic functions.[4] Transplanting autogeneic MSCs together with HSCs has also facilitated
the restoration of hematogenesis and decreased the incidence of complications arising from transplantation in
Asia-Pacific Journal of Chemical Engineering
patients with breast cancer who have been treated using
radiotherapy.
As a source of HSCs, umbilical cord blood (UCB)
offers many advantages when compared to other
alternatives such as the BM and peripheral blood
(PB).[5] UCB contains mesenchymal tissue stem cells,
and more primitive and abundant HSCs with poorer
immunogenicity, fewer antibodies against heterogenetic
antigens, less numbers of mature T-cells and a much
easier collection and conservation protocols. On the
other hand, it also contains a higher proportion of
CD34+ CD38− and CD34+ CD33− cellular populations,
lower chances for the occurrence of graft-versus-host
disease, more extensive resources, etc. All these factors term the fields relating to umbilical CB a hot spot.
Cultivating and harvesting both HSCs and MSCs at the
same time in a culture system could have a significant
impact on UCB clinical practices, which could then be
stored in CB banks.
Very few studies focusing on the co-culture of HSCs
and MSCs isolated from CB have been reported. Combining microcarriers with bioreactors, Fan et al . investigated the co-cultivation of UCB-HSCs and UCBMSCs using spinner flasks and a rotating wall vessel bioreactor (RWVB) in the absence of serum, by
adding an exogenous cytokine cocktail (composed of
SCF 15 ng mL−1 , FL 5 ng mL−1 , TPO 6 ng mL−1 ,
IL-3 15 ng mL−1 , G-CSF 1 ng mL−1 and GM-CSF
5 ng mL−1 ) and encapsulating the supporting stroma
cells within calcium alginate (CA) beads.[6] The results
have shown that the best amplification took place in the
RWVB, with the following outcome: after 12 days of
culture, the number of nucleated cells (NCs) was amplified by (3.7 ± 0.3)-fold, CFU-Cs by (5.1 ± 1.2)-fold,
CD34+ CD45+ CD105− (HSCs) by (5.2 ± 0.4)-fold and
CD34− CD45− CD105+ (MSCs) by (13.9 ± 1.2)-fold.
On the other hand, research on the co-culture of bone
marrow-derived HSCs and MSCs is still at a very
early stage. Chen et al . managed to successfully expand
BM-HSCs and BM-MSCs by 8- and 29-fold, respectively, by applying high doses of cytokine combinations (SCF 50 ng mL−1 , IL-3 10 ng mL−1 and IL-6
10 ng mL−1 ) in the RWVB.[7] However, Chen et al .
have followed a suspension co-culture protocol of HSCs
and MSCs, leading to difficult harvesting and separation procedures at the end of the culture period.
Moreover, up to our knowledge there has not been
reported until now any research on the co-culture of
HSCs and MSCs using human autologous CB serum.
HSCs and MSCs have suspended and adhesive growth
biological characteristics, for which both suspension
and dynamic adhesive surfaces should be provided
in vitro to simulate the in vivo microenvironment. The
strategy of combining the adhesion surface properties of microcarriers to a suitable automated bioreactor
with volume space for cell growth would be a feasible solution in the view of the authors. Sephadex
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
CO-CULTURE OF HEMATOPOIETIC STEM CELLS
(cytodex-3) microcarriers have been drawing a great
attention particularly in the three-dimensional dynamic
culture of adherent cells due to their favorable surface
adherence.
In addition, compared with the universally used
FBS, human autologous serum (HAS) isolated from
CB could provide a better nutritive source for cells,
while avoiding immune reactions and eliminating the
risk of cross-species contaminations that are a common
concern in the use of bovine or other animal-derived
serums for clinical applications.
In this research, the co-culture of UCB-HSPCs and
UCB-MSCs was carried out under serum-free condition
integrating microcarrier and bioreactor technologies and
using HAS in replacement of FBS. The cultured UCBHSPCs and UCB-MSCs were easily separated after their
ex vivo co-culture. Finally, to optimize the use of HAS,
different concentrations (2.8, 5.6, 8.3 and 11.1%) were
used and their influence toward the expansion of both
cell types was studied, while different concentrations of
FBS (5, 10, 15 and 20%) were used as control groups.
EXPERIMENTAL
Isolation of UCB-MNCs
UCB samples were obtained from normal full-term
deliveries after informed consent from parents. MNCs
were isolated from UCB by the Ficoll-Hypaque method
as described before.[8] Briefly, 4 mL of Ficoll
(1.077 g mL−1 ) was added into a 15-mL sterile plastic centrifuge tube. CB was diluted by same volume
of Ca+ - and Mg+ -free phosphate-buffered saline (PBS)
before gently dropping onto the surface of Ficoll within
the tube (using a proportion of 1 : 1–2 between the
Ficoll and UCB volumes). UCB-MNCs were isolated
by density gradient centrifugation over Ficoll and centrifuged horizontally at 2500 rpm for 25 min at room
temperature. Then, the MNCs at the interface were collected and washed twice with PBS, centrifuged twice
at 1000 rpm for 5 min and finally resuspended in
Iscove’s modified Dulbecco’s medium (IMDM, Gibco,
USA).
Isolation of human autologous serum from
cord blood
HAS was isolated from the CB samples by the FicollHypaque method as described before.[8] A sterile plastic centrifuge tube of 15 mL containing Ficoll and the
diluted CB from the previous step were centrifuged horizontally at 2500 rpm for 25 min at room temperature.
The top supernatant, termed HAS, was collected and
added directly to the culture medium for later use in
the co-culture.
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
841
842
K. SONG et al.
Pretreatment of cytodex-3 microcarriers
The desiccated cytodex-3 microcarriers were immersed
in Ca+ - and Mg+ -free PBS (50–100 mL g−1 ) overnight
in a siliconizing glass container at 37 ◦ C, and the
mixture was occasionally stirred. After discarding the
supernatant, the microcarriers were washed twice with
Ca+ - and Mg+ -free PBS, then refilled with 10 mL of
PBS and finally steam-sterilized at 115 ◦ C for 15 min
for use in the subsequent experiments.
Cellular counting
The cellular densities and cell numbers of freshly isolated and cultured cells were assessed using a hemacytometer.
Static culture
This experiment required seven cord blood units collected from full-term delivery newborns, from which
HAS and MNCs were separated at the same time.
Six of these samples were used to perform the coculture protocols described as follows. For the HAS
co-culture, freshly isolated UCB-MNCs were seeded
into a 24-well plate pre-covered with cytodex-3 microcarriers at a cellular density of 2 × 106 cells mL−1 ,
supplemented with 20% HAS and the plates were then
incubated under a 100% humidified atmosphere composed of 5% CO2 and 20% O2 at 37 ◦ C for 12 days.
The plate was observed under an inverted microscope
and the cultured cells counted every 24 h of culture. For the FBS co-culture system (control group),
freshly isolated UCB-MNCs were treated similarly as
for the HAS culture but medium was supplemented with
20% FBS.
The last cord blood unit was operated as follows:
freshly isolated UCB-MNCs were seeded into a 96well plate pre-covered with cytodex-3 microcarriers at
a density of 2 × 106 cells mL−1 , and HAS was added
at different concentrations to the medium (5, 10, 15
and 20%). A control group for each concentration of
HAS used, in which the same amount of FBS was
added to the media (5, 10, 15 and 20%) instead of
HAS, was also prepared. Here, we noted that this HAS
(concentrations used above) had been diluted by PBS
and anticoagulant agent at a certain percentage after
the Ficoll-Hypaque separation process, we therefore
converted those diluted concentrations to the actual
concentrations in the later applications, i.e. 2.8, 5.6,
8.3 and 11.1%. For each experimental group, ten wells
were used and supplemented with 100 µL of media per
well.
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pacific Journal of Chemical Engineering
Flow cytometry analysis of the CD34+ cellular
population
The percentage of CD34+ cells in the isolated and
cultured MNCs was determined by flow cytometry. Approximately, 1.0 × 106 of freshly separated or
expanded hematopoietic cells were washed once in
PBS containing 1% FBS, and afterwards resuspended
in 100 µL of this solution and stained with 10 µL
of anti-CD34-PE or 10 µL of anti-CD34-FITC (BD
Pharmingen, USA). These were then incubated at 4 ◦ C
in the dark for 20 min, washed again with PBS, resuspended in 1 mL of PBS containing 0.1% sodium azide
and finally assayed with an FACSCalibur and analyzed
using CellQuest software (BD, USA).
Measurement of cellular viability using CCK-8
This experimental work has also included the use of a
Cell Counting Kit-8 (CCK-8) kit to examine the activity
of UCB-MSCs on the microcarriers. Suspension cells
from random wells of the 96-well plate used were gently
aspirated everyday, then the microcarriers were washed
and the supernatant discarded after the microcarriers had
sedimentated. This procedure was repeated for two or
three times. Afterwards, each treated well was refilled
with 100 µL of IMDM and added 10 µL of CCK-8,
followed by an incubation period for 3 h at 37 ◦ C. After
discarding the supernatant, the optical density (OD) of
MSCs on the microcarriers was assayed through an
enzyme-labeling instrument tool.
Multilineage differentiation potential of
UCB-MSCs
MSCs cultured for 6 days on the cytodex-3 microcarriers were harvested for multilineage differentiation analysis. The harvested cells were plated in 24-well plates
at a density of 5 × 104 cells mL−1 (1 mL per well),
and cultured in IMDM containing 10% FBS for 3 days.
After this, inducing medium for the osteogenic and adipogenic lineages was added to each well according to
the experimental design, and the media changed every
3 days over a period of 3 weeks of culture. The expression of alkaline phosphatase (ALP) was observed in
the early osteogenic induction after 1 week, while calcified nodules were observed by Von-Kossa staining
after 2 weeks of culture. Moreover, intracellular lipid
droplets could also be observed through Oil-Red staining after 2 weeks of culture.[9 – 11]
Osteogenic-inducing medium consisted of IMDM
containing 10% FBS, 50 µM ascorbate (Sigma), 10 mM
β-glycerophosphate (Sigma) and 0.1 µM dexamethasone (Sigma). Medium for adipogenesis was composed
of IMDM containing 10% FBS, 1 µM dexamethasone
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
CO-CULTURE OF HEMATOPOIETIC STEM CELLS
(Sigma), 0.1 mM 3-isobutyl-1-methylxanthine (IBMX)
(Wako), 10 µM insulin and 0.2 µM indomethacin.
Statistical analysis
All experiments were performed in triplicate. Results
are expressed as the mean ± SD. The statistical significance of differences within each group was evaluated by
the t-test analysis using the software Origin7.0 (OriginLab Corporation, USA).
RESULTS
Achievement ratio of UCB-MSCs primary
culture
UCB-MSCs collected from five different CB units
were successfully cultivated, with a recovering ratio
of up to 71.4% in the HAS group, while for the
FBS group it was also possible to harvest UCB-MSCs
from their corresponding five samples. However, it
was not possible to recover UCB-MSCs from the
remaining groups, one of which being handled after
24 h of collection of the CB unit. The quality of
the MNCs could have been compromised during this
period.
Taking into account the efficiency of the cellular
attachment onto the microcarriers and the growth behavior of the cells, HAS isolated from the CB units could
satisfactorily sustain the ex vivo expansion of both
UCB-HSCs and UCB-MSCs, as shown in Fig. 1A. The
adhesion status and growth behavior of the UCB-MSCs
on the microcarriers of cytodex-3 were found to be satisfactory. From Fig. 1B, it is possible to observe that the
cultures treated with 11.1% HAS had a similar outcome
as those treated with 20% FBS, showing that 11.1%
HAS from CB could replace the use of 20% FBS, while
still promoting adhesion and a high growing rate of the
UCB-MSCs on the cytodex-3 microcarriers throughout
the culture process.
CD34+ flow cytometry analysis of primary
MNCs
Flow cytometry analysis of the freshly isolated MNCs
in terms of their CD34+ phenotype is shown in Fig. 2.
The negative control group, without the addition of
anti-CD34 antibody, had a positive ratio of P 2 =
0.4%, whereas that of test group containing anti-CD34
antibody was P 2 = 1.1%. The difference between the
CD34-positive ratios from both groups was regarded as
the contents in CD34+ cells (UCB-HSCs) from fresh
MNCs, which was about 0.7%. In this experiment, it
was possible to harvest a total of 5.28 × 107 MNCs in
the primary isolation, of which around 3.7 × 105 cells
were UCB-HSCs.
Adhesion of UCB-MSCs to cytodex-3
microcarriers
Figure 3 shows the morphology of the adhesive UCBMSCs on the cytodex-3 microcarriers after 10 days of
culture. Figure 3A–D represents the results obtained
for the groups treated with different concentrations of
HAS: 2.8, 5.6, 8.3 and 11.1%, respectively, whereas
8
B
C
Density of NCs (X106)
A
During the culture of the first six samples, the
cellular population was examined every 24 h through
a hemacytometer, whose results showing the change
in the cellular density in the cultures are plotted in
Fig. 1C. It can be seen that the change profile of the
cultures treated with a concentration of 11.1% HAS
was similar to that of the group containing 20% FBS.
The HAS group reached its peak value after 120 h of
culture at a density of 4.5 × 106 cells mL−1 , whereas
the group treated with 20% FBS reached its maximum
at 3.5 × 106 cells mL−1 after 72 h of culture, both of
which being followed by a 48-h platform stage. No
statistically significant differences were found between
both groups (P < 0.05).
7
11.1% HAS
20% FBS
6
5
4
3
2
1
0
24
48
72
96
120
144
Culture time (h)
Figure 1. UCB-MSCs adhered to microcarriers cultured with autologous serum (A) or FBS (B) and change of the cell
density (C) during the culture process. This figure is available in colour online at www.apjChemEng.com.
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
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844
K. SONG et al.
Asia-Pacific Journal of Chemical Engineering
Figure 2. Flow cytometric analysis of UCB-derived HSCs [control group (CG) and experimental group (EG)]. This figure is
available in colour online at www.apjChemEng.com.
Figure 3. UCB-MSCs adhered to microcarriers (A, autoserum 2.8%; B, autoserum 5.6%;
C, autoserum 8.3%; D, autoserum 11.1%; E, FBS 5%; F, FBS 10%; G, FBS 15%; H, FBS
20%). This figure is available in colour online at www.apjChemEng.com.
Fig. 3E–H represents the FBS group, whose serum percentages in the media were 5, 10, 15 and 20%, respectively. It was found that UCB-MSCs adhered to almost
all the cytodex-3 microcarriers with excellent spreading
status in eight experimental groups, but the cell number on the microcarriers from these experimental groups
was different, and the number increased along with the
enhancement of serum concentration regardless of HAS
or FBS used.
Expansion of total MNCs
Under different concentrations of HAS or FBS and
refilling 10 µL of fresh medium every other day, the
changes in the cellular density of the suspension cultures
in the 96-well plate culture system are shown in Fig. 4A
(HAS group) and 4B (FBS group). The effects of different HAS concentrations in the culture media toward the
cellular density were not significant. It was shown, however, that it increased slightly with increasing concentrations of HAS. All groups treated with HAS reached
their peak value after 4 days of culture, and were
2.9 (±0.1), 3.17 (±0.25), 3.47 (±0.4) and 3.9 (±0.1)
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
×106 cells mL−1 for the concentrations of 2.8, 5.6, 8.3
and 11.1%, respectively, corresponding to expansions
of (1.45 ± 0.05)-fold, (1.58 ± 0.13)-fold, (1.73 ± 0.2)fold and (1.95 ± 0.05)-fold, respectively (Fig. 4C). On
the other hand, the influence of different concentrations of FBS toward the change of the density of
NCs was also not significant. The peak values reached
after 4 days of culture, as shown in Fig. 4B, were 3.6
(±0.53), 4.0 (±0.25), 3.7 (±0.17) and 3.97 (±0.2)
×106 cells mL−1 for the 5, 10, 15 and 20% groups,
respectively, with corresponding proliferation rates of
(1.8 ± 0.26)-fold, (2.0 ± 0.13)-fold, (1.85 ± 0.08)-fold
and (1.98 ± 0.1)-fold, respectively (Fig. 4D). The best
result outcome and the highest cell density were
observed on the group treated with 10% FBS, a different
behavior from that observed in the HAS-treated groups.
Figure 5 shows the comparison between the best
culture conditions using HAS and FBS, i.e. 11.1%
HAS and 10% FBS, respectively. The cellular density
obtained in the group treated with 10% FBS was slightly
higher than that in the 11.1% HAS group, although
with no statistically significant difference between them
(P < 0.05).
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
HAS 2.8%
HAS 5.6%
HAS 8.3%
HAS 11.1%
4.8
4.0
3.2
2.4
1.6
4.8
B
4.0
3.2
2.4
1.6
0
1
2
3
4
5
6
Time (day)
7
8
9
0
3.0
1
2
3
4
5
6
Time (day)
7
8
9
3.0
HAS 2.8%
HAS 5.6%
HAS 8.3%
HAS 11.1%
2.5
2.0
EXpansion fold of NCs
EXpansion fold of NCs
FBS 5%
FBS 10%
FBS 15%
FBS 20%
5.6
A
The density of NCs
(×106 cell/ml)
5.6
Density of NCs
(×106 cell/ml)
CO-CULTURE OF HEMATOPOIETIC STEM CELLS
1.5
1.0
0.5
FBS 5%
FBS 10%
FBS 15%
FBS 20%
2.5
2.0
1.5
1.0
0.5
0.0
0.0
0
1
2
3
4
5
6
Time (day)
7
9
8
0
1
2
3
4
5
6
Time (day)
7
8
9
Figure 4. Change of the cell density of NCs cultured with autologous serum (A) or FBS (B) and expansion
fold of NCs cultured with autologous serum (C) or FBS (D). This figure is available in colour online at
www.apjChemEng.com.
FBS 10%
HAS 11.1%
Density of NCs (×106)
5.6
4.8
4.0
3.2
2.4
1.6
0
1
2
3
4
5
6
7
8
9
Time (day)
Figure 5. Change of the cell density of MNCs. This figure
is available in colour online at www.apjChemEng.com.
Evaluation of the expansion of the CD34+
population (UCB-HSCs)
Flow cytometry analysis was performed on the freshly
isolated cells before culture and then on the suspension
cells collected after 3 and 6 days of culture in order to
characterize their CD34+ phenotype (HSCs). The best
expansion outcome of UCB-HSCs was observed in the
group treated with 5.6% HAS, as shown in Fig. 6A,
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
where the contents in CD34+ cells (HSCs) gradually
increased with time, achieving an expansion of around
(1.88 ± 0.33)-fold when compared to day 0. However,
the percentage of CD34+ cells in the cultured cells was
lower than that in the primary cells for other groups.
Figure 6B shows that the group with the best results in
terms of FBS was the one treated with a concentration
of 10%, although its contents in CD34+ cells initially
increased with time, but afterwards started to decrease,
achieving a final expansion of (4.48 ± 0.9)-fold when
compared to day 0.
Figure 6C compares the CD34+ population expansion between the groups treated with 5.6% HAS and
10% FBS. From the diagram, it is possible to observe
that the effects of a concentration of 10% FBS in the
culture media toward the ex vivo culture of CB MNCs
are slightly better than those for a concentration of
11.1% HAS.
Activity analysis of UCB-MSCs on the
microcarriers
Figure 7 shows the variation of the activity of UCBMSCs on the microcarriers under different serum concentrations. Figure 7A represents the results obtained
for the groups treated with HAS, while Fig. 7B for
the FBS-treated groups. It is clear that there were no
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
845
Asia-Pacific Journal of Chemical Engineering
5
4
A
HAS 2.8%
HAS 5.6%
HAS 8.3%
HAS 11.1%
3
2
1
0
0
6
FBS
FBS
FBS
FBS
5
4
B
5%
10%
15%
20%
3
2
1
0
6
3
Time (day)
Expansion fold of CD34+ (HSCs)
6
Expansion fold of CD34+ (HSCs)
K. SONG et al.
Expansion fold of CD34+ (HSCs)
0
3
Time (day)
6
6
5
C
**
FBS 10%
HAS 5.6%
4
3
2
1
0
0
3
Time (day)
6
Figure 6. Expansions of UCB-derived HSCs cultured with autologous serum (A) or FBS (B) and expansions of UCB-derived
HSCs (C). This figure is available in colour online at www.apjChemEng.com.
1.0
HAS
HAS
HAS
HAS
0.8
1.0
A
2.8%
5.6%
8.3%
11.1%
B
FBS 5%
FBS 10%
FBS 15%
FBS 20%
0.8
0.6
0.6
OD
OD
846
0.4
0.4
0.2
0.2
0.0
1
2
3
4
5
6
7
Time (day)
8
9
10
1
2
3
4
5
6
7
Time (day)
8
9
10
Figure 7. OD value of UCB-MSCs adhered to microcarriers cultured with autologous serum (A) or
FBS (B). This figure is available in colour online at www.apjChemEng.com.
statistically significant differences in terms of the optical density (OD) values for the different HAS groups,
although the 11.1% HAS group behaved slightly better
than the other groups (P < 0.05). It was also observed
that the OD value initially increased and then declined
with time. The value reached its peak after 3 days of
culture in the 11.1% HAS group, while the other groups
reached their maximum value at day 4. On the other
hand, and as shown in Fig. 7B, the OD was not significantly different amongst the different groups treated
with FBS. The 20% group behaved slightly better than
the others, although with no statistically significant difference (P < 0.05). The OD value of the 10% group initially increased and afterwards declined, having reached
its peak (0.5 ± 0.01) at the end of 4 days of culture. The
OD of the other FBS groups has also initially increased
but then started to slightly decline from day 5, reaching
their maximum peak at day 7. The OD values obtained
after the culture period were 0.48 ± 0.04, 0.53 ± 0.07
and 0.6 ± 0.1, for the 5, 15 and 20% concentration of
FBS, respectively.
Figure 8A shows the comparison of the OD values
between the 11.1% HAS group and the 20% FBS group.
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
The OD obtained for the FBS group was just slightly
higher than that for the HAS group. Performing a ttest of the two groups in terms of their OD values,
no statistically significant differences were found (P <
0.05), as shown in Fig. 8B.
Multipotential differentiation of MSCs on the
cytodex-3 microcarriers
UCB-MSCs were shown to multipotentially differentiate into the adipose, bone and cartilage lineages. To
induce the differentiation toward the cartilage lineage,
it is required a large number of mesenchymal-like
cells that surpassed the number of harvested UCBMSCs in this experiment, thus such inducing differentiation experiment could not be performed. Through
this analysis, it was observed that the multipotential
differentiation of the cultured MSCs attached to the
cytodex-3 microcarriers in culture systems with different serum concentrations had no statistically significant
differences between the HAS- and FBS-treated groups
(P < 0.05).
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
CO-CULTURE OF HEMATOPOIETIC STEM CELLS
1.0
0.8
N.S.
A
HAS 11.1%
FBS 20%
B
0.6
OD
OD
0.6
0.4
0.4
0.2
0.2
0.0
0.0
1
2
3
4
5 6 7
Time (day)
8
9
10
HAS 11.1%
FBS 20%
Figure 8. Comparison of OD value of UCB-MSCs adhered to microcarriers (A, B). This
figure is available in colour online at www.apjChemEng.com.
Figure 9. ALP staining for osteogenic differentiation (A, autoserum 2.8%; B, autoserum
5.6%; C, autoserum 8.3%; D, autoserum 11.1%; E, FBS 5%; F, FBS 10%; G, FBS 15%; H,
FBS 20%). This figure is available in colour online at www.apjChemEng.com.
After 1 week of osteogenic induction, staining with
ALP has allowed the identification of pitchy particles in
the cytoplasm of the cells (Fig. 9). For the adipogenic
differentiation, potential assessment Oil-Red staining
was performed after 2 weeks of culture, although without any culture systems showing lipid droplets. It is
worth to note that it is not universally accepted that
UCB-MSCs have the potential to differentiate into adipose cells. Kern et al . compared the differentiation
potential of mesenchymal stem cells obtained from different sources (BM, CB and grease source) toward the
osteogenic, chondrogenic and adipogenic lineages, having found that all 11 samples of cord blood-derived mesenchymal stem cells did not show any evidence of adipogenic potential.[12] On the other hand, Change et al .
have also considered that the UCB-MSCs were not
sensitive enough to differentiate into adipose cells.[13]
Kern et al . believed that the conversion of mesenchymal tissue stem cells toward the adipogenic lineage
depended on the aging level of the stem cells, as
adipose cells exist in adult BMs and adipose tissue
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
while not found in infant BMs. Moreover, Moerman
et al . found that with the aging process of mesenchymal tissue stem cells, the potency toward osteogenesis
declined while that toward the adipogenic lineage was
enhanced.[14] In conclusion, the lower adipose induction potential of the UCB-MSCs used in this study
can be explained by their more primitive origin than
their BM or any other adult tissue source counterpart.
After 3 weeks of ossification induction, Von-Kossa
staining has shown that mineralized nodules and black
calcified stroma were formed in the cellular cytoplasm,
which stained for AgNO3 (Fig. 10). Although the black
nodules on the microcarriers were not very clear due to
the very low contents in terms of primary UCB-MSCs,
the stained calcified stroma can still be observed.
DISCUSSION
This investigation focused mainly on the development
and optimization of a protocol to substitute traditional
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
847
848
K. SONG et al.
Asia-Pacific Journal of Chemical Engineering
Figure 10. Von-Kossa staining for osteogenic differentiation (A, autoserum 2.8%; B,
autoserum 5.6%; C, autoserum 8.3%; D, autoserum 11.1%; E, FBS5%; F, FBS 10%; G,
FBS 15%; H, FBS 20%). This figure is available in colour online at www.apjChemEng.com.
FBS with HAS to promote the in vitro co-culture of
UCB-MSCs and UCB-HSCs. It was possible to successfully harvest UCB-MSCs from five out of seven CB
samples. The adhesive morphology of UCB-MSCs on
cytodex-3 microcarriers was typically spindle shaped,
and the achievement ratio of primary MNCs was 71.4%,
which is consistent with the 71.2% obtained by Zhang
et al .[15]
This experiment furthermore inspected the influence
of either HAS or FBS at different concentrations in
the cell culture media toward the cell growth and
adhesiveness of primary UCB-MSCs to the cytodex3 microcarriers, and the fold expansion of UCB-HSCs
through flow cytometry and cellular quantification analysis. The results showed that UCB-HSCs and UCBMSCs obtained from the same CB unit could be successfully cultured and simultaneously harvested in a
serum-only co-culture system using microcarriers. On
the other hand, it was also shown that CB-HAS could
efficiently replace traditional FBS by promoting similar expansion results at very low concentrations. On
the basis of this data, it was clear that different serum
concentrations had a slight influence toward the expansion of total NCs. However, the best results were
obtained for a concentration of 11.1% HAS, while the
optimal concentration of FBS was found to be 10%,
with no statistically significant differences between both
groups (P > 0.05). Moreover, through the analysis by
flow cytometry it was possible to observe that a concentration of 5.6% HAS promoted the best expansion
of UCB-HSCs, while 10% FBS was the best in the
control groups. The CCK-8 analysis has also shown
that there were no statistically significant differences
between these two groups in terms of their expansion
potential, although the fold expansion of UCB-HSCs
was slightly higher for increasing serum concentrations,
i.e. the concentration of blood serum was shown to have
a low impact on the expansion results, confirming that
the relatively low concentration of 5.6% CB-HAS was
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
an optimal choice for the in vitro co-culture of UCBHSCs and UCB-MSCs (considering the availability of
HAS).
The optical density change of NCs in suspension has
indicated that the expansion of NCs was not very high,
with the group treated with a concentration of 11.1%
HAS expanding by (1.95 ± 0.05)-fold, while the 10%
FBS group achieving (2.0 ± 0.13)-fold expansion. The
following two factors could be in the origin of this
observation:
1. A high inoculating cell density: the number of
mesenchymal stem cells in the harvested UCBMNCs was quite low, of around from 0.05 to
2.8 × 106 MNCs. Hence, it was required to use a
high cell seeding density (2 × 106 cells mL−1 ), so
there could be enough UCB-MSCs adhering onto the
microcarriers and that way guaranteeing the success
of co-culture.
Owing to space limitations, it was difficult to promote a high expansion of the NCs in vitro at such a
high cell seeding density. Certain experimental groups
were treated with a medium-diluted method, hence setting their maximum cell density at 1.5 × 106 cells mL−1
to further promote the expansion of HSCs. To simultaneously harvest UCB-MSCs and UCB-HSCs after the
primary culture, a high NC seeding density was found to
be a better choice; however, as discussed this compromises the expansion rates. Subsequent works, intending
to study the influence of these conditions toward the
optimization of cellular expansion, could in fact use a
lower cell seeding density in the following serial subcultivation.
2. Culture system with lack of space for cellular
growth: the volume of the 96-well plate culture system was limited to about 100 µL, an important factor
that has indeed restricted and maybe influenced the
expansion potential of the cells. Moreover, other disadvantages, including the presence of concentration
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
Asia-Pacific Journal of Chemical Engineering
gradients and the accumulation of metabolic products due to the batch conditions used, could have
also restricted the outcome of this experiment.
Although the expansion results of UCB-MNCs and
UCB-HSCs were not very satisfying, requiring further
investigation, it was shown in this work that CB-HAS
can sustain the ex vivo co-culture of UCB-MSCs and
UCB-HSCs and be a feasible alternative to animalderived serums, ensuring the safety requirements for
translating these stem cell techniques into clinical
practices. Following to the present work, the co-culture
of UCB-MSCs and UCB-HSCs within spinner flasks
and/or RWVBs with three-dimensional dynamic culture
microenvironments should be further investigated.
CONCLUSIONS
In this paper, we have shown that HAS obtained through
density gradient centrifugation could effectively replace
animal-derived FBS by providing nutrients and other
biological molecules for the ex vivo co-culture of UCBHSCs and UCB-MSCs. The successfully recovering
rate of primary UCB-MSCs could be promoted to
around 71.4%, and sustained adhesive cell growth
onto cytodex-3 microcarriers. Moreover, in the 96well plate serum-only culture system, UCB-MSCs and
UCB-MSCs could be simultaneously cultivated and
harvested, with the best dosage of autologous serum
found to be 5.6% in the supplied media.
CO-CULTURE OF HEMATOPOIETIC STEM CELLS
new teacher foundation of Ministry of Education
(20070141055). Mr Hugo Macedo is also grateful to
the Portuguese Fundação para a Ciência e Tecnologia
for his PhD grant number SFRH/BD/28138/2006.
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Acknowledgements
This work was supported by the National Science
Foundation of China (30670525, 30700181) and the
 2010 Curtin University of Technology and John Wiley & Sons, Ltd.
Asia-Pac. J. Chem. Eng. 2011; 6: 840–849
DOI: 10.1002/apj
849
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