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Journal of Cellular Biochemistry 117:815827 (2016)

Freezing of Fresh Wharton0s Jelly From Human Umbilical

Cords Yields High Post-Thaw Mesenchymal Stem Cell
Numbers for Cell-Based Therapies
Chui-Yee Fong, Arjunan Subramanian, Arijit Biswas, and Ariff Bongso*
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System,
National University of Singapore, Kent Ridge 119228, Singapore

ABSTRACT
Some cord blood banks freeze entire pieces of UC (mixed cord, MC) which after post-thaw yields mixed heterogeneous populations of
mesenchymal stem cells (MSCs) from all its microanatomical compartments. Freezing of such entire tissues results in sub-optimal post-thaw
cell recovery because of poor cryoprotectant diffusion and intracellular ice-formation, heat and water transport issues, and damage to
intercellular junctions. To develop a simple method of harvesting pure homogeneous MSCs for cord blood banks, we compared the post-thaw
behavior of three groups of frozen UC tissues: (i) freshly harvested WJ without cell separation; (ii) MSCs isolated from WJ (WJSC); and (iii) MC,
WJ, and WJSC produced high post-thaw cell survival rates (93.52  6.12% to 90.83  4.51%) and epithelioid monolayers within 24 h in
primary culture whereas post-thaw MC explants showed slow growth with mixed epithelioid and broblastic cell outgrowths after several
days. Viability and proliferation rates of post-thawed WJ and hWJSC were signicantly greater than MC. Post-thaw WJ and WJSC produced
signicantly greater CD24 and CD108 uorescence intensities and signicantly lower CD40contaminants. Post-thaw WJ and WJSC
produced signicantly lesser annexin-V-positive and sub-G1 cells and greater degrees of osteogenic and chondrogenic differentiation
compared to MC. qRT-PCR analysis of post-thaw MC showed signicant decreases in anti-apoptotic gene expression (SURVIVIN, BCL2) and
increases in pro-apoptotic (BAX) and cell cycle regulator genes (P53, P21, ROCK 1) compared to WJ and WJSC. We conclude that freezing of
fresh WJ is a simple and reliable method of generating large numbers of clinically utilizable MSCs for cell-based therapies. J. Cell. Biochem.
117: 815827, 2016. 2015 Wiley Periodicals, Inc.

KEY WORDS:

CHARACTERIZATION; CRYOPRESERVATION; HUMAN UMBILICAL CORD; WHARTONS JELLY; WHARTONS JELLY STEM CELLS; MIXED

CORD; FREEZE-THAW CELL SURVIVAL RATES

he bone marrow and adult organs have been the conventional

sources of mesenchymal stem cells (MSCs) for cell-based
therapies because they can be harvested from the same patient with
no immunorejection [Pittenger et al., 1999; Campagnoli et al., 2001;
Bara et al., 2014; Jung et al., 2015]. However, they have the
disadvantages of painful invasive harvest with risk of infection and
donor morbidity, inadequate cell numbers, and short-lived stemness
properties both in vitro and as patients grow older [Bongso and Fong,
2013].
Human umbilical cord blood (UCB) from the umbilical blood
vessels is an alternative source of MSCs but their existence has been

controversial. Although some reports have shown the presence of

MSCs in human UCB [Musina et al., 2007; Secco et al., 2008], others
have reported that the typical features of MSCs in UCB were their low
counts per volume and low proliferation rates [Lee et al., 2004]. Some
groups reported that they could not isolate MSCs from UCB
[Mareschi et al., 2001] while others [Wexler et al., 2003] claimed that
cord and peripheral adult blood were not rich sources of MSCs.
Several groups have isolated MSC populations from the individual
microanatomical tissue compartments of the human UC such as the
perivascular region [Sarugaser et al., 2005, 2009; Farias et al., 2011],
Wharton0 s jelly (WJ) [Fong et al., 2007; Troyer and Weiss, 2008;

The authors declare that they have no conict of interests.

Chui-Yee Fong and Arjunan Subramanian are co-rst authors.
Grant sponsor: Singapore National University Health System (NUHS); Grant sponsor: Aspiration Fund (New Idea);
Grant number: R-174-000-155-720; Grant sponsor: Aspiration Fund (Partner); Grant number: R-174-000-156-720.
*Correspondence to: Dr. Ariff Bongso, Research Professor, Department of Obstetrics and Gynaecology, Yong Loo Lin
School of Medicine, National University Health System, NUHS Tower Block, Level 12, 1E, Lower Kent Ridge Road,
Singapore 119228.
E-mail: obgbongs@nus.edu.sg
Manuscript Received: 9 September 2015; Manuscript Accepted: 9 September 2015
Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 14 September 2015
DOI 10.1002/jcb.25375  2015 Wiley Periodicals, Inc.

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Yang and Chao, 2013], subamnion [Gonzalez et al., 2010; Kita et al.,
2010; Reza et al., 2011], and amnion [Illancheran et al., 2007; Mihu
et al., 2009; Diaz-Prado et al., 2011]. In a comprehensive review
Conconi et al. [2011] reported that many studies showed that the
phenotypic, characterization, and differentiation proles of the
MSCs derived from these various compartments were different. We
conrmed these ndings in a recent study [Subramanian et al.,
2015]. However, some groups grow entire UC pieces containing all
the compartments as explants in culture and harvest mixed
heterogeneous MSC populations from the peripheral cell outgrowths
of the explants (mixed cord, MC) for their studies [Lu et al., 2006;
Pereira et al., 2008; Qiao et al., 2008; Secco et al., 2009; Capelli et al.,
2011; Bosch et al., 2012; Zhang et al., 2012b]. The generation of
MSCs from entire UC pieces is time-consuming as the cell
outgrowths take time to become conuent in primary culture. Serial
passaging is required to generate sufcient cell numbers from MC
explant cultures [Subramanian et al., 2015]. It is well known that
prolonged manipulation of cells in culture results in unstable
epigenetic chromosomal changes that make the cells unsuitable for
clinical application [Ben-David and Benvenisty, 2011]. It was also
reported that MSCs derived from entire UC pieces were incapable of
osteogenic differentiation even after exposure to potent osteoinductive substances such as 1.25-dihydroxy-vitamin D3 [Majore
et al., 2011] unlike MSCs derived from the WJ [Subramanian et al.,
2015]. In spite of such disadvantages of MC cultures some cord blood
banks freeze entire UC segments for later post-thaw recovery of
MSCs perhaps because it is less time-consuming and easy. Freezing
of entire UC segments has the disadvantage that the cryoprotectant is
unable to penetrate and preserve the interior parts of the frozen
tissue resulting in necrosis and release of toxins that may induce
genetic and behavioral changes of the cells [Dhanasekaran and
Reddy, 2008; Bueno et al., 2010; Djuwantono et al., 2011;
Chatzistamatiou et al., 2014]. Thus overall, MSCs derived from
fresh and frozen MC cultures may not be ideal for cell-based
therapies because of their heterogeneity, potential genomic alterations after prolonged manipulation in culture and competition in
differentiation along a desired lineage as the cells originate from the
different compartments of the UC.
The WJ compartment was shown to be the most attractive source
of clinically utilizable MSCs [Subramanian et al., 2015]. The
gelatinous matrix of the WJ is clearly visible and loosely attached
to the inner walls and umbilical blood vessels of the UC when it is cut
open making its separation easy without contamination by cells
from the other parts of the UC. The WJ contains in its matrix a large
number of individually buried stellate-shaped cells with stemness
properties (4.6  106 cells/cm of UC) [Fong et al., 2010].
These human WJ stem cells (WJSC) possess high proliferation
rates with short population doubling times, long-lasting stemness
properties in vitro, are clonogenic, can be differentiated into several
desirable tissues, are non-tumorigenic, and are tolerated well in
transplantation settings [Bongso and Fong, 2013]. The availability of
such large numbers does not necessitate passaging and in vitro
manipulation to generate sufcient numbers for clinical application
thus eliminating the risk of in vitro-induced epigenetic chromosomal
changes [Ben-David and Benvenisty, 2011]. To date several clinical
trials have been successfully carried out with WJSCs for the

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FREEZING OF FRESH WHARTONS JELLY FROM HUMAN UMBILICAL

treatment of a variety of diseases [Wu et al., 2011; Zhang et al.,

2012a; Hu et al., 2013; Lv et al., 2013].
Besides optimal functional outcome, efcient clinical application
of MSCs requires simple, rapid, and reliable derivation methods,
minimal in vitro manipulation and high freeze-thaw survival rates
with maintenance of stemness properties post-thaw [Gong et al.,
2012; Balci and Can, 2013; Hendijani et al., 2014]. To achieve these
objectives for cord blood banks we carried out a rigorous comparative
evaluation of the freezing and post-thaw behavior of three groups of
UC tissues: (i) pure gelatinous WJ harvested fresh from the UC without
any cell separation (WJ); (ii) MSC populations separated from the WJ
(WJSC); and (iii) entire UC segments (mixed cord, MC).

MATERIALS AND METHODS

HISTOLOGY
The human UC was xed in 10% neutral buffered formalin (Sigma, St
Louis, MO) for 24 h at room temperature and embedded in parafn.
Histological sections of 5 m thickness were then cut using a
microtome and mounted on Superfrost Plus slides (Practical
Mediscience, Singapore). The sections were de-waxed, stained
with Harris Hematoxylin and Eosin (Sigma), and examined using
bright eld optics (Nikon, Instruments, Tokyo).
HARVESTING OF FRESH WHARTONS JELLY (WJ), WHARTONS JELLY
MSCs (WJSC) AND ENTIRE CORD SEGMENTS (MC) FOR FREEZING
Ethical approval with written informed patient consent was obtained
from the Ministry of Health Domain Specic Review Board (DSRB)
for the collection and use of human UCs. The UCs were collected at
birth in sterile containers containing Hank0 s balanced salt solution
(HBSS, Invitrogen Life Technologies, Carlsbad, CA). Each cord was
cut into 2 cm pieces and equal numbers of pieces divided into three
groups (Groups 13). The UC pieces in Groups 1 and 2 were
separately cut open lengthwise with sterile forceps and curved
scissors. The cut open pieces were inverted face down in sterile
60 mm plastic Petri dishes containing 2 ml of enzymatic solution
comprised of collagenase type I (2 mg/ml), collagenase type IV
(2 mg/ml) and 100 IU of hyaluronidase (Sigma) in DMEM medium
(Invitrogen). The Petri dishes were then incubated at 37C in a 5%
CO2 in air atmosphere for 45 min to allow loosening of the gelatinous
WJ. The gelatinous WJ was then washed in stem cell culture
medium comprised of 80% DMEM high glucose supplemented with
20% FBS (Invitrogen), 16 ng/ml basic broblast growth factor
(Millipore Bioscience Research Agents, Temecula, CA), 1% nonessential amino acids, 2 mM L-glutamine, 0.1 mM b-mercaptoethanol, 1% insulin-transferrin-selenium (ITS) and antibiotic/antimycotic mixture [penicillin (50 IU), streptomycin (50 mg/ml)]
(Invitrogen) to remove any traces of enzymes.
For Group 1 (WJ), the pure gelatinous WJ as it is (without
separation of the MSCs from its gelatinous matrix) was transferred to
cryovials for freezing. For Group 2 (WJSC), MSCs were rst separated
from the gelatinous WJ using the derivation protocol of Fong et al.
[2010] and then transferred to cryovials for freezing. For Group 3
(MC), the 2 cm UC pieces were cut into smaller pieces of 23 mm each
and the entire pieces transferred to cryovials for freezing.

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FREEZING OF WJ, WJSC, AND MC

Since the same volume of WJ was used in the WJ and WJSC groups,
each of these groups had approximately the same number of cells
frozen (4.25  0.35  106) with or without the gelatinous matrix for
each replicate. It was not possible to nd out the cell numbers for MC
as entire UC pieces were frozen. The WJ, WJSC, and MC pieces was
slowly mixed with cell recovery freezing medium (Invitrogen) and
transferred into 1.5 ml cryogenic vials and the vials frozen in a slow
stepwise manner using a programmable slow-freezing machine
(Planer Series III, NY) from room temperature to 30C at 1C/min,
held for 10 min, then to 150C at 15C/min, held for 10 min and
then nally plunged into liquid nitrogen (196C).
THAWING OF WJ, WJSC, AND MC
After 30 days of storage in liquid nitrogen, the vials containing WJ,
WJSC, and MC were thawed by placing them in a water bath at 37C.
The contents of each cryovial were then diluted with fresh stem cell
culture medium in a slow drop-wise manner, centrifuged at 300 g for
5 min and the supernatant was discarded. The pellets were then
processed as follows.
The WJ pellet that comprised of intact gelatinous WJ post-thaw
was syringed through an 18 G needle a few times to loosen the
gelatinous WJ and dislodge the stem cells. The cell suspension was
then centrifuged at 300g for 5 min, supernatant discarded; cell pellet
resuspended in fresh stem cell culture medium and then seeded into
sterile 100 mm tissue culture petri dishes [Becton Dickinson (BD), NJ]
for culture. An aliquot of cells were taken to calculate the total live
cell numbers using trypan blue vital staining.
The WJSC pellet that comprised of only WJSCs without the
gelatinous matrix was resuspended in fresh stem cell culture
medium and then seeded into sterile 100 mm plastic tissue culture
Petri dishes (BD, NJ). An aliquot of cells were taken to calculate the
total live cell numbers using trypan blue vital staining.
The MC pellet was washed with fresh stem cell culture medium
and the individual cord pieces placed in 100 mm tissue culture Petri
dishes containing stem cell culture medium and grown as explant
culture [Bosch et al., 2012; Zhang et al., 2012a]. The dishes of all
three groups were incubated at 37C in a 5% CO2 in air atmosphere.
POST-THAW CELL MORPHOLOGY
The post-thawed cell morphological behavior and duration to attain
conuence was monitored and photographed at primary culture and
passages 1, 3, and 5 for the cells in the three groups using inverted
phase contrast optics (Nikon Instruments, Tokyo).
POST-THAW CD MARKER ANALYSIS
Post-thawed cells from all three groups at primary culture, passages
1, 3, and 5 were subjected to CD marker analysis. The cells were rst
dissociated using trypsin (TrypLETM Express, Invitrogen) for 35 min
at 37C in a 5% CO2 in air, then washed in phosphate buffered saline
(PBS) and blocked with 10% normal goat serum (NGS) (Invitrogen) to
prevent non-specic binding. The cells were then incubated with
mouse monoclonal primary antibodies for a series of CD markers
viz., CD14, CD24, CD29, CD40, CD44, CD73, CD90, CD108, HLAABC, and HLA.DR (Biolegend, San Diego, CA) for 30 min. This was
1
followed by incubation with Alexa Fluor 488 (1:750) goat anti-

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mouse secondary antibody (Invitrogen) for 30 min. The cells were

nally washed in PBS (), re-suspended in 10% NGS and ltered
using a 70 mm nylon strainer (BD, NJ) to remove any cell clumps and
analyzed using a CyAnTM ADP Analyzer (Beckman Coulter,
Fullerton, CA).
POST-THAW CELL VIABILITY AND PROLIFERATION
Post-thawed cells from all three groups at primary culture, passages
1, 3, and 5 were subjected to cell viability and proliferation assays.
The cell viability MTT assay was performed using a MTT reagent kit
[3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]
(Sigma) according to the manufacturer0 s instructions. Briey,
10 ml MTT reagent (nal concentration of 0.5 mg/ml) was added
to 100 ml of medium bathing the cells in wells of tissue culture plates
and the plates incubated for 4 h until a purple precipitate was visible.
The medium was then removed and 100 ml of the detergent reagent
was added into the each well and incubation carried out in the dark
for 2 h. Absorbance at 570 nm was spectrophotometrically measured
using a microplate ELISA reader (mQuant, BioTek and Winooski, VT)
with a reference wavelength of 650 nm.
The cell proliferation BrdU assay was performed using a kit and
according to manufacturer0 s instructions (Cell Signaling, MA). The
cells from each group were rst seeded at a density of 5  103 cells/
well in a 48-well tissue culture plate (NUNC, Rochester, NY). Briey,
the cells were incubated for 124 h with the BrdU solution followed
by removal of the medium and incubation for 30 min with the xing/
denaturing solution. Subsequently, the plate was incubated for 1 h
with BrdU detection antibody solution followed by three washes
with the wash buffer. The HRP-conjugate solution was then added
and incubated for 30 min followed by similar washing steps and
incubation with the substrate solution for 30 min. The enzymatic
reaction was nally stopped and the products quantied by
measuring absorbance at 450 nm using a microplate ELISA reader
(mQuant-BioTek, Winooski, VT).
POST-THAW IMMUNOCYTOCHEMISTRY
Post-thawed cells from all three groups were xed with 100% cold
ethanol for 5 min, washed with PBS and blocked with 10% NGS
(Invitrogen) for 1520 min at room temperature. The cells were then
incubated with mouse monoclonal primary antibodies for Ki-67
(Biolegend, San Diego, CA) for 1 h. This was followed by incubation
1
with Alexa Fluor 488 (1:750) goat anti-mouse secondary antibody
(Invitrogen) for 30 min. The cells were washed in PBS and stained
with 40 -6-diamidino-2-phenylindole (DAPI; 0.5 mg/ml) (Molecular
probes, Invitrogen Life Technologies, Carlsbad, CA) for 5 min at
room temperature. Finally, the cells were washed once with PBS,
examined and photographed using a confocal microscope.
POST-THAW CELL CYCLE ANALYSIS
Cell cycle analysis was compared between the post-thawed cells
from all three groups using ow cytometry. Briey, the cells were
trypsinized, washed with PBS and xed with ice-cold 70% ethanol
for 2 to 3 h. The xed cells were then washed with PBS and stained
with 50 mg/ml propidium iodide (PI) (Promega, WI) in PBS
containing 0.1% Triton X-100 (Bio-Red, CA) and 50 mg/ml
RNAse-A (Ambion, TX). The cells were then ltered using a

FREEZING OF FRESH WHARTONS JELLY FROM HUMAN UMBILICAL

817

70 mm nylon strainer to remove any cell clumps and analyzed using

a ow cytometer (Epics-Altra, Beckman Coulter, CA).
POST-THAW ANNEXIN V-FITC ASSAY
The annexin V-FITC assay was carried out on the post-thawed cells
of all three groups to evaluate apoptosis rates. Briey, the cells were
dissociated with trypsin, washed once with PBS () and then with
Annexin-V binding buffer (1) (Promega). The cells were then
stained with 5 ml Annexin V-FITC (Promega) and counterstained
with propidium iodide (1 mg/ml) (Promega) at room temperature for
15 min. The cells were ltered using a 70 mm nylon strainer to
remove any cell clumps and analyzed using a CyAnTM ADP Analyzer
(Epics-Altra, Beckman Coulter).
POST-THAW ADIPOGENIC DIFFERENTIATION
For adipogenic differentiation, post-thawed cells from all three
groups were seeded (10  104 cells/dish) into 6-well tissue culture
plates and incubated at 37C in a 5% CO2 atmosphere for 24 h to
allow cell attachment. The medium was then changed to adipogenic
induction medium containing DMEM- low glucose (4.5 g/L)
(Invitrogen) supplemented with 10% FBS, 1% penicillin/streptomycin, 0.01 mg/ml insulin (Invitrogen), 1 mM dexamethasone (Sigma),
0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX) (Sigma) and 0.2 mM
indomethacine (Sigma). The cells were maintained for 28 days in the
induction medium, with medium changes every 2 days. The cells
were then xed with 4% paraformaldehyde for 10 min, rinsed with
PBS and post-xed with 60% isopropanol for 5 min. The cells were
then stained with Oil Red O for 5 min, rinsed with distilled water and
counter-stained with hematoxylin (Sigma). The stained cells were
visualized using bright eld optics (Nikon).
POST-THAW OSTEOGENIC DIFFERENTIATION
For osteogenic differentiation, the post-thawed cells from all three
groups were seeded (10  104 cells/dish) into 6-well tissue culture
plates and incubated at 37C in a 5% CO2 atmosphere for 24 h to
allow attachment. The medium was then changed to osteogenic
medium contain DMEM medium-low glucose (4.5 g/L) (Invitrogen)
supplemented 5% FBS, 0.17 mM L-ascorbic-acid (Sigma), 100 nM
dexamethasone, 1% penicillin/streptomycin, and 10 mM b-glycerophosphate (Sigma). The cells were cultured for 28 days with fresh
changes of medium every 2 days. Osteogenic mineralization was
then evaluated by Von Kossa staining. Briey, the cells were rinsed
with PBS and xed in 3.7% formaldehyde solution (Sigma) for
10 min at room temperature. They were then washed with distilled
water and stained in 1% silver nitrate solution (Sigma) under UV
light for 60 min. The cells were then washed again with distilled
water, treated with 3% sodium thiosulfate (Sigma) for 5 min, and
counterstained with 1% nuclear fast red (Sigma) for 5 min. The
stained cells were washed with distilled water and photographed
under bright eld optics (Nikon).
POST-THAW CHONDROGENIC DIFFERENTIATION
For chondrogenic differentiation, the post-thawed cells from all
three groups were seeded (10  104 cells/dish) into 6-well tissue
culture plates and incubated at 37C in a 5% CO2 atmosphere for 24 h
to allow attachment. The medium was then changed to chondrogenic

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FREEZING OF FRESH WHARTONS JELLY FROM HUMAN UMBILICAL

medium containing DMEM medium-low glucose (4.5 g/L) (Invitrogen) supplemented with 1% penicillin/streptomycin, 1% insulintransferrin-selenium (ITS), 0.17 mM L-ascorbic-acid, 100 nM dexamethasone, 1 mM sodium pyruvate, 0.35 mM proline, and 10 ng/ml
transforming growth factor beta-3 (TGF-3) (Sigma). The cells were
cultured for 28 days with fresh changes of medium every 2 days. The
cells were then disassociated with trypsin (Invitrogen), washed with
PBS () and cytospun directly on to glass slides using CyotospinTM
(Thermo Scientic, Barrington, IL) at 200 g for 5 min. The cytospun
slides were stained with 0.5% Alcian Blue (Sigma) for 30 min at room
temperature, rinsed with tap water, and then counter-stained with
0.1% Nuclear Fast Red (Sigma) for 5 min. The stained slides were
examined under bright eld optics (Nikon).
POST-THAW QUANTITATIVE REAL TIME POLYMERASE CHAIN
REACTION (qRT-PCR)
Total RNA from post-thawed cells of all three groups was extracted
using TRIzolTM reagent (Invitrogen) and its quality and quantity
measured using a NanodropTM spectrophotometer (Nanodrop
technologies, Wilmington, DW). All RNA samples were treated
with DNase-I prior to rst strand cDNA synthesis with random
hexamers using the SuperScriptTM rst strand synthesis system
(Invitrogen). Primer sequences were taken from earlier published
studies and are summarized in Table I. qRT-PCR analysis was
performed with the ABI 7500 Fast Real-Time PCR System (Applied
Biosystems, Foster City, CA) using SYBR green as previously
described [Gauthaman et al., 2007] and relative quantication was
performed using the comparative CT (2-DDCT) method.
STATISTICAL ANALYSIS
The results of each assay were compared and analyzed between the
three groups using one-way ANOVA with Bonferroni0 s multiple
comparison post hoc analysis using the statistical package for Social
Sciences (SPSS 13). The results were expressed as mean  SEM from
three different replicates for individual assays and a value of
P < 0.05 was considered to be statistically signicant.

RESULTS
HISTOLOGY
Hematoxylin and Eosin stained cross-sections of ve different UCs
showed that the human UC comprised of a central region of two
arteries and one vein buried in a WJ matrix which in turn was
enclosed by an inner subamniotic membrane and outer amniotic
membrane. The WJ matrix had the largest volume and surface area.
Around the blood vessels a thin slightly darker staining perivascular
area of the WJ could be recognized. Thus, ve different regions or
compartments could be recognized in the human UC. Starting from
the outside these compartments included the amnion, subamnion,
WJ, perivascular area, and umbilical blood vessels (Fig. 1A).
MORPHOLOGY OF WJ, WJSC AND MC BEFORE FREEZING
The WJ recovered in the rst group from all ve cords contained the
perivascular area and was a transparent gelatinous substance that
could be easily separated from the umbilical blood vessels, subamnion

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TABLE I. The Genes and Primer Sequences Used for Quantitative

Real Time PCR

Gene

Primer sequence

CEBP b
PPAR g
FABP4
OCN
OPN
BSP
COL2A1
COMP
FMOD
BAX
BCL2
SURVIVIN
P53
P21
ROCK 1
GAPDH

F:5 -ACTTCAGCCCGTACCTGGAG-30
R:50 -GAGAAGAGGTCGGAGAGGAAGT-30
F:50 -GGCTTCATGACAAGGGAGTTTC-30
R:50 -AACTCAAACTTGGGCTCCATAAAG-30
F:50 -TTGACGAAGTCACTGCAGATGA-30
R:50 -CAGGACACCCCCATCTAAGGT-30
F:50 -CCCAGGCGCTACCTGTATCAA-30
R:50 -GGTCAGCCAACTCGTCACAGTC-30
F:50 -ACAGCCACAAGCAGTCCAGATT-30
R:50 -TGCTCATTGCTCTCATCATTGG-30
F:50 -CCAGAGGAAGCAATCACCAAA-30
R:50 -TTGAGAAAGCACAGGCCATTC-30
F:50 -GTGACAAAGGAGAGGCTGGA-30
R:50 -ACCTCTAGGGCCAGAAGGAC-30
F:50 - GGAGATCGTGCAGACAATGA-30
R:50 -GAGCTGTCCTGGTAGCCAAA-30
F:50 -CAACCAGCTGCAGAAGATCC-30
R:50 -CAGAAGCTGCTGATGGAGAA-30
F:50 -TGCTTCAGGGTTTCATCCAG-30
R:50 -GGCGGCAATCATCCTCTG-30
F:50 -GGCTGGGATGCCTTTGTG-30
R:50 -CAGCCAGGAGAAATCAAACAGA-30
F:50 -ACCAGGTGAGAAGTGAGGGA-30
R:50 -AACAGTAGAGGAGCCAGGGA-30
F:50 -GCGCACAGAGGAAGAGAATC-30
R:50 -CTCTCGGAACATCTCGAAGC-30
F:50 -CAGCGACCTTCCTCATCCAC-30
R:50 -GAGAAACGGGAACCAGGACA-30
F:50 -GAAGAAAGAGAAGCTCGAGAAGAAGG-30
R:50 -ATCTTGTAGCTCCCGCATCTGT-30
F:50 -GCACCGTCAAGGCTGAGAAC-30
R:50 -GGATCTCGCTCCTGGAAGATG-30

and amnion without contamination of cells from these compartments.

Under the stereomicroscope (magnication 20) circular shaped cells
with prominent nucleoli (WJSCs) were observed buried in the oating
fresh gelatinous WJ matrix (Fig. 1B). When separated from the
gelatinous matrix, the fresh WJSCs recovered in the second group
were individual cells with a short broblastic or stellate morphology.
The third group (MC) comprised of tiny cut-open segments (2 mm
each) of entire UC.
POST-THAW MORPHOLOGY OF FROZEN WJ, WJSC, AND MC
After thawing, the WJ appeared healthy and maintained its translucent
gelatinous texture and the cells that were buried in its matrix had intact
cell membranes and nuclei with minimal cryoinjury. Enzymatic
separation and syringing of the cells from the gelatinous matrix was
easy and the cells attached readily to plastic dishes within 24 h and
produced islands of stellate-looking cells (Fig. 1C). The cells from the
WJSC group were also intact after freeze-thaw and grew readily in
culture. The frozen intact whole cord pieces from the MC group
appeared unhealthy after thawing with dark necrotic cores. When these
cord pieces were grown as explants on plastic they did not show cell
outgrowths within the rst 24 h (Fig. 1D) but started showing a few cell
outgrowths several days later followed by slow growth thereafter. The
time taken to separate the cell outgrowths and expand the cells was
much longer than for cell outgrowths from unfrozen fresh MC cultures.
POST-THAW CELL SURVIVAL RATES
Using Trypan Blue staining, the WJ and WJSC groups showed
high mean  SEM viable post-thaw cell survival rates

Fig. 1. (A) H & E stained cross-section of human UC showing the presence of two arteries and one vein buried in a smooth gelatinous WJ matrix which was enclosed by a thin
amniotic membrane (amnion). (B) Circular clusters of cells with prominent nucleoli (WJSCs) were visible within the fresh WJ matrix. Inset shows high magnication of the WJSCs.
(C) Cells from the post-thaw WJ attached readily to plastic culture dishes forming islands of epithelioid cells within 24 h. (D) Post-thaw MC explants did not attach to plastic and
did not show cell outgrowths within 24 h of primary culture. Note the centre of the MC explant showing dark necrotic areas.

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819

(90.83  4.51% to 93.52  6.12%) compared to MC (Fig. 2A).

FACS analysis showed that there were no signicant differences
in the MSC CD signature markers (CD29, CD44, CD73, CD90,
and HLA,abc, CD14, HLA.DR) of cells between post-thaw WJ
and WJSC (Fig. 2B).
POST-THAW MORPHOLOGY OF CELLS FROM WJ, WJSC, AND MC
In primary culture, the cells from the post-thaw WJ and WJSC
showed islands of epitheliod cells on day 6 (Fig. 3Aa, c) which
became completely conuent by day 12 (Fig. 3Ab, d) whereas the
post-thaw MC explants showed poor growth with no cell islands on
day 6 (Fig. 3Ae) and a few broblast-like cells growing out from the
peripheral regions of the explants on day 12 (Fig. 3Af). On day 12 in
primary culture the cell numbers were signicantly greater in postthaw WJ (6.82  1.26  106) and WJSC (6.91  1.64  106) compared to post-thaw MC (2.64  0.61  106) (Fig. 3B).
POST-THAW CELL VIABILITY AND PROLIFERATION
The results of the MTT assay showed that the cells isolated from postthaw WJ and WJSC showed signicantly greater cell viability rates
at passage 1 (P1) (1.48  0.23% and 1.65  0.0.13%), P3
(2.13  0.18% and 1.92  0.18%), and P5 (2.26  0.21% and
2.31  0.25%) compared to cells from post-thaw MC (Fig. 4A). The
results of the BrdU assay showed that proliferation rates of the cells
isolated from post-thaw WJ and WJSC were signicantly greater at
P1 (2.63  0.25% and 2.48  0.19%), P3 (2.94  0.20% and
3.18  0.15%), and P5 (3.62  0.18% and 3.46  0.18%) compared
to cells from post-thaw MC (Fig. 4B). Immunohistochemistry
analysis of the cells isolated from post-thaw WJ and WJSC showed
strong positive staining for the proliferative marker (Ki-67)
compared to cells from post-thaw MC (Fig. 4C).

Fig. 2. (A) The WJ and WJSC groups showed high post-thaw viable cell
survival rates (Trypan blue staining) ranging from 90.83  4.51% to
93.52  6.12%. (B) The expression levels of the typical MSC CD stemness
markers seen in fresh unfrozen WJSC were retained in the cells of the postthawed WJ and WJSC groups.

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POST-THAW CD MARKER ANALYSIS

Flow cytometric analysis of cells from all three post-thaw groups
showed that they were positive for the CD29, CD44, CD73, CD90,
CD105, and HLAabc markers (98.03  0.61% to 99.65  0.17%) and
negative for CD14, CD34, CD45, and HLA.DR (4.98  0.49% to
9.26  0.86%) with no signicant differences between the groups.
However, the mean  SEM uorescence intensities for percentages
of CD24 and CD108 markers were signicantly greater in the cells
derived from the post-thaw WJ and WJSC groups (67.52  9.70 to
84.26  9.56) compared to the cells from the MC group (26.39  7.88
to 47.08  9.53) (Fig. 5Aa, b). Also, the CD40 marker percentages
were signicantly greater for post-thaw MC cells (67.46  8.7%)
compared to post-thaw cells from WJ (29.08  4.3%) and WJSC
(27.91  5.1%) (P < 0.05) (Fig. 5Ac).
POST-THAW ANNEXIN V-FITC AND CELL CYCLE ANALYSIS
Annexin V-FITC analysis showed signicantly greater apoptosis
signals for the cells from the post-thaw MC (6.93  1.26%)
compared to cells from post-thaw WJ and WJSC (1.46  0.67%
to 1.71  0.55%) (Fig. 5Ba-d). The cells from all three post-thaw
groups showed normal cell cycle proles with no signicant
differences between them. However, post-thaw MC showed higher
percentages of cells at sub-G1 (9.76  1.14%) compared to cells
from post-thaw WJ and WJSC (6.83  0.91% to 7.04  0.69%)
(Fig. 5Cac).
POST-THAW CELL DIFFERENTIATION
The cells recovered from all three post-thaw groups could be
differentiated into adipogenic, osteogenic, and chondrogenic
lineages. Adipogenic colonies stained with Oil Red O were similar
size and there were no signicant differences in staining intensity
between the three groups (Fig. 6Aac). However, the cells from the
post-thaw WJ and WJSC groups that were exposed to osteogenic
differentiation medium showed the greatest number of Von Kossapositive stained cells and the greatest staining intensity of
osteocytic nodules compared to cells from the MC group
(Fig. 6Bac). Cells from the post-thaw WJ and WJSC groups
that were exposed to the chondrogenic differentiation medium
showed greater numbers of cells that were positive for Alcian blue
compared to cells from the MC group (Fig. 6Cac).
The comparative scores for the expression levels of the adipocyte,
osteocyte, and chondrocyte genomic markers showed that
expression was signicantly greater for osteocyte and chondrocyte
differentiation for cells recovered from the post-thaw WJ and
WJSC groups compared to MC with no differences in adipocyte
differentiation between groups (Table II).
POST-THAW QRT-PCR ANALYSIS
qRT-PCR analysis for adipogenic, osteogenic, and chondrogenic
genomic markers conrmed that the cells from all three post-thaw
groups differentiated into these three lineages. There were no
signicant differences in the expression levels for the adipogenic
marker genes (CEBPb, FABP4, and PPARg) between the three
groups (Fig. 7AC). However, the expression levels of the
osteogenic marker genes [osteocalcin (OCN), osteopontin (OPN),
and bone sialoprotein (BSP)] were signicantly greater for cells

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Fig. 3. (A) Phase-contrast inverted optical images showing epitheliod morphology of cell islands from post-thaw WJ and WJSC groups on day 6 in primary culture (a, c) and
complete conuent epitheliod monolayers on day 12 (b, d) whereas the MC group did not show islands of cell outgrowths on day 6 (e) but showed a few long broblast-like cells
growing out of the peripheral regions of the explants by day 12 (f). (B) The post-thaw viable cell numbers in the WJ and WJSC groups were signicantly greater than those in the
MC group in primary culture on day 12.

Fig. 4. (A, B) The MTT and BrDU assays showed signicantly greater cell viability and proliferation rates for post-thaw WJ and WJSC compared to MC at passages 1, 3, and 5
(P < 0.05). (C) Cells isolated from post-thaw WJ and WJSC showed stronger positive staining for the cell proliferative marker (Ki-67) compared to cells from post-thaw MC.

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821

Fig. 5. (A a,b) Fluorescence-activated cell sorting (FACS) analysis showed that the uorescence intensities for the CD24 and CD108 markers were signicantly greater in the
cells derived from post-thaw WJ and WJSC groups compared to cells from post-thaw MC (P < 0.05). (c) The cells derived from post-thaw MC showed signicantly greater
percentages of CD40 cell contaminants compared to post-thaw WJ and WJSC (P < 0.05). (Bad) Annexin V-FITC analysis showed greater apoptosis signals for the post-thaw
MC group compared to post-thaw WJ and WJSC groups. (Cac) The cells from post-thaw MC showed higher percentages of cells at sub-G1 compared to cells from post-thaw WJ
and WJSC.

from the post-thaw WJ and WJSC groups (4.68.06 fold) compared

to MC (Fig. 7DF). Similarly, the expression levels of the
chondrogenic marker genes [collagen type II (COL2A1), cartilage
oligomeric matrix protein (COMP), and bromodulin (FMOD)] were
signicantly greater for cells from the post-thaw WJ and WJSC
groups (27.3549.72 fold) compared to MC (Fig. 7GI). qRT-PCR
analysis showed that there were no signicant differences in the
expression levels of the pro-apoptotic genes (SURVIVIN, BCL2),
anti-apoptotic gene (BAX) and cell regulator genes (P53, P21,
ROCK 1) in post-thawed cells between WJ and WJSC groups
(Fig. 8A-F). However, the expression levels of the anti-apoptotic
genes (SURVIVIN and BCL2) were signicantly decreased
(0.740.88 fold) in cells from post-thaw MC compared to cells
from post-thaw WJ and WJSC (P < 0.05) (Fig. 8A, B) and the proapoptotic and cell regulator genes (BAX, P53, P21, and ROCK
1) were signicantly increased (0.651.55 fold) in cells from postthaw MC compared to WJ and WJSC (P < 0.05) (Fig. 8CF).

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DISCUSSION
It has been reported that the CD24, CD108, and CD40 markers
reliably discriminate between bona de MSCs and non-stem cell
mesenchymal cells [Wetzig et al., 2013]. Increased expression of
CD24 and CD108 markers conrmed the presence of actual MSCs
while increased expression of CD40 cells conrmed the presence of
non-MSC broblast contaminants [Wetzig et al., 2013]. Fresh
unfrozen cultured MC explants were shown to generate mixed
heterogeneous populations of epitheliod and broblast-like cells
originating from the ve different compartments of the UC that were
positive for the conventional MSC CD markers (CD29, CD44, CD73,
CD90, CD105, and HLA-ABC) and CD24, CD40, and CD108
[Subramanian et al., 2015]. The results of the present study showed
that post-thaw MC cultures had greater numbers of CD40 cells and
lesser numbers of CD24 and CD108 MSCs suggesting that such
CD40 contaminants were more resilient to the freeze-thaw process

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Fig. 6. (Aac) The cells from the post-thaw WJ, WJSC, and MC groups that were exposed to the adipogenic differentiation medium showed positive Oil Red O staining with no
signicant differences in positively stained cell numbers between the three groups. (Ba-c and Ca-c) However, the cells from post-thaw WJ and WJSC groups that were exposed to
the osteogenic and chondrogenic differentiation media showed greater cell numbers that were positive for Von Kossa and Alcian blue staining respectively compared to postthaw MC.

and these may have been the broblast-like cell outgrowths that
survived the freeze-thaw process. It is also possible that certain MSC
populations from specic compartments were favored in their
freeze-thaw survival. Since the post-thaw WJ and WJSC groups
yielded homogeneous populations of signicantly greater numbers
of cells that were not only positive for the conventional MSC CD
markers but also CD24 and CD108, they would be more ideal than
MC for research and clinical application.
In a recent comparative study on the characterization of fresh
unfrozen cells between WJ, perivascular, subamnion, amnion, and
MC cultures we observed that the cells from the WJ had the greatest
osteogenic and chondrogenic differentiation potential compared to
the rest [Subramanian et al., 2015]. The results of the present study
showed similar differences in differentiation potential between
frozen-thawed WJ and MC further conrming the benets of
freezing WJ or WJSC over MC.

TABLE II. Scoring of Adipogenic, Osteogenic, and Chondrogenic

Differentiation of Cells Derived From Post-Thaw WJ, hWJSC, and
MC

Differentiation
Adipogenic
Osteogenic
Chondrogenic

WJ

WJSC

MC

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It has been reported that Rho and ROCK 1 are activated when cells
undergo stress causing cytochrome C release to activate caspase
mediated apoptosis [Gauthaman et al., 2010]. High expression levels
of ROCK I were shown to lead to stress-induced apoptosis and
increased expression of BAX was associated with accelerated
apoptotic cell death [Gauthaman et al., 2010; Wu et al., 2011].
Such stress-related changes may appear to have occurred during the
freezing of entire UC segments (MC) resulting in the decreased
expression of the anti-apoptotic genes (SURVIVIN and BCL2) and
increased expression of the pro-apoptotic and cell regulator genes
(BAX, P53, P21, and ROCK 1).
The gelatinous human WJ is considered an extracellular matrix
(ECM) composed of an insoluble three-dimensional (3D) brillary
network of collagen and glycoprotein microbrils impregnated with
mucopolysaccharides (hyaluronic acid and chondroitin sulfate). The
high thaw-survival rates and minimal freezing damage of MSCs
observed in the fresh WJ group of the present study was probably due
to the protective effects of the mucopolysaccharides and the 3D
topography of the gelatinous matrix that allowed proper diffusion of
DMSO, optimum biophysical and physicochemical changes for ice
crystallization, and optimum mass and heat transport processes.
It is quite common to assume that conventional cryopreservation
procedures can be applied universally for cells, tissues, and organs.
Cryopreservation of entire cord segments irrespective of their size
appears to have many limitations as observed in the results of the

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823

Fig. 7. (AC) qRT-PCR analysis showed that there were no signicant differences in the expression levels of the adipogenic marker genes (CEBPb, FABP4, and PPARg) between
post-thaw WJ, WJSC, and MC. (DF) However, the expression levels of the osteogenic marker genes OCN, OPN, and BSP were signicantly greater in cells from post-thaw WJ and
WJSC compared to MC (P < 0.05). (G-I) Similarly, the expression levels of the chondrogenic marker genes COL2A1, COMP, and FMOD were signicantly greater in cells from
post-thaw WJ and WJSC compared to cells from MC (P < 0.05).

present study. There is ample evidence to suggest that the intrinsic

responses of cells to cryopreservation are different if the cells are
lying individually in a liquid or gelatinous milieu as compared to
being part of a tissue. Intracellular ice formation kinetics indicates
that the biophysical parameters governing water transport and
intracellular nucleation dynamics during freezing are different for
individual cells as compared to cells in tissues. Intracellular ice
formation requires a higher degree of cytoplasmic super-cooling and
is affected by cell-cell and cell-extracellular matrix interactions
[Larese et al., 1992; Yarmush et al., 1992]. In tissue freezing, mass
transport processes such as addition and removal of cryoprotectants
before and after freezing and redistribution of biological water
during freeze-thaw are also very important. These processes are
compromised in whole tissue cryopreservation compared to cell
cryopreservation. It has been reported that during the freezing of
multilayer tissues such as skin, the interior cell layers tend to retain
more water than cells in the surface layer. The same would be true
when freezing entire cord segments as opposed to individual cells

824

FREEZING OF FRESH WHARTONS JELLY FROM HUMAN UMBILICAL

buried in the WJ. The cells in the core of the UC pieces may resist
freeze-induced dehydration [Bischof et al., 1993].
In addition to water transport it is also important to consider the
dynamics of cryoprotectant diffusion in and out of tissues during the
addition and removal of cryoprotectants. Longer exposure times to
cryoprotectants were shown to be associated with higher probabilities of cell toxicity [Carpenter and Dawson, 1991]. The spatial
distribution of DMSO in tissues during cryoprotectant loading was
measured and it was found that there was a signicant lag
(approximately 1 h) for DMSO incorporation into the interior of a
1 cm3 sample of porcine myocardium compared to DMSO uptake at
the periphery of the sample with corresponding spatial concentration gradients of approximately 1 M cm1 [Carpenter and
Dawson, 1991]. Thus, when entire UC segments are frozen there
may be signicant differences in cryoprotectant concentration
between the surface and interior of the tissue. Consequently, there
may be a situation where surface cells are exposed to toxic
concentrations of cryoprotectant in order to attain the necessary

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Fig. 8. (AF) qRT-PCR analysis showed that there were no signicant differences in the expression levels for the anti-apoptotic (SURVIVIN, BCL2), pro-apoptotic (BAX) and cell
regulator (P53, P21, ROCK 1) genes between post-thaw WJ and WJSC. (AB) However, SURVIVIN and BCL2 were signicantly decreased in post-thaw MC compared to post-thaw
WJ and WJSC and (CF) BAX, P53, P21, and ROCK1 were signicantly increased in post-thaw MC compared to WJ and WJSC (P < 0.05).

cryoprotectant concentration in the interior of the tissue. Conversely, if care is taken not to harm surface cells during
cryoprotectant addition, inner cells may have too low concentrations of cryoprotectant and thus not be sufciently protected
against freezing [Karlsson and Toner, 1996].
There are also heat transport limitations in whole tissue
cryopreservation compared to single cell cryopreservation. Due
to the macroscopic size of the specimen and its nite thermal
conductivity, it is generally more difcult to achieve rapid cooling
and warming rates in tissues compared to cell suspensions because
of large thermal gradients from the surface to the interior of the
tissue [Diller, 1992]. It should also be noted that there are many
biological structures in tissues that may be sensitive to
cryopreservation damage. Intercellular and cell-matrix junctions
which are important for the normal physiological functions of
multicellular tissues were suggested as potential targets of injury
during tissue cryopreservation and it was shown that intercellular
junctions are affected by prolonged exposure to freezing temperatures [Armitage et al., 1994].
One of the drawbacks in the use of MSCs for cell based therapies is
insufcient numbers. Since the post-thaw survival rates of MSCs in
both the WJ and WJSC groups were high and given the fact that
WJSCs have high proliferation rates, the freezing of fresh WJ or
WJSCs may be more suitable than freezing entire cord segments (MC)
for cell based therapies. The slow growth and recovery of cell
outgrowths from post-thaw MC explants require time, labor, and
prolonged manipulation to scale up adequate MSC numbers and may
result in epigenetic chromosomal changes in the cells making them
unsuitable for clinical application [Ben-David et al., 2011]. Previous
studies have reported that when entire UC segments were frozen for
periods of 6 weeks to 6 months, the post-thaw recovery of healthy

JOURNAL OF CELLULAR BIOCHEMISTRY

MSCs from the frozen tissue fragments was unsuccessful at all time
points [Chatzistamatiou et al., 2014]. The cryopreservation of fresh
WJ is simple and fast and large thaw-survival numbers of WJSCs
could be produced with minimal manipulation without the need
prolonged culture. This is coupled with the fact that programmed
slow-freezing of WJSCs (where the cooling temperature of the cells is
brought down slowly from 37C to -130C and the cells then plunged
into liquid nitrogen) yields high thaw-survival rates. The freezing of
fresh WJ eliminates the steps required for separation of WJSCs
before freezing, unlike the WJSC group, and is therefore a simple and
efcient adjunct protocol to UCB storage for cord blood banks for
future use of autologous or allogeneic MSCs for regenerative
medicine purposes.

AUTHORS CONTRIBUTIONS
Chui-Yee Fong: Conceptualization and design of experiments,
writing, applying, and obtaining grant funding, analysis of data,
editing manuscript, Arjunan Subramanian: Execution of experiments, analysis of data, writing manuscript, Arijit Biswas: Seeking
written informed patient consent and obtaining IRB approval,
collection of patient material, Ariff Bongso: Conceptualization and
design of experiments, editing grant application, writing and editing
manuscript

ACKNOWLEDGMENTS
This research is supported by the Singapore National University
Health System (NUHS) Aspiration Fund (New Idea) (R-174-000-155720) and Aspiration Fund (Partner) (R-174-000-156-720) grants.

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825

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