ORIGINAL RESEARCH

A new standardized clinical-grade protocol for banking human

umbilical cord tissue cells
Raffaella Fazzina,1* Andrea Mariotti,2* Annabella Procoli,2* Daniela Fioravanti,3 Paola Iudicone,3
Giovanni Scambia,2 Luca Pierelli,3,4 and Giuseppina Bonanno2

BACKGROUND: Mesenchymal stromal cells (MSCs)

isolated from human umbilical cord tissue (UCT) can be
considered the perfect candidates for cell-based
therapies and regenerative medicine. UCT-derived MSCs
can be cryogenically stored in cell banks and expanded
as needed for therapeutic uses.
STUDY DESIGN AND METHODS: We developed a
new method for UCT-MSC isolation, cryopreservation,
and expansion, following all criteria required by a stem
cell bank. UCT-MSCs were isolated either by manual
dissociation (MM) or by a semiautomatic dissociation
system (SAM). In both protocols UCTs were treated
enzymatically using Type IV collagenase good
manufacturing practices (GMP) graded and
hyaluronidase (medicinal product). Isolated UCT-MSCs
were cryopreserved and analyzed after thawing for
phenotype; for proliferation rate; and for their osteogenic,
adipogenic, and chondrogenic differentiation capabilities.
RESULTS: We found that SAM reduced the time of
tissue enzyme exposure and enabled us to obtain a
homogeneous single-cell suspension deprived of tissue
fragments. The isolated cells in both groups showed high
expression of MSC markers CD105, CD73, and CD90
and similar differentiation capabilities, phenotype, and
proliferation potential. Moreover, the final yield of MSCs
was comparable between the two techniques.
CONCLUSION: In this study, we have established a
reliable and standardized protocol to isolate UCT-MSCs
from UCT for cell banking purposes. Processing the
whole umbilical tissue with GMP-graded enzymes using
a semiautomatic dissociator allowed us to obtain a
single-cell suspension product with a known number of
isolated cells that can be cryopreserved right after
isolation and thawed as needed for expansion and
clinical use.

esenchymal stromal cells (MSCs) have

gained considerable attention over the past
decades as promising candidates for novel
clinical application. MSCs are multipotent
precursors identified by the expression of the surface antigens CD105, CD73, and CD90; the absence of hematopoietic markers CD34, CD45, CD14, and HLA-DR; and the
ability to differentiate into osteoblasts, adipocytes, and
chondroblasts in vitro.1 MSCs were initially isolated from
marrow by their tight adherence to plastic and their
capacity to form single cell colonies.2 For many years marrow has been considered the principal source of MSCs but
nowadays MSCs can be isolated from many other adult
tissues such as adipose tissue, dental pulp, amniotic fluid,

ABBREVIATIONS: DT 5 doubling time; GMP 5 good

manufacturing practices; MM 5 manual dissociation
method; MSC(s) 5 mesenchymal stromal cell(s); PDT 5
population doubling time; PL 5 platelet lysate; SAM 5
semiautomatic dissociation method; UCT 5 umbilical cord
tissue; UCTC(s) 5 umbilical cord tissue cell(s).
From the 1InScientiaFides Foundation, Republic of San
Marino; and the 2Institute of Gynecology and Obstetrics,
Sacred Heart Catholic University; 3Immunohematology and
Transfusion Medicine, San Camillo Forlanini Hospital; and the
4

Department of Experimental Medicine, Sapienza University,

Rome, Italy.
Address reprint requests to: Luca Pierelli, Immunohematology and Transfusion Medicine, San Camillo-Forlanini Hospital,
Rome, Italy; e-mail: lpierelli@me.com.
This work was supported by

the

InScientiaFides

Foundation.
*These authors contributed equally to this work.
Received for publication March 15, 2015; revision received
June 14, 2015; and accepted June 23, 2015.
doi:10.1111/trf.13277
C 2015 AABB
V

TRANSFUSION 2015;00;0000
Volume 00, Month 2015 TRANSFUSION 1

FAZZINA ET AL.

placenta, umbilical cord blood, and umbilical cord tissue

(UCT).3-5
UCT represents a novel source of MSCs, easily accessible with a noninvasive procedure, with no risks for the
donor and without ethical concerns.6 Moreover, several
studies have been demonstrated that human UCT-MSCs
have multipotent properties, between embryonic and
adult stem cells, since they are also able to differentiate
into neural progenitors, cardiomyocytes, skeletal myocytes, and hepatocytes.3,4,7-15 Furthermore, UCT-MSCs
showed a faster proliferation rate, compared to those
found in adult tissues, and a low immnogenicity, which
makes them an attractive candidate both for autologous
and for allogenic cellbased therapies.4,15,16 Additionally,
since UCT-MSCs are collected from a neonatal tissue,
they are expected to carry less nuclear and mitochondrial
mutations than cells isolated from aged individuals.17
For these multiple capabilities UCT-MSCs have been
employed in many preclinical and clinical trials for the
treatment of different pathologies such as Type I diabetes,
spinal cord injury, retinal disease, cerebral ischemia, ulcerative colitis, hepatic cirrhosis, Duchenne muscular dystrophy, amyotrophic lateral sclerosis, hereditary cerebellar
ataxia, cardiomyopathy, rheumatoid arthritis, multiple
sclerosis, and liver failure16,18,19 (www.clinicaltrials.com).
Moreover, because of their immunomodulatory, homing,
and trophic properties, as well as their ability to release
cytokines and growth factors, UCT-MSCs are believed to
support tissue regeneration and wound healing.20-23
Thus, in view of their rapidly increasing applications,
the idea of banking UCT-MSCs precursors, that is, UCT
cells (UCTCs), appears very interesting. However, for
banking purposes the use of standardized protocols for
UCTCs isolation, as a prerequisite to subsequent MSCs
expansion, remains a major issue, since it requires that all
procedures, from tissue collection to final cell product,
must be processed following the guidelines for good manufacturing practices (GMP).24,25 Moreover, to set up a clinical grade isolation protocol for UCTCs, it is important to
take into account that the isolation, manipulation, and
cryopreservation techniques may impact the yield and
quality of isolated cells.
Several groups have described two different strategies
to isolate UCT-MSCs: by the explant method or by enzymatic digestion. In the explant approach small pieces of
cord are seeded onto the surface of a culture dish, letting
MSC precursors migrate directly from tissue to the plastic
of the flask, while the enzymatic one uses collagenase,
hyaluronidase, or trypsin, alone or in combination with,
to separate the UCTCs from the connective tissue.8,26,27
Although the digestion requires a higher degree of
manipulation compared to the explant method, it seems
to be the more appropriate for banking, since it ends up
in a single-cell suspension containing a known number of
UCTCs, which can be cryopreserved immediately after
2

TRANSFUSION Volume 00, Month 2015

isolation and thawed as needed for subsequent MSCs

expansion without modifying their properties.25 In contrast, when the explant method is chosen, cell culture
must be performed to obtain single cells to cryopreserve,
which requires a more prolonged process time.
Some authors also described the alternative to freeze
down directly small pieces of UCT, although their data
showed that the postthaw processing does not always
result in MSC recovery.28 Additionally, data about viability
and recovery of cells isolated from long-term frozen tissue
has not been reported yet.
Regarding the blood vessels contained in the UCT, it
has been proven that their removal is not necessary to
obtain nascent MSCs. In fact, many articles have shown
that MSCs isolated either from whole cord tissue or from
specific cord regions, such as Whartons jelly, arteries,
vein, and cord lining, exhibit similar characteristics.6,27,29
Moreover, it is important to consider that the dissection of
distinct cord regions is more labor-intensive and timeconsuming than processing the entire cord piece, whereas
for banking purposes it is necessary to reduce manipulation steps.30 Thus, the aim of our study was to set up a
new and reliable standardized protocol for isolation and
cryopreservation of UCTCs as a privileged source of
MSCs.
In this study we attempted to compare two different
UCTC isolation methods, using either manual dissociation
(MM) or semiautomatic dissociation (SAM) by the gentleMACS tissue dissociator (Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany), to obtain an homogeneous cell suspension of UCTCs that can be cryopreserved immediately
after manipulation and stored for long-term periods for
future expansion of MSCs to be applied for clinical
purposes.

MATERIALS AND METHODS

UCT processing and UCTC collection
Ten umbilical cords were collected after Cesarean deliveries with the patients written consent at the Department
of Gynecology and Obstetric of the Catholic University of
Rome. The investigation was reviewed and approved by
the Ethical Committee of Catholic University of Sacred
Heart of Rome. UCTs were stored in sterile saline solution
(0.9% wt/vol sodium chloride) at 48C and processed
within 48 hours of collection. Sterility was assessed before
and after manipulation using an automated blood culture
system to detect both aerobic and anaerobic and fungal
contamination (BACTEC 9240, Becton Dickinson, San
Jose, CA). Whole cord was washed with sterile saline solution to remove blood and blood clots from the outer layer
and immersed in 0.05% sodium hypochlorite (Angelini,
Rome, Italy) for 2 minutes. Twenty grams of UCT was cut
into small segments with a sterile scalpel and divided in

UMBILICAL CORD MESENCHYMAL STROMAL CELLS

two equal parts. Two types of isolation methods were

used: half of the tissue was processed by MM and the
other half was processed using a SAM (gentleMACS), following the manufacturers instructions. The gentleMACS
dissociator is a bench-top instrument for the semiautomated dissociation of tissues into single-cell suspensions.
The system is supplied with a specific program for the
cord tissue dissociation and has been validated by the
manufacturer, obtaining the CE mark, for each preset
processing procedure and in our laboratory on a previous
series of experiments (eight experiments) in which the
gentleMACS dissociator never yielded a number of UCTCs
lower than 75% of that obtained with a parallel MM.
Briefly, every segment was cut into small pieces and
immersed in a solution containing phosphate-buffered
saline (PBS) with calcium and magnesium, 1 IU/mL
collagenase NB6 GMP grade (Serva Electrophoresis, Heidelberg, Germany), and 1 IU/mL hyaluronidase (Bioindustria, Laboratorio Italiano Medicinali, Novi Ligure, AL,
Italy). In the MM the tissue fragments were incubated for
2.5 hours at 378C without agitation. For the SAM process,
tissue fragments were first mechanically dissociated with
gentleMACS, following the manufacturers instructions,
and then incubated for 1.5 hours at 378C without agitation. After digestion, the cell suspension was filtered
through a 100-mm cell strainer (Miltenyi Biotec), washed
with PBS with 0.5% human albumin (Grifols, Sant Cugat
del Valles, BCN, Spain) and 0.02 mol/L ACDA, and centrifuged at 1500 rpm for 10 minutes.

centrates as previously described.31 Culture medium was

replaced every 3 days. Cells were harvested at confluence
(P0), by trypsin-EDTA treatment (Euroclone), counted
with a Neubauer chamber, and then reseeded at appropriate concentration for next propagation steps. After 90% of
confluency, usually within 1 week, cells were harvested
and used for phenotypic analysis and for differentiation
tests. Cells were expanded until P2. Proliferation rate was
determined by calculating total cell yield and population
doubling time (PDT). The total number of cells at each
passage was calculated as a ratio of number of cells harvested to number of cell seeded, multiplied by the total
number of cells from the previous passage. Total yield of
UCT-MSCs at P2 was calculated per grams of processed
UCT. DTs were calculated from cell counts with the
equation
DT : T log2=logY logX ;
where (X) indicates cells at seeding and (Y) indicates the
harvested cells.

Statistical analysis
All comparisons were performed with the t test. Data
obtained by parallel growth experiments using MM or
SAM were compared during culture passages in terms of
DT, and total cell yield, by a paired two-tailed t test. A p
value of less than 0.05 was considered significant.

Phenotypic analysis by flow cytometry

Cryopreservation
Cell suspensions obtained after tissue digestion were centrifuged at 329 3 g for 10 minutes and counted using trypan blue assay. Total cells (UCTCs) were resuspended in
low-glucose GMP-grade Dulbeccos modified Eagles
medium (DMEM; LystarFish, Cernusco S/N, Milan, Italy)
with 2% human albumin and 10% GMP-grade dimethyl
sulfoxide (LystarFish) and frozen using a controlled-rate
cooling program (218C/min) with a controlled-rate
freezer (Planer Kryo 50-16, Cryo Solutions, Hertogenbosch, the Netherlands). Cells were stored in liquid nitrogen for at least 1 week.

UCTC culture and UCT-MSC propagation

and proliferation
UCTCs were thawed in a water bath at 378C and immediately resuspended in prewarmed complete medium. Cell
viability was determined by trypan blue assay. All cells
were plated in low-glucose GMP-grade DMEM (ListarFish)
supplemented with 10% human platelet lysate (PL; trademarked as Mesengen and kindly provided by Futura ReLife srl Rome. Italy), 4 IU/mL heparin, and 2 mmol/L Lglutamine (Euroclone, Pero, MI, Italy). PL was obtained by
pools of pathogen-inactivated human donor platelet con-

For phenotypic analysis UCT-MSCs were harvested and

stained for 15 minutes at room temperature with the following monoclonal antibodies (MoAbs): MSC phenotyping kit (anti-human CD105-PE, CD90-FITC, CD73-APC,
CD14/CD20/CD34/CD45-PerCP, Miltenyi Biotec) antihuman CD166-PE, anti-human HLA-DRPE-Cy7 (BD
PharMingen, Heidelberg, Germany), VE-cadherinAPC
(Biolegend, San Diego, CA), CD31-FITC (Biolegend), or
with the appropriate fluorochrome-conjugated, isotypematched irrelevant MoAbs to establish background fluorescence. Cells were washed with PBS, centrifuged at
287 3 g for 5 minutes and resuspended in PBS for analysis.
Samples were run through a flow cytometer (FACSCalibur,
Becton Dickinson) with standard equipment. Data were
analyzed with computer software (CellQuest, Becton
Dickinson).

Osteogenic, chondrogenic, and adipogenic

differentiation
The UCT-MSCs osteogenic differentiation was induced
using NH OsteoDiff medium (Miltenyi Biotec) according
to manufacturers instructions. Briefly, 5 3 104 cells were
resuspended in NH OsteoDiff medium, plated in 35-mm
cell culture dishes, and incubated at 378C. Cell culture
Volume 00, Month 2015 TRANSFUSION 3

FAZZINA ET AL.

Fig. 1. (A) Umbilical cord (UC) dissociation methods. UCT was divided in two equal parts and processed in parallel by two methods: the MM and the SAM based on the gentleMACS technology. (B) UCTC yield after thawing. Cell recovery after thawing of cell
suspensions obtained by SAM and MM. Results are represented as mean 6 SEM. Statistical analysis did not show a significant difference (p > 0.05).

medium was replaced every 3 days. After 10 days of culture cells were fixed with a citrate-acetone-methanol solution, stained with alizarin red S (Sigma-Aldrich, St Louis,
MO) according to manufacturers instructions. The UCTMSCs differentiation into chondrogenic lineage was performed using NH ChondroDiff medium (Miltenyi Biotec)
according to manufacturers instructions. Briefly, a micromass culture of UCT-MSCs was prepared resuspending
2.5 3 105 cells in NH ChondroDiff medium (Miltenyi Biotec) into a polypropylene conical tube. Tubes were incubate at 378C and culture medium was replaced every 3
days. On Day 24 differentiated chondrocytes, which
formed three-dimensional clusters, termed chondrocyte
nodules, were fixed in 4% formalin and incubated overnight at room temperature. Nodules were dehydrated
with ethanol dilution series followed by 30 minutes of
incubation in xylol and then included in paraffin at 588C.
Three-micrometer-thick tissue sections were obtained
with a microtome. Chondrocyte differentiation was
detected by observing toluidine blue (BioOptica, Milan,
Italy) staining of extracellular matrix.
The UCT-MSCs adipogenic differentiation was
induced using NH AdipoDiff medium (Miltenyi Biotec)
according to manufacturers instructions. Briefly, 5 3 104
cells were resuspended in NH AdipoDiff medium and
plated in 35-mm cell culture at 378C . Cell culture medium
was replaced every 3 days. After 2 to 3 weeks of culture,
4

TRANSFUSION Volume 00, Month 2015

cells were washed twice with deionized H2O and stained

with oil red O (Sigma-Aldrich) to detect adipocytes.

Karyotype analysis
For karyotype analysis UCT-MSCs were cultured for 90
minutes using the above-mentioned culture medium with
the addiction of colchicine (Sigma-Aldrich) at a final concentration of 0.03 mg/mL. Cells were then harvested and
treated with 0.075 mol/L hypotonic potassium chloride
(Sigma-Aldrich) for 20 minutes at 378C and then fixed in
acetic acid:methanol at a 1:3 ratio. Slides were then dried
for 24 hours at 608C and then incubated for 24 hours at
room temperature. For G-banding, slides were treated
with 0.25% trypsin for 55 to 60 seconds, rinsed in saline
solution, and stained with 5% Giemsa for 2 minutes. Cytogenetic analysis was performed with an analysis system
(Cytovision Image, Olympus, Shinjuku, Tokyo, Japan).

RESULTS
Two different isolation methods, using either MM or SAM,
were used for UCTC isolation (Fig. 1A). Digestion of UCT
with SAM presented many advantages compared to
the MM. The SAM procedure required a lower enzyme
incubation time than MM, 1 hour 30 minutes instead of
2 hours 30 minutes, to homogenize cord tissue. Moreover,
after gentleMACS dissociation, the cell suspension obtained

UMBILICAL CORD MESENCHYMAL STROMAL CELLS

Fig. 2. Morphology of UCT-MSCs in culture. Spindle-shaped adherent cells with MSC morphology were observed in UCTC culture
isolated by SAM (A) and MM (B) on Day 10 (microscope examination at 2003). At confluence UCT-MSCs obtained with both
methods (C, D) were distributed in a homogeneous monolayer (microscope examination at 1003). UCT-MSCs obtained by SAM
and MM reached confluence, respectively, on Days 18.9 6 5.5 and 18.1 6 3.7.

was more homogeneous and deprived of tissue fragments,

which resulted in a faster and easier filtration of the
digested tissue. Indeed, the digestion and filtration steps
were performed in half the time in the SAM procedure compared to MM (2 hr rather than 3.5 hr), which is very important in the perspective of a stem cell bank procedure.
The number of primary cells isolated was evaluated
by trypan blue exclusion assay after both procedure
types. Cell yield was comparable between the two methods and showed a high variability, ranging from 1 3 106
to 10 3 106 cells/g tissue, which is probably due to the
presence of blood cells, fibroblasts, endothelial cells,
and stromal cells of cord tissue, as well as to the individual variability within human samples. Within this heterogeneous population of cells, the MSC fraction was not
detectable by flow cytometric analysis, most likely due
to the low number of MSCs. All primary cells obtained
from 10 g of UCT were frozen using a clinical-grade system, stored in liquid nitrogen, and thawed after 1 to 12
weeks to evaluate the MSC number and expansion rate.

Cell recovery after thawing was evaluated by trypan blue

exclusion assay, showing a mean of primary cells of 1.2
3 105 cells/g for SAM and 0.74 3 105 cells/g for MM
(Fig. 1B). Sterility assay was performed before and after
processing cord tissue and resulted in 95% of negativity
for aerobic or anaerobic and fungal contaminants. The
sterility test was performed as a quality control (QC) for
the whole process, including the collection procedure,
since cord tissues are collected in the operating room
during Cesarean deliveries, which represents a major
risk factor for bacterial and fungal contamination
because the tissues may be exposed to the air. In fact,
the only positive microbiologic culture was found before
processing; thus, it was most likely contaminated during
collection. However, the microbiologic culture was negative after processing, confirming the effectiveness of tissue disinfection with 0.05% sodium hypochlorite for low
microbial load removal. To evaluate the yield and the
growth kinetics of UCT-MSCs isolated from the UCT, all
cells were thawed and plated in low-glucose DMEM
Volume 00, Month 2015 TRANSFUSION 5

FAZZINA ET AL.

Fig. 3. (A) Comparison of cell proliferation capability and

cumulative cell yield of cultured UCT-MSCs. The proliferation ability of UCT-MSCs derived from UCTCs, isolated by
SAM and MM, was measured in terms of DT. Data are shown
as means 6 SEM of 10 umbilical cords processed. (B) Cumulative cell yield at P2. The number of UCT-MSCs harvested at
P2 per gram of UCT processed with the two different methods were compared. Results are represented as mean 6 SEM.

supplemented with PL, which is a human fetal bovine

serum substitute already known to promote growth and
proliferation of MSCs without altering their phenotypical
and functional characteristics.31 As shown in Fig. 2, small
colonies of spindle-shaped adherent cells with MSC morphology start to appear, both in the SAM and in the MM
cultures, after 10 days of culture and reached confluence,
respectively, on Day 18.9 6 5.5 and Day 18.1 6 3.7. UCTMSCs were expanded for at least two subculture passages.
No differences in morphology and cell shape were observed
between the two cultures during passages. Regarding the
proliferation capacity of UCT-MSCs derived from SAM and
MM procedures, Fig. 3A shows the results in terms of PDT
mean obtained from all samples and calculated between P1
and P2. No significant differences in proliferation rate were
observed between UCT-MSCs obtained from SAM and
MM, with PDT means, respectively, of 1.82 and 1.51. The
cumulative yield of UCT-MSCs at the end of three passages
(P0-P2) had a great variability within samples in both dissociation methods, ranging between 0.76 3 106 and 14.2 3
106 per gram for the SAM and between 2.74 3 106 and 13.8
3 106 per gram for the MM (data not shown). This could
depend more on intrinsic properties of the umbilical cords
or interdonor variability than on the method. However, as
depicted in Fig. 3B, the mean of UCT-MSCs isolated per
6

TRANSFUSION Volume 00, Month 2015

gram of cord tissue was comparable between the two procedures, resulting in 6.5 3 106 MSCs/g for the MM and 6.13
3 106 MSCs/g for the SAM, that, taking into account that
the total weight of the umbilical cord can range from 20 to
100 g, is a number of cells that may be suitable for cell therapy. The cell surface antigen profile of UCT-MSCs either by
SAM or by MM was determined and compared by flow
cytometric analysis. Cells derived from both isolation methods exhibited a typical MSC immunophenotype as defined
by International Society of Cellular Therapy. The UCTMSCs were strongly positive for the MSCs markers CD73,
CD105, CD90, and CD166 with similar intensities expression between the two procedures, while they were negative
for the hematopoietic markers CD34, CD45, CD14, and
CD20 and for the HLA-DR antigen (Fig. 4). Moreover, since
we processed the whole UCT without blood vessel removing, we investigated the presence of residual endothelial
cells in the culture. Flow cytometric analysis showed the
absence of typical endothelial antigens such as CD31 and
VE-cadherin confirming the selectivity of culture medium
for MSCs. No significant differences were found in antigen
expression between the two populations of MSCs. When we
analyzed and compared the ability of UCT-MSCs, SAM versus MM, to differentiate into the three mesenchymal lineages, we found that both MSC populations had the ability
to differentiate toward adipocytes, osteoblasts, and chondrocytes, as shown in Fig. 5. Indeed, after 10 days under
osteogenic stimuli UCT-MSCs showed typical osteogenic
morphologic features and calcium deposits revealed by alizarin red S staining, while the unstimulated UCT-MSCs did
not present Ca21 deposits. In the adipogenic induction,
many large, flattened, often oval cells appeared in the culture, showing small lipid droplets accumulation positive for
oil red O staining, while the nonstimulated cells maintained
their original spindle-shaped morphology and the formation of lipid granules was not observed (Fig. 5). The chondrogenic potential was evaluated using a pellet micromass
system. Under these conditions UCT-MSCs showed cell
condensation in nodule-like structures and a high intensity
of glycosaminoglycans production from cell aggregates,
revealed by metachromatic toluidine blue staining. No
micromass formation was observed in nonconditioned
UCT-MSCs. No visible difference in staining was noted in all
three differentiation assays between the two populations.
Karyotype analysis was performed as a QC for genetic stability of MSCs-UCT cells isolated with both MM and SAM protocol. Analysis of 20 metaphases confirmed that no
significant chromosomal aberrations were found in MSCs
from either method (Fig. 6).

DISCUSSION
UCT has gained in importance in the past years as a novel
source of MSCs for tissue engineering and regenerative
medicine applications. UCT-MSCs present many

UMBILICAL CORD MESENCHYMAL STROMAL CELLS

Fig. 4. Flow cytometric analysis of UCT-MSC phenotype at P1. (A) Comparison of MSC marker and endothelial antigen expression of
MM- and SAM-derived UCT-MSCs measured by flow cytometry. Results are expressed as mean 6 standard error deviation. (B) Histograms showing the expression of MSC-positive and -negative surface antigens of UCT-MSCs isolated with SAM and MM (two representative experiments are shown).

advantages compared to MSCs isolated from other adult

tissues, due to the noninvasive collection procedure,
their higher proliferation ability, their low immnogenicity, the multilineage differentiation ability, and their
immunomodulatory and trophic properties.13-15,17 For
these characteristics UCT-MSCs are currently under
evaluation in several clinical trials for neural, cardiac,
hepatic, and immunologic diseases. In this scenario, the
idea of banking the UCTCs appears very interesting.
However, for banking purpose the UCTC isolation
method become of critical importance since it must
maximize cell yield without compromising cell features.
Moreover, the development of cellular therapeutics
requires the use of standardized products for cell processing and culture and the compliance with GMP.24,25 To
set up an efficient and standardized procedure for isolation and banking of UCTCs as privileged source of
MSCs, we developed a new semiautomated protocol
based on gentleMACS technology. We chose to perform
cell isolation by processing the whole UCTs using enzy-

matic digestion with collagenase and hyaluronidase,

without removing blood vessels. Indeed, as shown by
several reports, the time-consuming and labor-intensive
dissection of the cord into specific regions is not necessary to obtain a valuable population of UCT-MSCs, since
enzymatic digestion of whole cord provides cells with
MSC-like properties similar to those isolated from separated cord regions.6,29,30 In our protocol, we combined
the enzymatic digestion with a semiautomated dissociation, which allows a consistent homogenization of tissue, lowering the time of enzyme exposure required.
Nevertheless, enzymatic digestion is one of the most
critical steps since excessive and prolonged digestion
might affect cell viability. We compared the SAM to the
MM based on tissue fragmentation by surgical scalpel.
Our results showed that the mechanical device reduced
considerably the processing time and tissue handling. In
fact, the isolation process was performed in less than
half the time with SAM compared to MM, allowing us to
save 1.5 hour for each sample processed, which is very
Volume 00, Month 2015 TRANSFUSION 7

FAZZINA ET AL.

Fig. 5. Differentiation potential of UCT-MSCs isolated by SAM and MM. Representative images of undifferentiated UCT-MSCs (A,
D, G) and UCT-MSCs isolated with SAM and MM, harvested at P1, and induced to differentiate into osteogenic (B, C), adipogenic
(E, F), and chondrogenic lineages (H, I). A-C, magnification 2003; D-F, magnification 4003; G, magnification 2003; H, I, magnification 403.

Fig. 6. Karyotype analysis G-banding analysis of UCT-MSCs isolated with MM (A) and SAM protocol (B). Normal karyotype
2n 5 23, autosomal and sex chromosomes.

important in the perspective of a stem cell bank procedure. Regarding cost analysis, both procedures required
use of the same materials, except for the gentleMACS
8

TRANSFUSION Volume 00, Month 2015

instrument used for SAM. However, considering the

labor-time costs, the SAM was remarkably more
convenient.

UMBILICAL CORD MESENCHYMAL STROMAL CELLS

Moreover, the digested tissue after SAM resulted in a

homogeneous cell suspension, deprived of tissue fragments and ready to be cryopreserved. Regarding the primary UCTCs obtained after digestion, we found that the
number of total viable cells was comparable between the
two methods and showed a high variability, probably due
to the presence of heterogeneous cells derived from cord
tissue, as well as to the individual variability within human
samples. Within this heterogeneous population of cells, the
MSC fraction was not detectable by flow cytometric analysis, most likely due to the low number of MSCs. In fact,
data regarding the characterization of freshly isolated
MSCs from tissues such as marrow and cord tissue has
been hampered by the scarcity of cells within the tissue
(i.e., 0.001%-0.01% in marrow) and by its limited availability in large quantity. In contrast, analysis of uncultured
MSCs has been reported for adipose tissue, due to the
abundant quantity available and the high percentage of
these cells within the tissue, which allow extensive analysis.
Moreover, it has been demonstrated that these cells change
remarkably their phenotype very early during cell culture.
In particular, after expansion in vitro, these progenitor cells
showed an increased expression of MSC-related markers,
such as CD105 and CD90, and the loss of HSC marker
CD34.32,33 Another point to be considered is that the UCTC
suspension includes several cell subpopulations, some of
which share the same markers. Thus, the native MSC fraction cannot be easily identified within the primary UCTCs.
We wanted then to investigate whether this UCTC
suspension could be cryopreserved directly after digestion
to have a ready to use cell suspension for potential MSC
isolation and expansion. Thus, all the UCTCs isolated
were frozen using a clinical-grade system and subsequently thawed to evaluate UCT-MSC recovery, expansion
rate, and phenotypic and functional characteristics. Postthaw cells were plated in low-glucose DMEM with PL
added, which is a serum-free supplement that can be
used to support MSC clinical-scale cultures without
affecting their antigenic and functional properties.
UCT-MSCs isolated by SAM and MM start to appear
after 10 days of culture and were expanded for at least two
subculture passages to evaluate their proliferation capacity.
Our results showed no differences in proliferation capabilities between the UCT-MSCs obtained from UCTCs isolated with both methods. PDT showed similar duration for
both the two conditions, ranging from 1.82 to 1.51 days, for
SAM and MM, respectively. Additionally, the mean of UCTMSCs isolated per gram of UCT was comparable between
the two procedures, resulting in 6.13 3 106 MSCs/g for the
SAM and 6.5 3 106 MSCs/g for the MM.
Our results indicate that the UCT is a likely source of
MSCs that can potentially provide a suitable number of
cells for cell therapy, taking into account that the total
weight of an umbilical cord can range from 20 to 100 g and
from each gram of tissue it is possible to obtain at least 6 3

106 MSCs after only two passages. The investigation of the

phenotypical and functional characteristics of the UCTMSCs showed that UCT-MSCs obtained from both isolation methods exhibited a typical MSC immunophenotype
as defined by International Society of Cellular Therapy. As
depicted in Fig. 4, UCT-MSCs were strongly positive for
CD73, CD105, CD90, and CD166, while they were negative
for the hematopoietic markers (CD34, CD45, CD14, CD20)
and for the HLA-DR antigen. Moreover, these UCT-MSCs
did not show expression of endothelial antigens (CD31 and
VE-cadherin), confirming the absence of residual endothelial cells in the culture. No significant differences were
found in antigen expression between UCT-MSCs derived
from SAM or MM. Additionally, both UCT-MSCs population showed the ability to differentiate toward adipocytes,
osteoblasts, and chondrocytes.
In this study we compared two different isolation
methods, using either MM or SAM, to obtain an homogeneous cell suspension of UCTCs that can be cryopreserved
immediately after manipulation and stored for long-term
periods for subsequent clinical applications. For the first
time, a mechanical device was used to isolate a source of
MSCs from UCT. The semiautomated technique resulted in
a faster and more standardized procedure for isolation and
banking of UCTCs, as source of mesenchymal progenitor
cells. Our results showed that UCTCs obtained using the
semiautomated procedure can be cryopreserved immediately after processing without compromising the proliferation capabilities, phenotype, and multipotency properties
of UCT-MSCs. The potential benefit of the semiautomated
procedure is manyfold and could help to standardize and
streamline the processing of UCT for banking purposes.
ACKNOWLEDGMENTS
We are grateful to Dr Angelitti Maria Rosaria, Bertolini Marina,
and Dr Carla Ruscio for karyotype analysis.

CONFLICT OF INTEREST
The authors have disclosed no conflicts of interest.

REFERENCES
1. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for
defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
Cytotherapy 2006;8:315-7.
2. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guineapig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:
393-403.
3. Nombela-Arrieta C, Ritz J, Silberstein LE. The elusive nature
and function of mesenchymal stem cells. Nat Rev Mol Cell
Biol 2011;12:126-31.
Volume 00, Month 2015 TRANSFUSION 9

FAZZINA ET AL.

4. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in

health and disease. Nat Rev Immunol 2008;8:726-36.
5. Secunda R, Vennila R, Mohanashankar AM, et al. Isolation,
expansion and characterisation of mesenchymal stem cells
from human bone marrow, adipose tissue, umbilical cord
blood and matrix: a comparative study. Cytotechnology 2014
[Epub ahead of print].
6. Mennan C, Wright K, Bhattacharjee A, et al. Isolation and
characterisation of mesenchymal stem cells from different
regions of the human umbilical cord. Biomed Res Int 2013;
2013:916136.7. Yang H, Xie Z, Wei L, et al. Human umbilical cord mesenchymal stem cell-derived neuron-like cells rescue memory deficits and reduce amyloid-beta deposition in an AbPP/PS1
transgenic mouse model. Stem Cell Res Ther 2013;4:76.
8. De Bruyn C, Najar M, Raicevic G, et al. A rapid, simple, and
reproducible method for the isolation of mesenchymal stromal cells from Whartons jelly without enzymatic treatment.
Stem Cells Dev 2011;20:547-57.
9. Qian Q, Qian H, Zhang X, et al. 5-Azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated
kinase. Stem Cells Dev 2012;21:67-75.
10. Kocaefe C, Balci D, Hayta BB, et al. Reprogramming of human
umbilical cord stromal mesenchymal stem cells for myogenic
differentiation and muscle repair. Stem Cell Rev 2010;6:512-22.
11. An SY, Han J, Lim HJ, et al. Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cordderived mesenchymal stem cells. Tissue Cell 2014;46:127-35.
12. Anzalone R, Lo Iacono M, Corrao S, et al. New emerging
potentials for human Whartons jelly mesenchymal stem
cells: immunological features and hepatocyte-like differentiative capacity. Stem Cells Dev 2010;19:423-38.
13. Troyer DL, Weiss ML. Whartons jelly-derived cells are a
primitive stromal cell population. Stem Cells 2008;26:591-9.
14. Fong CY, Chak LL, Biswas A, et al. Human Whartons jelly
stem cells have unique transcriptome profiles compared to
human embryonic stem cells and other mesenchymal stem
cells. Stem Cell Rev 2011;7:1-16.
15. Weiss ML, Anderson C, Medicetty S, et al. Immune properties of human umbilical cord Whartons jelly-derived cells.
Stem Cells 2008;26:2865-74.
16. Kim DW, Staples M, Shinozuka K, et al. Whartons jellyderived mesenchymal stem cells: phenotypic characterization and optimizing their therapeutic potential for clinical
applications. Int J Mol Sci 2013;14:11692-712.
17. Nagamura-Inoue T, He H. Umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical
utility. World J Stem Cells 2014;6:195-202.
18. Bongso A, Fong CY. The therapeutic potential, challenges and
future clinical directions of stem cells from the Whartons jelly
of the human umbilical cord. Stem Cell Rev 2013;9:226-40.
19. El Omar R, Beroud J, Stoltz JF, et al. Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal

10

TRANSFUSION Volume 00, Month 2015

stem cell-based therapies? Tissue Eng Part B Rev 2014;20:

523-44.
rcia RN, Simo
~ es SI, et al. The role of human
20. Santos JM, Ba
umbilical cord tissue-derived mesenchymal stromal cells
(UCX(R)) in the treatment of inflammatory arthritis. J Transl
Med 2013;11:18.
21. Martins JP, Santos JM, de Almeida JM, et al. Towards an
advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue:
quality and safety data. Stem Cell Res Ther 2014;5:9.
22. Shohara R, Yamamoto A, Takikawa S, et al. Mesenchymal
stromal cells of human umbilical cord Whartons jelly accelerate wound healing by paracrine mechanisms. Cytotherapy
2012;14:1171-81.
23. Amable PR, Teixeira MW, Carias RB, et al. Protein synthesis
and secretion in human mesenchymal cells derived from
bone marrow, adipose tissue and Whartons jelly. Stem Cell
Res Ther 2014;5:53.
rst D, et al. GMP-compliant isola24. Fekete N, Rojewski MT, Fu
tion and large-scale expansion of bone marrow-derived
MSC. PLoS One 2012;7:e4325525. Silva CP. Isolation of human umbilical cord mesenchymal
stem/stromal cells from a stem cell bank perspective: an
integrated overview. IJPCBS 2014;4:31-410.
26. Salehinejad P, Alitheen NB, Ali AM, et al. Comparison of different methods for the isolation of mesenchymal stem cells
from human umbilical cord Whartons jelly. In Vitro Cell Dev
Biol Anim 2012;48:75-83.
27. Li DR, Cai JH. Methods of isolation, expansion, differentiating
induction and preservation of human umbilical cord mesenchymal stem cells. Chin Med J (Engl) 2012;125:4504-10.
28. Chatzistamatiou TK, Papassavas AC, Michalopoulos E, et al.
Optimizing isolation culture and freezing methods to preserve Whartons jellys mesenchymal stem cell (MSC) properties: an MSC banking protocol validation for the Hellenic
Cord Blood Bank. Transfusion 2014;54:3108-20.
29. Ishige I, Nagamura-Inoue T, Honda MJ, et al. Comparison of
mesenchymal stem cells derived from arterial, venous, and
Whartons jelly explants of human umbilical cord. Int J Hematol 2009;90:261-9.
30. Hendijani F, Sadeghi-Aliabadi H, Haghjooy Javanmard S.
Comparison of human mesenchymal stem cells isolated by
explant culture method from entire umbilical cord and
Whartons jelly matrix. Cell Tissue Bank 2014;15:555-65.
31. Iudicone P, Fioravanti D, Bonanno G, et al. Pathogen-free,
plasma-poor platelet lysate and expansion of human mesenchymal stem cells. J Transl Med 2014;12:2832. Boquest AC, Shahdadfar A, Frnsdal K, et al. Isolation and
transcription profiling of purified uncultured human stromal
stem cells: alteration of gene expression after in vitro cell
culture. Mol Biol Cell 2005;16:1131-41.
33. Baer PC, Geiger H. Adipose-derived mesenchymal stromal/
stem cells: tissue localization, characterization, and heterogeneity. Stem Cells Int 2012;2012:812693.