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Scandinavian Journal of Clinical and LaboratoryInvestigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Comparison of clinical grade human plateletlysates for cultivation of mesenchymal stromalcells from bone marrow and adipose tissue
Morten Juhl, Josefine Tratwal, Bjarke Follin, Rebekka H. Søndergaard, MariaKirchhoff, Annette Ekblond, Jens Kastrup & Mandana Haack-Sørensen
To cite this article: Morten Juhl, Josefine Tratwal, Bjarke Follin, Rebekka H. Søndergaard, MariaKirchhoff, Annette Ekblond, Jens Kastrup & Mandana Haack-Sørensen (2016): Comparison ofclinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bonemarrow and adipose tissue, Scandinavian Journal of Clinical and Laboratory Investigation, DOI:10.3109/00365513.2015.1099723
To link to this article: http://dx.doi.org/10.3109/00365513.2015.1099723
Published online: 11 Jan 2016.
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SCANDINAVIAN JOURNAL OF CLINICAL & LABORATORY INVESTIGATION, 2015http://dx.doi.org/10.3109/00365513.2015.1099723
ORIGINAL ARTICLE
Comparison of clinical grade human platelet lysates for cultivation ofmesenchymal stromal cells from bone marrow and adipose tissue
Morten Juhla, Josefine Tratwala, Bjarke Follina, Rebekka H. Søndergaarda, Maria Kirchhoffb, Annette Ekblonda,Jens Kastrupa and Mandana Haack-Sørensena
aCardiology Stem Cell Centre, The Heart Centre, Rigshospitalet, Copenhagen University Hospital; bDepartment of Clinical Genetics,Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
ABSTRACTBackground: The utility of mesenchymal stromal cells (MSCs) in therapeutic applications forregenerative medicine has gained much attention. Clinical translation of MSC-based approachesrequires in vitro culture-expansion to achieve a sufficient number of cells. The ideal cell culturemedium should be devoid of any animal derived components. We have evaluated whether humanPlatelet Lysate (hPL) could be an attractive alternative to animal supplements. Methods: MSCs frombone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donorswere culture expanded in three different commercially available hPL fulfilling good manufacturingpractice criteria for clinical use. BMSCs and ASCs cultured in Minimum Essential Medium Eagle-alpha supplemented with 5% PLT-Max (Mill Creek), Stemulate� PL-S and Stemulate� PL-SP (COOKGeneral Biotechnology) were compared to standard culture conditions with 10% fetal bovineserum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiationpotential were analyzed. Results: Regardless of manufacturer, BMSCs and ASCs cultured in hPLmedia showed a significant increase in proliferation capacity compared to FBS medium. In general,the immunophenotype of both BMSCs and ASCs fulfilled International Society for Cellular Therapy(ISCT) criteria after hPL media expansion. Comparative genomic hybridization measurementsdemonstrated no unbalanced chromosomal rearrangements for BMSCs or ASCs cultured in hPLmedia or FBS medium. The BMSCs and ASCs could differentiate into osteogenic, adipogenic, orchondrogenic lineages in all four culture conditions. Conclusion: All three clinically approvedcommercial human platelet lysates accelerated proliferation of BMSCs and ASCs and the cells meetthe ISCT mesenchymal phenotypic requirements without exhibiting chromosomal aberrations.
ARTICLE HISTORY
Received 21 May 2015Revised 5 September 2015Accepted 21 September 2015Published online24 December 2015
KEYWORDS
Platelet lysate, mesenchymalstromal cells, adipose tissue-derived stromal cells, cellculture, clinical application
Introduction
In recent years, developments and progress in stem cell
technology have given rise to new therapeutic strategies
for different degenerative diseases. Mesenchymal stro-
mal cells (MSCs) are a rare and quiescent population
that can be isolated from several tissue sources, which
have gained much attention for regenerative medicine
because they hold no ethical concerns, are capable
of self-renewal, can differentiate to a variety of cell
lineages, in addition to trophic and immunosuppressive
effects [1]. MSCs from bone marrow (BMSCs) are the
most well-defined, and have been tested in several
clinical trials for a wide range of therapeutic applications
[2,3]. However, MSCs isolated from adipose tissue,
adipose tissue-derived stromal cells (ASCs), have been
suggested as an ideal cell source for regenera tive
medicine, as adipose tissue is an abundant and readily
accessible site for isolation of cells suitable for regen-
erative medicine applications [4,5]
The potential clinical applications of autologous and
allogeneic MSCs include treatment of conditions with
limited treatment options. As such, MSCs are being used
as experimental treatment of as diverse pathologies as
cardiovascular disease, liver disease, and autoimmune
conditions [2,3]. Clinical translation of cell-based
approaches often requires clinical grade in vitro culture-
expansion to achieve sufficient therapeutic number of
cells [6], and although no single standard protocol for
the culture expansion of MSCs exists, fetal bovine serum
(FBS) has been the most commonly used medium
supplement, which significantly contributes to growth
of a variety of cell types and the rapid development of
clinical cell-based therapeutics. While FBS is still widely
CONTACT Mandana Haack-Sørensen, PhD [email protected] Cardiology Stem Cell Centre, Rigshospitalet, Copenhagen UniversityHospital, DK-2100 Copenhagen Ø, Denmark
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used for cell culture and accepted by
regulatory authorities, its use in clinical settings is
associated with the risk of disease transmission from
animals to humans [7–9] and the possible complications
associated with animal supplements may restrict such
regulatory status. Moreover, there is a concern with a
large batch-to-batch variation which can affect the
quality of cell lots generated for banking [7,10]. This
concern is echoed by guidelines issued by the highest
authorities, such as from FDA and EMA, urging a
substitution from material of animal origin to human
whenever possible [11,12]. To achieve more standar-
dised and reproducible manufacturing processes proto-
cols refraining from using animal sera should be
developed [7,10,13].
Efforts have been made to replace animal sera
supplements and to design more standardised and
well-defined serum-free formulations. However, custom
media development is often needed based on the cell
type, source, and species, which would make it an
expensive and impractical option for large-scale cell
expansion [14–17].
Human platelet lysate (hPL), which is typically
prepared from pooled platelet-rich plasma by lysis
through freeze-thaw cycles, sonication, or activation,
has proven to be a very effective cell culture additive
[9,18–21]. However, hPL is often prepared in small
portions at different local university hospitals, and the
small batches reveal significant lot-to-lot variability.
We consider it important and necessary to use a
simple, standardised and uniform culture medium addi-
tive produced under GMP for culture expansion of MSCs
in vitro for clinical use. In the present study, three
different commercial hPL products were studied for
expansion of both BMSCs and ASCs. These hPL products
are manufactured with proper documentation at an
industrial scale in accordance with GMP standards from a
large number of pooled human platelet donors, result-
ing in a low lot-to-lot variability, high consistency and
purity. The cultivation efficacy of the three different hPL
media was compared to FBS medium with regard to cell
proliferation, immunophenotype, differentiation poten-
tial, and genomic stability.
Materials and methods
Media and supplements
Lymphoprep (1077 g/cm3, Medinor, Denmark);
Phosphate-Buffered Saline (PBS) (Gibco, Life
Technologies); Collagenase NB4 (Serva GmbH,
Germany); Hank’s Balanced Salt Solution (HBSS) (with
CaCl2 and MgCl2) (Gibco, Life Technologies); Minimum
Essential Medium Eagle Alpha (aMEM) without
Ribonucleosides and Deoxyribonucleosides, (Gibco, Life
Technologies); Penicillin/Streptomycin (Gibco, Life
Technologies); Heparin (1000 IE/mL, Amgros); irradiated
Fetal Bovine Serum (FBS) (Gibco, Life Technologies);
TrypLE� Select (Gibco, Life Technologies); Human plate-
let lysate (PLTMax) (Mill Creek Life Sciences); Stemulate
(PL-S) (COOK General Biotechnology); Stemulate (PL-SP)
(COOK General Biotechnology). All three hPL have been
approved for manufacturing cells for human clinical use.
Experimental design
ASCs were isolated from lipoaspirate obtained from
three healthy female donors (age between 32 and 47
years; mean age 40 years). BMSCs were obtained from
bone marrow aspirate from three healthy donors, one
male and two females (age 20–25 years; mean age 22
years). The use of ASCs and BMSCs from healthy
volunteers was approved by the National Ethical
Committee protocol no. H-3-2009-119. All donors
signed the informed consent.
The experimental set-up is illustrated in Figure 1. The
isolated mononuclear cells (MNCs) from bone marrow
and stromal vascular fraction (SVF) from adipose tissue
were cultured in four different GMP-compliant media
containing 5% hPL or 10% FBS. BMSCs and ASCs P0, P1,
and P5 were characterized and used for different
analyses.
Bone marrow preparation and MNC isolation
A total of 50 mL bone marrow aspirate was obtained
from the iliac crest by needle aspiration under local
anesthesia. MNCs were harvested by gradient centrifu-
gation on Lymphoprep, as described previously [22],
washed with PBS and counted using NucleoCounter�
NC-100� (Chemometec, Denmark) according to manu-
facturer’s instructions.
Lipoaspirate preparation and SVF isolation
Approximately 100 mL lipoaspirate was obtained from
liposuctions of subcutaneous abdominal fat performed
under local anesthesia. SVF isolation was performed
according to Zuk et al., with some modification [23]. The
lipoaspirate was washed twice with PBS to remove
residual blood. The adipose tissue was digested by
incubation with 0.6 PZ U/mL collagenase NB4 dissolved
in HBSS (diluted to a concentration of 2 mM Ca2+) at
37 �C for 45 min under constant rotation. The collage-
nase was neutralized with complete medium and the
suspension was filtered through a 100 mm mesh (Cell
Strainer, BD Falcon), centrifuged at 1200 g for 10 min at
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room temperature, re-suspended, and the resulting
isolated SVF was counted using NucleoCounter� NC-
100�.
Cell culture
Primary cell cultures of BMSCs and ASCs were estab-
lished by seeding 2� 107 MNCs/T75-flask (Nunc, Thermo
Scientific, Denmark) and 4.5� 106 SVF/T75-flask, respect-
ively, in complete medium containing Minimum
Essential Medium, MEM Alpha (aMEM), 1% Penicillin/
Streptomycin and with four different supplements:
(1) 5% Human platelet lysate (PLTMax), 10 IU heparin;
(2) 5% Human platelet lysate (hPL-S), 10 IU heparin;
(3) 5% Human platelet lysate (hPL-SP);
(4) 10% FBS.
hPL is comprised of plasma with fibrinogen and other
clotting factors, therefore heparin must be added to
prevent gelatinization. COOK General Biotechnology
produces Stemulate� pooled human platelet lysate in
two different versions, heparin-required PL-S and a
heparin-free PL-SP, where some of the clotting factors
have been removed and addition of heparin is not
required. Some aggregation did occur in PLTMax and PL-
S, which was removed prior to addition to medium.
The cells were incubated under standard conditions at
37 �C in humidified atmosphere with 5% CO2. The
medium was changed 5 days and 2 days after initial
seeding of BMSCs and ASCs to discard non-adherent
cells, and subsequently every 3–4 days.
When the culture reached a confluence level of
approximately 90%, cells were washed with PBS,
detached with 3 mL TrypLE� Select for 10 min at
37 �C, and neutralized with 7 mL complete medium. The
suspension was centrifuged at 300 g for 5 min at room
temperature, counted, and passaged with 3.5� 105
cells/T75-flask or seeded at different cell densities for
experimental set-ups.
To determine the cell yield for ASCs and BMSCs at
passage 0 and 1, cells were counted from three T75
flasks from each culture conditions of each donor. For
analysis of chromosomal stability, cells were cultured for
5 passages.
Cell proliferation
10,000 ASCs and BMSCs at passage 1 (P1) were seeded in
12-well plates (NUNC, Termo Fisher Scientific) in tripli-
cates. Number of cells was determined at days 1, 2, 3, 5,
and 7 using a Burker-Turk counting chamber. To assess
population doubling (PD), the following formula was
used: PD¼ ln (N/N0)/ln 2, where N is the harvested cell
number at day 7 and N0 is the seeded cell number.
Flow cytometry analysis
Primary culture-expanded BMSCs and ASCs were evalu-
ated and analysed by flow cytometry after initial seeding
(passage 0, P0) and following first passage (P1). Cells were
harvested by incubation for 5–10 min at 37 �C with 3 mL
TrypLE per T75 flask. The cells were washed with 7 mL
FACS-PBS mixture (FACS-PBS (Hospital pharmacy,
Copenhagen, Denmark), 1% EDTA (Hospital pharmacy,
Copenhagen, Denmark), and 10% new born calf serum
(Gibco, Life Technologies)), and centrifuged for 5 min at
300 g. Afterwards, the cell pellet was re-suspended in a
suitable volume FACS-PBS mixture and distributed to
Figure 1. Flowchart of the experimental design. MNCs and SVF were isolated from bone marrow and lipoaspirate, then cultured topassage 5 (P5) in four different complete culture media: PLTMax and PL-S, which required addition of heparin; PL-SP, no heparinrequired, and FBS.
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FACS tubes (Falcon, BD) with or without antibodies. The
cells were incubated for 30 min at room temperature with
the antibodies shown in Table I. Afterwards the cells were
washed twice with FACS-PBS mixture and fluorescence
was measured on a Navios flow cytometer (Beckman
Coulter) using a six-colour protocol. The protocol was
developed with manual compensation, isotypic controls,
and Fluorescence Minus One. Viability was determined by
addition of 1 mL SYTOX blue 5 min prior to analysis
(SYTOX�, Invitrogen, Life Technologies). Dead cells and
doublets were excluded from the final analysis. Data was
analyzed using Navios software and Kaluza (Beckman
Coulter).
Comparative genomic hybridization array
Genomic stability was determined comparing BMSCs
and ASCs at P1 and P5 using array Comparative Genomic
Hybridization (CGH). Passage one cells were used for
baseline controls. DNA was extracted using the QIAGEN
Genomic-tip 20/G or 100/G (Qiagen, Hilden) according
to the manufacturer’s protocol. Array CGH was per-
formed using the Agilent SurePrint G3 Human CGH
Microarray kit 8� 60K (design ID 021924) with 41 Kb
overall median probe spacing (Agilent Technologies).
Practical resolution was approximately 164 Kb. Donor
DNA and DNA of a sex-matched control (0.5 mg of each)
was labelled with Cy3-dUTP and Cy5-dUTP, respectively
(CGH labelling kit for oligo arrays, Enzo Life Sciences).
Labeled products were purified by Amicon Ultra 30 K
filters (Millipore). Hybridization was performed according
to the protocol provided by Agilent (Protocol v6.3,
October 2010). Donor and control DNA was pooled and
hybridized with 2 mg of Human Cot-I DNA at 65 �C with
rotation for 24 h. Arrays were analyzed using an Agilent
SureScan Microarray scanner and the Agilent Feature
Extraction software (v11.5), and results were presented
by Agilent Genomic Workbench (v.7.0).
Differentiation assays
The osteogenic, adipogenic, and chondrogenic differen-
tiation capacity of BMSCs and ASCs (P1) was determined
using StemPro differentiation kit (Gibco, Life
Technology), according to the manufacturer’s protocols.
For osteogenic differentiation, 10,000 ASCs or BMSCs/
well in 12-well plates were incubated in osteogenic
induction medium (StemPro Osteocyte/Chondrocyte
Differentiation Basal Medium, StemPro Osteogenesis
Supplement, Penicillin/Streptomycin). For adipogenic
differentiation, 20,000 ASCs or BMSCs/well in 12-well
plates were incubated in adipogenic induction medium
(StemPro Adipocyte Differentiation Basal Medium,
StemPro Adipocyte Supplement, Penicillin/
Streptomycin). For chondrogenic differentiation, mul-
tiple 5 mL drops of 80,000 ASCs or BMSCs were incubated
in chondrogenic induction medium (StemPro Osteocyte/
Chondrocyte Differentiation Basal Medium, StemPro
Chondrogenesis Supplement, Penicillin/Streptomycin).
Cells were induced for 21 days, with medium changed
every 3–4 days. Control cells were incubated with
complete medium without supplement until confluent.
To detect the osteogenic differentiation, cells were
stained for calcium deposition with Alizarin Red S
(Sigma-Aldrich), adipogenic differentiation was evalu-
ated through the morphological appearance of lipids
droplets stained with Oil Red O (Sigma-Aldrich), and to
identify chondrogenic differentiation, glycosaminogly-
cans were stained with Alcian Blue 8GX (Sigma-Aldrich).
Statistics
Analyses were performed using IBM SPSS version 19.
Before analysis, cell count data were log transformed.
Data obtained and transformed are generally normally
distributed, according to the Shapiro-Wilk test. Levene’s
test for equality of variance was used. Cell proliferation
was compared using repeated measures ANOVA, while
PDs were compared using one way ANOVA. Graphs
and plots were made using Excel 2010 (Microsoft Inc.)
and IBM SPSS. Data are expressed as mean ± standard
error of mean (SEM). Significance was determined at
p50.05.
Results
Cell morphology, yield and proliferation
Bone marrow mesenchymal stromal cells. Initially, BMSCs
P0 obtained from the standard isolation and maintained
Table I. Panel of antibodies used to determine the immuno-phenotype of ASCs and BMSCs at passage 0 and 1.
Marker Fluorochrome Company
CD45 PC7 Beckman CoulterCD34 APC Beckman CoulterCD105 PE R&DCD90 FITC Beckman CoulterCD73 PE Beckman CoulterCD13 ECD Beckman CoulterCD166 PE BD BioscienceCD29 FITC Beckman CoulterHLA-DR FITC Beckman CoulterCD19 ECD Beckman CoulterCD14 PC7 Beckman CoulterCD106 FITC BD BioscienceCD31 FITC BD BioscienceCD36 FITC BD Bioscience
PC7, phycoerythrin-cyanin (PC7); APC, allophycocyanin; PE, phycoerythrin;ECD, phycoerythrin and Texas Red energy coupled dye; FITC, Fluoresceinisothiocyanate.
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in adherent culture conditions consisted of a more
morphologically heterogeneous population in all three
hPL-supplemented media compared to FBS medium.
Morphologies of the cells are displayed in Figure 2A.
Two distinct cell morphologies were observed in BMSCs
P0 flasks; one appeared as small, rounded, and flattened
cells, while the second was more spindle-shaped and
fibroblast-like. However, when BMSCs were passaged,
the cell population became more homogenous and the
morphology of the BMSCs P1 was uniformly spindle-
shaped in all four culture conditions.
The hPL-media promoted a higher proliferation rate,
resulting in confluence achieved in shorter time of
BMSCs P0 compared to FBS medium. The ratios between
BMSCs P0 harvested and MNC seeded (BMSC:MNC) were
0.23 in PLTMax, 0.19 in PL-S, 0.16 in PL-SP after 9 days in
culture, and 0.10 in FBS after 12 days in culture
(Figure 2B). Similarly, after passage, BMSCs in P1 had a
faster rate of proliferation in hPL-media, resulting in
higher PD. PLTMax and PL-S media increased the
proliferation even more than PL-SP medium within
same culture time. After 9 days in culture, PD for
BMSCs P1 was 3.6 in PLTMax, 3.5 in PL-S, 2.8 in PL-SP.
BMSCs P1 in FBS medium reached a similar confluence
after 14 days, with a PD of 3.
When examined over the course of 7 days, the BMSCs
P1 in 12-well plates showed the same pattern as
observed in T75 flasks (Figure 3A). The proliferation of
Figure 2. Cellular morphology of BMSCs and ASCs at passage P0 and P1 culture expanded in four different complete media.Representative phase contrast images at 10� original magnification. Morphological differences are clarified by enlarging the image inthe corner of the pictures.
Table II. Seeding and calculated cell counts of BMSCs and ASCs cultured from P0 and P1 in different conditions. Results are expressedas the mean number (±SEM) from data obtained from three fat tissue and three bone marrow donors.
MNC BMSC P0 harvested Mean days BMSC:MNC BMSC P0 BMSC P1 harvested Mean daysn¼ 3 Seeded in culture ratio seeded in culture PD
PLTMax 2.0E + 07 4.55E + 06 ± 1.25E + 06 9 ± 1 0.23 3.5E + 05 4.30E + 06 ± 7.84E + 05 9 ± 2.3 3.6PL-S 2.0E + 07 3.84E + 06 ± 7.38E + 06 9 ± 1 0.19 3.5E + 05 4.00E + 06 ± 3.28E + 05 9 ± 2.3 3.5PL-Sp 2.0E + 07 3.27E + 06 ± 3.27E + 06 9 ± 1 0.16 3.5E + 05 2.50E + 06 ± 4.78E + 05 9 ± 2.3 2.8FBS 2.0E + 07 1.99E + 06 ± 1.99E + 06 12 ± 0.3 0.10 3.5E + 05 2.80E + 06 ± 3.77E + 05 14 ± 0 3.0
SFV ASC P0 harvested Mean days ASC:SVF ASC P0 ASC P1 harvested Mean daysn¼ 3 Seeded in culture ratio seeded in culture PD
PLTMax 4.50E + 06 6.80E + 06 ± 3.66E + 05 7 ± 0 1.51 3.5E + 05 5.00E + 06 ± 5.11E + 05 7 ± 0 3.8PL-S 4.50E + 06 6.60E + 06 ± 1.06E + 06 7 ± 0 1.47 3.5E + 05 4.60E + 06 ± 7.38E + 05 7 ± 0 3.7PL-SP 4.50E + 06 5.20E + 06 ± 8.16E + 05 7 ± 0 1.16 3.5E + 05 4.10E + 06 ± 3.35E + 05 7 ± 0 3.5FBS 4.50E + 06 5.10E + 06 ± 1.08E + 06 7 ± 0 1.13 3.5E + 05 1.30E + 06 ± 3.66E + 05 23 ± 2.2 1.9
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BMSCs was significantly (p50.05) higher in PLTMax and
PL-S media, PD 4.57 and 4.51, respectively, compared to
both PL-SP and FBS with a PD of 3.38 and 2.7,
respectively (Figure 3B).
Adipose tissue-derived stromal cells. ASCs P0
cultured in FBS or hPL-supplemented media displayed
the characteristic MSC-like spindle-shape with less dis-
cernible morphological differences in the four cul-
ture conditions. After the first passage, ASCs P1
continued to have the spindle-shape in hPL-cultures,
while ASCs in FBS varied in cell size and shape and
the majority of cells became large and flattened cells
(Figure 2A).
After 7 days, the ratio between ASCs P0 harvested and
SVF seeded (ASC:SVF) was 1.51 in PLTMax, 1.47 in PL-S,
1.16 in PL-SP and 1.13 in FBS (Table II). Heparin-
containing PLTMax and PL-S media induced higher cell
growth ratio than non-heparin PL-SP medium and FBS
medium. When ASCs P0 were passaged, all three hPL-
media achieved a high PD level within 7 days; 3.8 in
PLTMax, 3.7 in PL-S, and 3.5 in PL-SP. ASCs cultured in
FBS medium proliferated very slowly and achieved a PD
1.9 after 3 weeks in culture (Figure 2B).
Seven days examination of ASCs P1 proliferation rate
in 12-well plates revealed that the three hPL-media
induced significantly higher proliferation compared to
FBS medium (Figure 3B). PDs for ASCs in hPL-media were
significantly (p50.05) higher than in FBS medium
(PLTMax¼ 5.85, PL-S¼ 5.88, PL-SP¼ 5.56 and
FBS¼ 1.64). In general, ASCs reached confluence at a
faster rate than BMSCs, indicating a more extensive
proliferative capacity.
Phenotypic characterization
Phenotypic characterization of BMSCs and ASCs was
carried out using flow cytometric analysis of cell surface
marker expression according to the International
Federation for Adipose Therapeutics (IFATS) and the
International Society for Cellular Therapy (ISCT) require-
ments [24–26]. The expression of the surface markers on
BMSCs and ASCs seemed to depend on culture condi-
tions and time.
Bone marrow mesenchymal stromal cells. BMSCs P0
cultured in hPL containing media expressed high levels
(�40–60%) of CD45, CD14, HLA-DR, CD31, and CD106
which are characteristic markers for monocytes and
endothelial cells (Figure 4A). MSC-associated markers,
such as CD73 and CD90, were expressed at a similar
level. Cells cultured with FBS exhibited higher levels of
the mesenchymal markers (�80%), and lower levels of
non-MSC markers (�20%). CD105 and CD13 were highly
expressed, and CD34 was expressed at very low levels in
all four culture conditions.
Figure 3. Proliferative potential. (A, B) Growth and population doubling (PD) of BMSCs P1 cultured in four different media for sevendays. (C, D) Growth and PD of ASCs P1 cultured in four different media for seven days. Significance was determined at p50.05; n¼ 3.
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BMSCs in P1 met the ISCT requirements, they were
uniformly positive for the MSC markers and did not
express CD45, CD34, HLA-DR, CD 19, CD14, and CD31 in
all four culture conditions (Figure 4C).
Adipose tissue-derived stromal cells. Freshly harvested
ASCs P0 almost uniformly expressed all MSC markers in
any of the four culture conditions. They were495%
positive for CD105, CD90, CD13, and CD29, and
expressed CD166 and CD73 at relatively low levels.
More than 80% of the cells were CD34 positive
(Figure 4B). After one passage, ASCs P1 became highly
homogeneous with495% positivity of all MSC asso-
ciated markers regardless of culture conditions. The
expression level of CD34 decreased dramatically after
passaging. Expression of CD45, HLA-DR, CD14, CD106,
and CD31 were almost the same in both P0 and P1
regardless of culture conditions. CD19 was higher
expressed (�30%) in ASCs P0 cultured in FBS medium
but not in hPL media. After one passage CD19 was very
low expressed in all cultures. The expression of second-
ary ASC marker CD36 generally vary between 40 and
60% for hPL-supplemented ASC cultures, with a slight
decrease once passaged. In FBS cultures, however, CD36
experienced an increase from around 40% in P0 to
around 80% in P1 (Figure 4B and 4D).
Genomic stability
ASCs and BMSCs were culture-expanded for five pas-
sages (only early passages are transplanted clinically) to
document genetic stability during proliferation. Array
CGH analysis of cells at P1 and P5 demonstrated that
ASCs and BMSCs expanded in vitro, in the presence of
hPL or FBS, did not show imbalanced chromosomal
rearrangements.
Differentiation capacity
The ability of ASCs and BMSCs to differentiate into tri-
lineage mesodermal cell types was investigated. Culture-
expanded BMSCs (Figure 5A) and ASCs (Figure 5B) in all
four media at passage one were capable of differentiat-
ing into osteoblasts, adipocytes, and chondrocytes as
evaluated by Alizarin Red S staining, Oil Red O staining
and Alcian Blue staining. Control cells were maintained
in the respective complete media and were all nega-
tively stained.
Discussion
To achieve therapeutic clinical results with MSCs, the
treatment necessitates adequate quantities of cells to be
generated by in vitro culture expansion. Various
processes have been developed to produce clinical
grade MSCs; however, no consensus has emerged on the
best clinical-grade MSC isolation and culture conditions,
including tissue sources, separation technique, or the
composition of cell culture media, cell seeding density
and number of cell population doubling [27].
In vitro culture conditions significantly impact the
proliferative capacity and function of expanded cells,
and preparation of a culture medium for the production
Figure 4. Immunophenotypic profile of culture-expanded BMSCs and ASCs in four different culture conditions. The mean percent ofCD expression of the panel of antigens which are expressed on (A, B) BMSCs P0 and MSCs P1 and (C, D) ASCs P0 and ASCs P1; n¼ 3.
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of MSCs for clinical application still remains a critical
issue [28]. FBS has been the most universally applicable
cell culture additive and has been implemented in many
existing culture protocols for the stimulation of cell
proliferation [10,29,30]. However, there are some con-
cerns about the use of FBS in human therapeutical
approaches. Besides batch-to-batch variability, there is
risk of immunoreactivity, contamination by pathogens,
or transmission of xenogeneic proteins such as the
prions causing Bovine Spongiform encephalopathy
[1,11,17,31,32].
In this study we aimed to evaluate whether
commercial clinical grade human platelet lysates could
substitute FBS in MSC culture expansion. We have
tested culture expansion of BMSCs and ASCs in four
different culture conditions, media containing differ-
ent commercially available hPL that have been manu-
factured at an industrial scale in accordance to GMP
or standard medium supplemented with a pretested
FBS batch. The hPL has been manufactured as
a batch from multiple donors pooled to secure
consistency.
Figure 5. Multilineage differentiation of culture expanded BMSCs and ASCs in either hPL or FBS. Cells were induced in three differentconditions for three weeks. (A) Differentiation of BMSCs (B) ASCs into osteoblast as evaluated by Alizarin Red S staining, inducedadipocytes stained by Oil Red O and chondrogenic potential demonstrated by Alcian Blue staining. Control cells were cultured in fourrespectively complete media; n¼ 3.
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The focus of our evaluation was BMSC and ASC
proliferation, phenotype, genomic stability, and differ-
entiation potential over several passages.
Our study confirms that hPL supports culture expan-
sion and proliferation of both BMSCs and ASCs signifi-
cantly better than FBS. Particularly, proliferation of
ASCs was substantially increased with hPL, which has
also been shown by some groups [13,20,31] but not
by others [33]. This could be due to variations in
the production of the used hPL products. This pre-
sent growth advantage was less pronounced for
BMSCs, which indicates that these cells may differ
in their nutritional requirements or in proliferation
capacity.
In general, heparin-requiring PLTMax and PL-S sup-
ported the growth of BMSCs and ASCs better than
heparin-free PL-SP. This was significant for BMSCs, with a
non-significant tendency for ASCs. However, PLTMax
and PL-S supplements led to more platelet aggregation/
residual clotting compared to PL-SP, which may pose
problems in some cultivation protocols if not thoroughly
dissolved or removed. No data suggests that the clots
affect cell phenotype or function, and the consequences
may be negligible. PL-SP is processed more during
manufacturing and some of the coagulation factors
could have been involved in platelet aggregations.
During this process, some of the growth factors may
also have been removed, which could explain why PL-SP
Figure 5. Continued.
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supported cell proliferation less than the two other hPL
products.
Commercially available hPL holds the promise of
creating a consistency which may be hard to achieve
from individually processed hPL at numerous separate
laboratories. In order to serve for medicinal purposes,
commercial hPL is placed under regulatory authorities
who will ensure the proper documentation. In addition,
this could lead to more elaborate guidelines not only for
manufactory, but also for active substances, etc. This
would render the use of hPL more harmonised, uniform,
and widespread.
For culture expansion of freshly isolated BMSCs P0
from MNCs, all three hPL media seemed to promote the
attachment of not only MSCs, but also monocyte-like
cells. These cells appeared small, rounded, and flattened,
and were not present in FBS-containing medium, where
only few colonies were observed within the first few days
in culture. The phenotypic data support the presence of
CD45, HLA-DR, and CD14 on BMSCs P0 cultured with hPL.
The MNC fraction are expected to contain a larger
proportion of monocytes and hematopoietic cells com-
pared to cultured BMSCs [24], but they usually do not
attach. Unlike our observation, Bieback et al. observed
less hematopoietic contamination in BMSCs culture at
passage 0 with hPL compared to FBS supplemented
media [20]. The hematopoietic contamination we found
in hPL cultures was only detectable in primary cultures P0.
Hereafter, populations became more homogenous and
the cells in all culture conditions expressed the MSC
markers defined as minimal criteria according to ISCT, also
including the ability to differentiate into osteoblasts,
adipocytes, and chondrocytes [26].
Culture expansion of adipose tissue-derived ASCs
was quite different from BMSCs. The morphology of
the ASCs P0 was highly uniform, with spindle-shaped
appearance in all four culture conditions. Proliferation
ceased for ASCs P1 in FBS medium in contrast to
hPL cultures, where proliferation was significantly
enhanced and comparable to expansion kinetics in P0.
Immunophenotypically, the SVF cells that initially
adhered to culture flasks were less contaminated with
hematopoietic cell population compared to BMSCs P0,
irrespective of medium. Accordingly, CD45, HLA-DR,
CD19, CD14, CD106, and CD31 were expressed at very
low levels. On the other hand, CD34 was very highly
expressed in all cultures, but subsequently, the expres-
sion level diminished dramatically after one passage,
which is consistent with reports of CD34 declining with
successive passages after initial plating of SVF [34,35].
Similar to other studies [36], we observed that CD73 and
CD166 were relatively low expressed in initial culture but
increased to a higher level after passaging. The cell
product achieved from all four GMP-compliant media
fulfilled the requirements of ASCs suggested by the
IFATS and ISCT including the ability to differentiate into
osteoblasts, adipocytes, and chondrocytes [24].
It is known that the frequency of stromal cells is
significantly higher within adipose tissue than in bone
marrow. Around 2% of the uncultured SVF contribute to
ASCs [37], while within bone marrow, only 0.001–0.01%
of the total extracted MNCs are BMSCs in culture [38].
Both cell types undergo many PD within the first
passage, but compared to the number of MNCs
seeded and BMSCs harvested after initial cultivation,
the BMSCs undergo more than 10 PDs compared to
ASCs with an estimate around only 6 PDs, according to
the cell yield results we got at passage 0. No chromo-
somal aberrations were found in CGH analysis of BMSCs
and ASCs at P5, which is in accordance with other
studies [4,31]. We did not evaluate the genomic stability
of ASCs and BMSCs above passage 5 because for clinical
trials, we only use ASCs and BMSCs at passage 2 or 3.
Twice the regular cultivation period should consolidate
genomic stability of the cells.
Efforts have been made to develop serum-free
formulations that will provide all nutrients and growth
factors that are essential to maintain physiological
functions and to facilitate cellular proliferation [39], yet
most of these serum replacements have failed to
support the growth of the cells [17]. Thus, autologous
and allogeneic human serum has been tested as
alternatives to animal sera for cell expansion; in some
studies human serum improve growth of cells while
others report growth arrest [40–43]. Nevertheless,
difficulties in collection, processing, and quality of the
autologous serum vary from patient to patient and
hinder standardisation of culture conditions.
Furthermore, the amount of autologous serum required
for sufficient expansion exceeds the amount a single
donor can provide [41].
In summary, substitution of FBS with hPL will rid the
manufacturing process of animal products, reduce the
required number of passages, and minimise the ex vivo
cultivation time, without compromising genomic stabil-
ity. Based on our extensive experience with FBS for
expansion of autologous BMSCs from bone marrow and
ASCs from adipose tissue for clinical use, we believe that
future clinical and non-clinical studies can benefit from a
change from FBS to hPL as a culture-expansion medium
supplement.
Conclusion
The proliferation of BMSCs and ASCs was greatly
enhanced when cultured with commercial hPLs
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compared to FBS, while the cells retained genomic
stability, multilineage differentiation potential, and sur-
face marker expression patterns according to ISCT
criteria.
Acknowledgements
The authors thank Sonja Kim Brorsen, Sofie Lykke Larsen, andHanne Rose for their technical assistance. We are grateful toAndreas Printzlau for supplying the liposuction aspirates andthe patients for consenting to participate. This work wassupported by Arvid Nilssons Foundation and Aase and EjnarDanielsens Foundation.
Declaration of interest
The authors report no conflict of interest. The authors alone areresponsible for the content and writing of the paper.
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