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Scandinavian Journal of Clinical and LaboratoryInvestigation

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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 Mandana.haack-soerensen@regionh.dk 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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