TRANSLATIONAL AND CLINICAL RESEARCH
Human Alternatives to Fetal Bovine Serum for the Expansion of
Mesenchymal Stromal Cells from Bone Marrow
KAREN BIEBACK,a ANDREA HECKER,a ASLI KOCAÖMER,a HEINRICH LANNERT,b KATHARINA SCHALLMOSER,c,d
DIRK STRUNK,c,e HARALD KLÜTERa
a
Institute of Transfusion Medicine and Immunology, German Red Cross Blood Service of Baden-WürttembergHessen, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Heidelberg, Germany; bDepartment
Hematology, Oncology and Rheumatology, Medical Clinic of the University Heidelberg, Heidelberg, Germany;
c
Stem Cell Research Unit Graz, Medical University of Graz, Graz, Austria; dUniversity Clinic of Blood Group
Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria; eUniversity Clinic of Internal
Medicine, Department of Hematology, Medical University of Graz, Graz, Austria
Key Words. Mesenchymal stromal cells
marrow
•
Fetal bovine serum
•
Platelet-derived factors
•
Pooled platelet lysate
•
Human serum
•
Bone
ABSTRACT
Mesenchymal stromal cells (MSCs) are promising candidates for novel cell therapeutic applications. For clinical
scale manufacturing, human factors from serum or platelets have been suggested as alternatives to fetal bovine serum (FBS). We have previously shown that pooled human
serum (HS) and thrombin-activated platelet releasate in
plasma (tPRP) support the expansion of adipose tissuederived MSCs. Contradictory results with bone marrow
(BM)-derived MSCs have initiated a comprehensive comparison of HS, tPRP, and pooled human platelet lysate
(pHPL) and FBS in terms of their impact on MSC isolation, expansion, differentiation, and immunomodulatory
activity. In addition to conventional Ficoll density gradient
centrifugation, depletion of lineage marker expressing cells
(RosetteSep) and CD2711 sorting were used for BM-MSC
enrichment. Cells were cultured in medium containing ei-
ther 10% FBS, HS, tPRP, or pHPL. Colony-forming units
and cumulative population doublings were determined,
and MSCs were maximally expanded. Although both HS
and tPRP comparable to FBS supported isolation and
expansion, pHPL significantly accelerated BM-MSC proliferation to yield clinically relevant numbers within the first
two passages. MSC quality and functionality including cell
surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar
in MSCs from all culture conditions. Importantly, spontaneous cell transformation was not observed in any of the
culture conditions. Telomerase activity was not detected in
any of the cultures at any passage. In contrast to previous
data from adipose tissue-derived MSCs, pHPL was found
to be the most suitable FBS substitute in clinical scale
BM-MSC expansion. STEM CELLS 2009;27:2331–2341
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Bone marrow (BM) is a complex tissue harboring hematopoietic stem and progenitor cells, endothelial cells, adipocytes,
osteocytes, and fibroblastoid stromal cells. On cell culture
expansion, BM can yield a multipotent precursor population.
These mesenchymal stromal cells (MSCs) have been assessed
in a variety of preclinical and clinical settings ranging from
regenerative medicine to immunological or hematopoietic
support [1]. With MSCs becoming established in the clinical
setting, issues have been raised regarding how to expand these
cells in large-scale good-manufacturing practice (GMP)-compliant protocols [2–4]. Most expansion protocols use a medium supplemented with fetal bovine serum (FBS). Serum
supplementation is practical because it provides the cells with
vital nutrients, attachment factors, and growth factors. However, the use of xenogenic serum is complicated because of
high lot-to-lot variability and is associated with a risk of
transmitting infectious agents and immunizing effects [5–7].
Regulatory guidelines aiming to minimize the use of FBS
have further reinforced an intensive search for possible
Author contributions: K.B.: Conception and design, financial support, administrative support, collection and assembly of data, data
analysis, manuscript writing; A.H.: Conception and design, collection and assembly of data, data analysis, manuscript writing, final
approval of the manuscript; A.K.: Conception and design, provision of study material; H.L.: provision of study material, collection of
data; K.S.: provision of study material, manuscript writing; D.S.: financial support, data interpretation, manuscript editing; H.K.:
financial and administrative support, final approval of the manuscript. K.B. and A.H. contributed equally to this work.
Correspondence: Karen Bieback, Ph.D., Institute of Transfusion Medicine and Immunology, German Red Cross Blood Service of
Baden-Württemberg-Hessen, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Ludolf-Krehl-Str. 13-17, 68167
Mannheim, Germany. Telephone: 49-621-383-9720; Fax: 49-621-383-9720;; e-mail: [email protected] Received
C AlphaMed
February 11, 2009; accepted for publication May 21, 2009; first published online in STEM CELLS EXPRESS June 4, 2009. V
Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.139
STEM CELLS 2009;27:2331–2341 www.StemCells.com
Human Alternatives to FBS for BM-MSC Expansion
2332
alternatives [8–10]. Most current clinical data have been
accomplished with MSCs having been expanded in FBS supplemented media without the appearance of major side
effects. In some cases, however, immunological reactions and
anti-FBS antibodies have been observed and considered as
having possibly affected the therapeutic outcome [7, 11].
A chemically defined standardized, xenogeneic antigenand serum-free media composition would be the preferential
solution for pharmaceutical scale manufacturing. Such a formulation allowing for both isolation and expansion has not
been achieved thus far [12]. Based on extensive demand, FBS
may also become scarce and expensive.
In the development of a cell-based medicinal product, any
change in the manufacturing process that impacts final product quality must show comparability or superiority [13].
Human blood products are already considered to represent
drugs and are produced accordingly, thus offering certain
advantages as potential FBS substitutes. Accordingly, a variety of human supplements have been postulated as alternatives to FBS to provide nutrients, attachment factors, and
especially growth factors. These include autologous or allogeneic human serum, human plasma, cord blood serum, human
platelet derivatives including platelet lysate, and platelet
released factors [3, 4, 14-24]. Analysis of platelet releasates,
lysates, and subcellular fractions has shown that numerous
bioactive molecules are stored within distinct platelet organelles including adhesive proteins, coagulation factors, mitogens, protease inhibitors, and proteoglycans [25]. Compared
with serum, buffy coat-derived platelet preparations are of
particular interest because they do not compete with erythrocyte and plasma preparation for the limited available blood
donations [26].
In a previous study, we evaluated a variety of platelet
activation protocols to obtain biologically active proteins to
isolate and expand MSCs. Thrombin-activated platelet releasate in plasma (tPRP) and human blood type AB serum (HS)
were found to be superior adjuvants in isolating and expanding human adipose tissue-derived MSCs (AT-MSCs) [27].
The efficiency of both HS and tPRP, but not of pooled human
platelet lysate (pHPL), in expanding AT-MSCs was notable in
contrast to previous reports on BM-MSCs [20, 28]. Consequently, in a recent study we compared the effects of these
three human alternatives on the isolation, expansion, differentiation, and immunomodulatory capacities, as well as the
immunophenotype of BM-MSCs using FBS as the standard
substitute. The experimental setup was expanded by additional
analysis of product purity, because our previous observations
showed reduced depletion of contaminating hematopoietic
cells in AT-MSCs cultured in human supplements. In this
new study, the standard Ficoll gradient density centrifugation
method was therefore compared with the enrichment of MSCs
by either depleting mature hematopoietic cells or by purifying
MSCs expressing CD271 (low affinity nerve growth factor receptor [LNGFR]) and cultivating the obtained mononuclear
cells in the four different supplements.
MATERIALS
AND
METHODS
Media and Supplements
Dulbecco modified Eagle’s medium low glucose (Lonza Group
Ltd., Basel, Switzerland, http://www.lonza.com), supplemented
with 4 mM L-glutamine (PAA, Coelbe, Germany, http://
www.paa.at), 50,000 units (U) penicillin/50,000 lg streptomycin
(PAA) served as basal medium in all instances. It was completed
with (a) 10% FBS (MSCGM Single Quots; Lonza Group Ltd.),
(b) 10% HS, (c) 10% tPRP, or (d) 10% pHPL.
Human AB Serum
HS was derived from whole blood donations of prescreened AB
blood group-typed donors. From each donor, whole blood was
drained into blood bags without anticoagulants and allowed to clot
overnight at 4 C. The serum was aliquoted and separated by centrifugation at 2,000g for 15 minutes. Subsequently, the supernatant
was aliquoted into 15-ml sterile tubes (Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com/en) and frozen at –30 C.
After thawing aliquots from at least five donors, HS was pooled
and sterilely filtered through 0.2-lm pore filters (Nalgene filtration
device; Nalgene Nunc International, Rochester, NY, USA, http://
www.nuncbrand.com). HS-supplemented medium was pretested to
maintain its mitogenic capacity over a period of at least 4 weeks.
Thus, HS medium was not freshly made for each individual use.
At least 10 different pools were checked to verify reproducibility.
Thrombin-Activated Platelet Releasate Plasma
Four whole blood donations of AB or O blood group-typed donors
were used to prepare one pooled platelet concentrate derived from
buffy coats. Instead of using an additive solution like T-Sol, the
pooled platelet concentrate was suspended in AB plasma of one
donor. Platelet counts ranged between 20 1011 and 30 1011
platelets per liter determined by CellDyn 3,200 (Abbott, Wiesbaden, Germany, http://www.abbott.de). Subsequently, the platelet
concentrate was activated by 1 U of human thrombin (Sigma
Aldrich, Hamburg, Germany, http://www.sigmaaldrich.com) [27].
The released factors were separated from the cellular debris by
centrifugation at 3,000g, followed by filtration through 0.2-lm
pores. By pooling two pooled platelet concentrates, tPRP finally
represented eight donors. Five-milliliter aliquots were stored at
–80 C. After thawing, the aliquot was centrifuged again for 5
minutes at 1,500g to remove any developing clots. To prevent in
vitro gel formation, 2 U of heparin (Heparin-Natrium-5000-ratiopharm; Ratiopharm, Ulm, Germany, http://www.ratiopharm.de)/ml
of medium was added before the tPRP. tPRP was shown to rapidly
lose mitogenic activity. A storage time exceeding 48 hours
resulted in extensive loss of mitogenic activity; thus, the medium
was prepared freshly for each individual use. To verify reproducibility, at least 11 different pools were applied.
Pooled Human Platelet Lysate
pHPL was prepared in Graz as previously described [3]. Briefly,
four buffy coat units of blood group O-typed donors were pooled
in AB plasma and centrifuged (340g, 6 minutes, 22 C). The platelet rich plasma (PRP) was leukocyte depleted by inline filtration
and was frozen at –30 C. After thawing at 37 C, at least 10 units
of freeze-thaw lysed human platelets were further pooled resulting in approximately 40-50 donations per batch to minimize donor variations. pHPL was aliquoted and stored at –30 C. Before
use in cell culture, pHPL was thawed and centrifuged at 4,000g
for 15 minutes, whereas only the supernatant was added to the
culture medium containing 2 U/ml of preservative-free heparin.
Reproducibility of pHPL effects was verified by using at least
seven different batches of pHPL identically prepared in Mannheim by pooling platelet concentrates from eight donors. Where
specified, tPRP and pHPL were prepared from one platelet concentrate split in two halves to directly compare both.
Isolation and Culture of BM-Derived MSCs
BM aspirates were harvested using an optimized bone marrow
harvesting technique [29]. Illiac crest bone marrow aspirates were
derived from 14 young healthy donors (median age 22) after having received informed consent. Mononuclear cells (MNCs) were
isolated from all heparinized BM aspirates by density gradient
centrifugation (Ficoll Paque, GE Healthcare, Uppsala, Sweden,
http://www.gehealthcare.com) as described elsewhere [30]. Independent of the cell number, the MNCs were split into equal subfractions and cultured within the respective basal medium
Bieback, Hecker, Kocaömer et al.
2333
Figure 1. Morphology of bone marrow
(BM)-mesenchymal stromal cells (MSCs)
and BM-MSC allocation to specific tests.
(A): Photomicrographs of one representative donor at primary culture at day 10 for
fetal bovine serum human serum, thrombinactivated platelet releasate in plasma, and
pooled human platelet lysate are shown in
rows. Columns reflect cells either isolated
using Ficoll density centrifugation, depletion of lineage positive cells by RosetteSep,
or CD271 selection followed by plastic adhesion. Magnification, 100. (B): The
scheme shows the total number BM samples and those used for the respective parallel tests. Numbers of BM samples were
reduced in subsequent passages because of
replicative senescence-induced growth retardation. Abbreviations: FBS, fetal bovine
serum; HS, pooled human serum; tPRP,
pooled thrombin-activated platelet-richplasma; pHPL, pooled human platelet
lysate.
supplemented with either FBS (n ¼ 14), HS (n ¼ 12), tPRP (n ¼
12), or pHPL (n ¼ 6) (Fig. 1B).
In n ¼ 6 BM samples, MSC enrichment using RosetteSep
(StemCell Technologies Inc, St. Katharinen, Germany, http://
www.cellsystems.de) was compared with Ficoll-only isolation.
The RosetteSep antibody cocktail (CD3, CD11b, CD14, CD16,
CD19, CD56, CD66b, and glycophorin A) crosslinks undesirable
cells and forms immunorosettes with red blood cells. These are
pelleted after Ficoll gradient centrifugation. In this case, the BM
aspirate was split into two equal aliquots before MNC isolation.
Resulting cells were cultured in media supplemented with FBS,
HS, or tPRP. On four other samples, CD271 (LNGFR) enrichment was performed in one half of the BM sample, whereas the
other half was split to perform Ficoll and RosetteSep separation.
CD271 sorting involved a magnetic bead-assisted preselection
(AutoMACS device; program ‘‘Possel D’’ [2 columns] and ‘‘Possel
S’’ [sensitive]) using CD271 microbeads (Miltenyi Biotec GmbH,
Bergisch Gladbach, Germany, http://www.miltenyibiotec.com).
Because purity reached, at best, 80%, a flow cytometric sorting followed the enrichment (BD FACS Vantage TM SE: sorter used for
flow cytometric cell sorting). This yielded a >99% CD271þ cell
population as assured by flow cytometric analysis (CD 271-FITC
and CD 271-PE; Miltenyi Biotec GmbH).
All cell cultures were incubated with the respective supplements at 37 C, 5% CO2 in a humidified atmosphere. In a standardized fashion, all nonadherent cells were removed 24 hours after
initial plating by media changes. The cells were cultured with
media changed twice weekly until reaching confluence of 7080%. At this time, cells were passaged using 1 trypsin-EDTA
(PAA). At each passage (p), cells were replated at a standard
density of 200 cells per cm2 at any subsequent passage.
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Proliferation Kinetics
Cells were passaged and counted once they reached a subconfluence of 70-80%. The population doubling (PD) rate was determined using the following formula [31]:
X¼
½log10ðNH Þ log10ðN1 Þ
log10ð2Þ
NH is the harvested cell number and N1 is the plated cell
number. The PD for each passage was calculated and added to
the PD of the previous passages to generate data for cumulative
population doublings (CPD).
In addition, the generation time (average time between two
cells doublings) of four BM within all media conditions was calculated at passage 1 (p1) and p4 using the following formula:
X¼
log2 Dt
logðNH Þ logðN1 Þ
The effects of heparin and thrombin, which are present in
tPRP and pHPL, were checked separately. BM-MSCs of two
donors were cultured in (a) 10% FBS, (b) 10% FBS þ 2 U heparin/ml, or (c) 10% FBS þ 1 U thrombin/ml for three passages.
No impact on MSC growth kinetics was observed within the
three passages.
Colony-Forming Unit-Fibroblast Assays
The colony-forming unit-fibroblast (CFU-F) assay in primary culture was determined for six donor BM, and colonies were
2334
counted after 10 days. Freshly isolated BM-MNCs derived from
the three different isolation methods and cultured within the four
different media were plated in duplicate in 6-well plates at densities of 1 104, 5 104, and 1 105 per well. On day 10, the
cell layer was fixed with methanol and stained with Giemsa solution (Merck, Darmstadt, Germany, http://www.merck.de). Individual colonies composed of at least 50 cells were counted. CFU-F
frequency was calculated based on the respective input cell number as CFU-F per 1 104 MNCs.
In Vitro Differentiation Potential
The adipogenic and osteogenic differentiation capacity of MSCs
was assessed at p2/p3 for all BM donors and for all culture conditions [27]. To detect the osteogenic differentiation, cells were
stained for calcium deposition using von Kossa stain. Adipogenic
differentiation was indicated by the morphological appearance of
lipid droplets stained with Oil Red O.
Flow Cytometry Analysis
Immunophenotypic analyses were performed on three BM-MSC
batches for all supplements and selected MSC samples derived
from the different isolation methods at p3.
The following mouse anti-human antibodies were used in
multiplexed flow cytometric analysis: CD105-FITC (clone 8E11;
Chemicon/Millipore, Schwalbach/TS, Germany, http://www.millipore.com), CD144-PE (TEA1/31; Beckman Coulter GmbH,
Krefeld, Germany, http://www.beckman.com), CD90-APC (5E10;
Becton Dickinson GmbH, Heidelberg, Germany, http://www.
bdeurope.com), CD106-FITC (51-10C9; Becton Dickinson
GmbH), CD146-PE (TEA-1/34; Beckman Coulter), CD34-PerCPCy5.5 (8G12; Becton Dickinson GmbH), CD133/1-APC (AC133;
Miltenyi), CD44-APC-Alexa750 (IM7; NatuTec, Frankfurt/Main,
Germany, http://www.natutec.de), CD15-FITC (HI98; Becton
Dickinson GmbH), CD45-FITC (HI30; Becton Dickinson
GmbH), CD3-FITC (UCHT1; Becton Dickinson GmbH),
CD235a-FITC (GA-R2; Becton Dickinson GmbH), CD14-FITC
(M5E2; Becton Dickinson GmbH), CD19-FITC (AE1; Diatec/
Dianova Hamburg, Germany, http://www.dianova.de), CD117-PE
(104D2; Becton Dickinson GmbH), CD33-PerCP-Cy5.5 (P67.6;
Becton Dickinson GmbH), CD31-APC (WM59; NatuTec), CD29APC-Cy7 (TS2/16 BioLegend/Biozol Eching b, München, Germany, http://www.biozol.de), CD73-PE (AD2; Becton Dickinson
GmbH), HLA-ABC-APC (G46-2.6; Becton Dickinson GmbH),
HLA-DR-PE-Cy7 (L243; Becton Dickinson GmbH), and 7-AAD
(Beckman Coulter) for dead cell exclusion. The samples were analyzed using the BD FACS-Canto II and DIVA software. Comparative analysis was performed with FlowJo Version 7.2.5 (Tree
Star, Inc., Ashland, OR, USA, http://www.treestar.com).
Inhibition of Phytohemagglutin-Induced
T-Cell Proliferation
To test the allostimulatory or inhibitory effect of MSCs on T-cell
proliferation, we co-cultured phytohemagglutin (PHA)-stimulated
MNCs derived from healthy human buffy coat preparations with
decreasing numbers of MSCs. Specifically, 1 104, 5 103, and
2.5 103 MSCs were preseeded as quadruplicates into wells of a
flat-bottom 96-well plate in Roswell Park Memorial Institute
1640 (PAA) medium containing 10% FBS. On the following day,
the medium was discarded, and 1 105 MNCs were added to each
well in RPMI medium containing 10% FBS and interleukin-2 (IL2; 20 U/ml ¼ 0.01 lg/ml; Roche Applied Science, Mannheim,
Germany, http://www.roche-applied-science.com). One half of the
wells were stimulated with PHA (2.5 lg/ml PHA-L; Roche
Applied Science) to induce T-cell proliferation. Controls included
nonstimulated, co-cultured MNCs, nonstimulated MNCs, and
stimulated MNCs without MSC co-culture. To simultaneously
quantify the cell number, viability, phenotype, and activation level
of T-cell subsets, we used a modified method established by
Nguyen et al. [32]. CD3, CD4, and CD8 antibodies were used to
distinguish T-cell subsets, whereas T-cell activation was measured
Human Alternatives to FBS for BM-MSC Expansion
by the assessment of CD71 expression. For T-cell quantification,
fluorescent microparticles with defined concentrations were used.
The absolute count of target cells was calculated on the basis of
the known bead concentrations using the following equation:
Total cells per microliter ¼
Numbers of cells measured
Number of fluorespheres measured
Flow count concentration:
After 5 days, the MNCs were harvested and stained with a
cocktail of the following reagents: flow-count fluorospheres
(Beckmann Coulter) to directly determine absolute cell counts,
anti-CD3-PE-Cy7 (Becton Dickinson GmbH), anti-CD4-PE (Becton Dickinson GmbH), anti-CD8-FITC (Becton Dickinson
GmbH), anti-CD71-APC (Becton Dickinson GmbH), and 7-AAD
(Beckmann Coulter) to exclude dead cells. The samples were analyzed using the FACS-Canto II and DIVA software. The percentage of inhibition of T cells was calculated by comparing control cultures stimulated with PHA in the absence of MSCs (¼0%
inhibition) to those in the presence of MSCs.
We used BM-MSC samples from three donors cultured in the
differing supplements. Because of the fact that, in two HS-MSC
cultures, T cells showed reduced viability (<70%) in unstimulated co-cultures because of unknown reasons, data for HS-MSCs
were not statistically evaluated.
Detection of Telomerase Activity
To detect potential telomerase activity, we used the Telo TAGGG
Telomerase PCR ELISA (Roche Applied Science) following the
manufacturer’s instructions. Samples of BM aspirates and BMMSCs at different passages cultured in the various supplements
were analyzed. Samples were regarded as telomerase negative if
the difference in absorbance after subtraction of the negative control was <0.2.
Human Cytokine Expression Profile
The cytokine profile of culture medium supplemented with 10%
FBS, HS, tPRP, or pHPL and that of 3-day MSC-conditioned medium was analyzed with a semiquantitative human cytokine antibody array that can detect 174 cytokines per experiment (RayBio
Human Cytokine Antibody Array G series 2000; Tebu-bio
GmbH, Berlin, Germany, http://www.tebu-bio.com). To minimize
variances, tPRP and pHPL were derived from the same initial
platelet concentrates.
Despite the human specificity of the array, we also tested
FBS, but finally interpreted only the conditioned medium. All
sample measurements were performed in duplicate according to
the manufacturer’s instructions. The signals were detected using a
laser scanner (GMS 418 array scanner; Affymetrix, Santa Clara,
CA, USA, http://www.affymetrix.com) and analyzed with array
vision version 7 (Imaging Research, Inc., St. Catharines, Canada,
http://www.imagingresearch.com). Signals were normalized using
positive, negative and internal controls included on the array. For
analysis, the internal negative controls were used to determine
the cut-off rate for a positive signal as 2 SD. Thus, signal intensity values of >2,000 were regarded as positive.
Statistical Analysis
Statistical tests were performed using SPSS 12.0 (SPSS, Inc.,
Chicago, IL, USA, http://www.spss.com) or SigmaPlot 11.0
(Systat Software, Inc., San Jose, CA, USA, http://www.systat.
com) statistical software. Data are represented as arithmetic mean
SD. Data were tested for normality and equal variance before
analysis. Statistical differences were calculated using analysis of
variance (ANOVA; or ANOVA on ranks if equal variance testing
failed) and t test (paired t test where applicable). Differences
were considered significant at *, p < .05 or **, p < .01.
Bieback, Hecker, Kocaömer et al.
2335
Figure 2. Effect of isolation strategies. (Top) Mean cumulative population doublings of bone marrow-mesenchymal stromal cells isolated using
Ficoll gradient centrifugation, RosetteSep, or CD271 sorting followed by plastic adhesion in medium supplemented with fetal bovine serum,
human serum, or pooled thrombin-activated platelet-rich-plasma. (Bottom) Cumulative days needed for each MSC culture to be passaged. Low
numerical values indicate high proliferative activity (for initial n, see Fig. 1B). *In comparison to Ficoll, p < .05 using analysis of variance and
paired t test. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma.
RESULTS
MSCs Isolation and Expansion
Effect of Isolation Strategies. Because our previous experiments with AT-MSCs resulted in a transient contamination with
hematopoietic cells in human supplement cultures, we applied
two MSC enrichment strategies. First, we depleted mature lineage marker expressing cells by rosetting to erythrocytes (RosetteSep). Second, we used magnetic combined with flow cytometric sorting of CD271-expressing cells to enrich MSCs.
Both enrichment strategies reduced contaminating round
and loosely adherent cells in the primary passage (Fig. 1).
Ficoll gradient-derived cultures supplemented with FBS had
experientially few contaminating cells indicated by the presence of small loosely adherent round cells reactive with antiCD45 (data not shown). These cells were easily depleted by
repetitive media changes that occurred in the primary culture.
RosetteSep very efficiently depleted the round contaminating cells in all culture conditions and yielded MSCs to be passaged 11.2 1.48 days after seeding compared with 15.42 4.46 days after seeding for Ficoll/FBS, respectively (p ¼ .01;
Fig. 2). A more rapid proliferation was observed by p3. However, this did not correlate with higher cumulative population
doublings. Up to p3 expansion kinetics of RosetteSepenriched cells showed a higher proliferation. Interestingly,
immunodepleted cells from some donors showed an earlier
onset of replicative senescence compared with Ficoll-isolated
cells from p4 on, indicated by reduced proliferation and morphologic changes. We observed differences in the effects of
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HS and tPRP on RosetteSep-enriched cultures. The cell increment in HS exceeded that of ficolled cells up to p4 (Fig. 2).
Proliferation rates were accelerated in HS, with a significant
increase only in p1.
FBS-supplemented cultures sorted for CD271þ cells
showed bacterial contamination in three of four cases. This
necessitated the abandonment of said cultures. Despite the
addition of the same concentration of penicillin/streptomycin,
parallel cultures using the human alternatives displayed no
bacterial outgrowth. This may suggest that human FBS alternatives might have intrinsic antibacterial components.
In summary, the experiments with CD271þ cells cultured
in FBS, HS, or tPRP showed that CD271þ cells tended to
grow in colonies during the entire culture period, never forming confluent monolayers. Expansion kinetics were delayed in
FBS-driven cultures of CD271þ cells after p3 (Fig. 2). This
corresponded to the low cell numbers yielded within each
passage. Cells stopped proliferation in p5 yielding maximum
24.91 CPD for FBS (n ¼ 1; 17.47 for HS, p4, n ¼ 1 and
21.92 for tPRP, p5, n ¼ 1).
Effect of Supplements. As indicated above, the culturing of
MNCs after density gradient centrifugation in FBS-supplemented medium yielded few contaminating hematopoietic
cells. In contrast, supplementing MNCs with HS or tPRP
resulted in variably high numbers of hematopoietic cells.
Interestingly, and unlike HS and tPRP, pHPL-supplemented
cultures were devoid of contaminating cells (Fig. 1).
Calculating the number of CFU-Fs showed a precursor
frequency of 1:25,000 MSCs/MNCs, which was not affected
2336
Human Alternatives to FBS for BM-MSC Expansion
Figure 3. Colony-forming unit-fibroblast of bone marrow (BM)-mesenchymal stromal cells (MSCs). Photomicrographs represent BMMSCs from one donor assessed after 10 days cultivated in fetal bovine
serum, pooled human serum, pooled thrombin-activated platelet-richplasma, or pooled human platelet lysate (magnification, 100). Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP,
pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human
platelet lysate.
by the use of different culture substitutes. However, the number of cells composing the colonies was larger in all of the
human supplements. Colonies in pHPL were densely packed
with very small spindle-shaped cells compared with only
loosely connected cells in FBS cultures (Fig. 3).
The comparison of the expansion rates of MSCs in FBS to
either HS- or tPRP-supplemented culture conditions showed no
significant differences (Fig. 4). However, cells cultured in HS
and tPRP decelerated proliferation from p4, reaching 14.46 3.46 (HS, n ¼ 3) and 18.47 2.92 CPD (tPRP, n ¼ 7) compared with 18.73 1.96 CPD (FBS, n ¼ 13). This correlated
well with an increased population doubling time. Beginning in
p1, the generation time of ficolled MSCs cultured in HS was
significantly prolonged with 3.41 1.23 days compared with
2.51 0.87 days in the FBS cultures. In p4, both HS and tPRP
showed an extended generation time (12.53 6.54 days for HS
and 12.72 7.39 days for tPRP) compared with FBS (3.72 0.44 days). Cells from one donor (HS) or two donors (tPRP)
expanded until p5. In contrast, 11 samples from a total of 14
donors cultured in FBS reached p5.
Consistently, cultures supplemented with pHPL yielded significantly higher expansion rates than cells in FBS, reaching a
maximum of 52.82 CPD (in p8; n ¼ 1 from initially six donors)
compared with 31.43 3.13 in FBS (in p7; n ¼ 8 from initially
14 donors, p ¼ .004). Calculating the generation time at p1 and
p4 yielded, in both cases, significantly reduced population doubling times: 1.27 0.23 days in p1 and 1.9 0.32 days in p4
compared with FBS (2.51 0.87 days in p1 and 3.72 0.44
days in p4; p ¼ .012 and p ¼ .004, respectively).
MSC Quality and Functionality
Immune Phenotype. Typical CD44, CD73, CD90, CD105,
CD146, and HLA-ABC surface marker expression was
detected in all MSC cultures at p3 despite a measurable donor
variance. CD29 was expressed on 44.46 9.49% of BMMSCs cultured in tPRP, whereas the other supplement yielded
Figure 4. Effect of supplements. (Top) Mean cumulative population
doublings of bone marrow-mesenchymal stromal cells (MSCs) isolated using Ficoll gradient centrifugation and cultivation either in fetal
bovine serum (FBS), pooled human serum, pooled thrombin-activated
platelet-rich-plasma, or pooled human platelet lysate. (Middle) Cumulative days needed for each MSC culture to be passaged (for initial n,
see Fig. 1B). (Bottom) Generation time of MSCs at p1 (black) and p4
(white) (n ¼ 4). *p < .05 and **p < .01 in comparison to FBS using
analysis of variance (ANOVA) or ANOVA on the ranks, respectively.
Abbreviations: FBS, fetal bovine serum; HS, pooled human serum;
tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled
human platelet lysate; CPD, cumulative population doublings.
significantly higher positivity; for example, in HS, 98.12 0.76%. CD29 mean fluorescence intensity was significantly
higher in HS (FBS, 967.38 476.55; HS, 2,176.07 416.98;
tPRP, 134.57 19.18; pHPL, 438.88 306.06). CD15,
CD33, lineage (CD45, CD3, CD235a, CD14, and CD19),
CD117, CD144, and HLA-DR showed less than 5% positivity. Selected antigens representing one donor are depicted in
Figure 5. No further statistically significant differences
between FBS and the other supplements were detected.
Bieback, Hecker, Kocaömer et al.
2337
Figure 5. Flow cytometric characterization of bone marrow (BM)mesenchymal stromal cells (MSCs).
Comparison of the expression of
surface proteins of ficolled BMMSCs cultured in fetal bovine
serum, pooled human serum,
pooled thrombin-activated plateletrich-plasma, or pooled human platelet lysate analyzed by flow cytometry. One representative donor and
typical MSC marker expression
are depicted in the overlay to the
unstained/control. For statistical analyses, n ¼ 3 BM-MSC donors
were paired assessed at passage 3.
Abbreviations: FBS, fetal bovine
serum; HS, pooled human serum;
tPRP, pooled thrombin-activated
platelet-rich-plasma; pHPL, pooled
human platelet lysate.
Other markers showed donor-dependent variable reactivity
perhaps influenced by the supplement used and/or the degree
of hematopoietic cell contamination: CD31 (FBS, 0.95 0.74;
HS, 0.71 0.31; tPRP, 5.5 4.48; pHPL, 19.12 10.78),
CD133 (FBS, 6.45 11.23; HS, 5.14 7.21; tPRP, 6.04 5.94; pHPL, 0.77 0. 89), and CD106 (FBS, 6.18 7.91; HS,
8.35 9.7; tPRP, 13.54 14.34; pHPL, 18.28 9.41).
Differentiation Potential. MSCs derived from all conditions
demonstrated differentiation toward the osteogenic and adipogenic lineage as assessed by von Kossa and Oil Red O staining (supporting information Fig. 1).
eration and activation. To study the impact of culture conditions on MSC inhibitory activity, the same donor MNCs were
used for all MSC samples. In co-culture controls without adding PHA, MSCs did not induce an alloreaction of the T cells
but rather a loss of T cells in the range of 10-20% compared
with the control. All MSCs independent of the culture conditions inhibited the PHA-driven T-cell proliferation and activation dose dependently (Fig. 6). Both CD4þ and CD8þ T-cell
subsets were similarly affected. MSCs cultured in the human
platelet-derived substitutes showed a tendency toward aggravated inhibitory activity at ratios of 1:10 and 1:20 that was
not statistically significant.
Inhibition of PHA-Induced T-Cell Proliferation
Telomerase Activity
We used a flow cytometric method to simultaneously quantify
mitogen-driven T-cell proliferation, subtypes, activation level,
and viability. T-cell stimulation by PHA led to strong prolif-
Telomerase activity was analyzed in MSCs at different passages to control the onset of spontaneous immortalization. We
never detected telomerase activity except for the primary BM.
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2338
Human Alternatives to FBS for BM-MSC Expansion
Figure 6. Immunomodulatory
capacity of bone marrow (BM)mesenchymal
stromal
cells
(MSCs). BM-MSCs, irrespective
of the supplement, mediated a
dose-dependent inhibition of
phytohemagglutin-induced T-cell/
CD3 stimulation. Proliferation of
the CD4 (T-helper) and CD8
(cytotoxic) subsets were similarly affected. Simultaneously,
we quantified the proportion of
activated T cells by means of
CD71 expression. Like T-cell
proliferation, T-cell activation
was inhibited dose dependently.
The overlay depicts CD71
expression of CD3þ cells. The
same buffy coat mononuclear
cells were used for all experiments. Three MSC batches were
paired assessed at passage 3.
Dose dependent differences were
observed compared to ratio 1:10
with p < .05 (* ¼ fetal bovine
serum (FBS), # ¼ thrombin-activated platelet releasate in plasma
(þPRP) and $ ¼ pooled human
platelety lysate (pHPL)). tPRPand pHPL MSCs-induced CD3
(plus CD4 for tPRP MSCs and
CD8 for pHPL MSCs) inhibition
did not differ significantly from
that at 1:10. Statistically reduced
inhibition was found at the ratio
1:40. A paired t test was used to
compare dose dependency and
analysis of variance to compare
culture supplements.
Here we could attribute the low telomerase activity to the
CD34þ proportion (data not shown).
Cytokine Content in Supplements
and Conditioned Medium
The cytokine content in the 10% supplemented media and the
conditioned media (CM) after 3 days of culture was evaluated. For these analyses, tPRP and pHPL were derived from
the same pools to eliminate donor-specific differences.
Because the cytokine array is human specific, data for the
FBS-containing medium were not interpreted (Fig. 7; supporting information Table 1).
Overall growth factor levels in FBS-CM were lower than
in any of the human supplemented cultures, indicating that
detectable levels are continuously present in the human supplements and remain unchanged including Acrp30 (adiponectin), angiogenin, CD14, glucocorticoid-induced tumor necrosis
factor receptor (GITR), platelet-derived growth factor (PDGF)
AB, and sgp130 (soluble gp130). Other cytokine levels
dropped during culture (because of consumption or degradation) such as epidermal growth factor, macrophage-derived
chemokine/CCL22, pulmonary and activation-regulated chemokine, PDGF-AA, and PDGF-BB. Insulin-like growth factor
binding protein (IGFBP)-3, interleukin 6, monocyte chemoattractant protein-1, macrophage stimulating protein (MSP)a,
osteoprotegerin, thrombopoietin, and tissue inhibitor of metalloproteinases-1 and -2 levels increased during culture, presumably because of production by MSCs.
tPRP differed from HS and pHPL with regard to a variety
of cytokines. In tPRP-CM, hepatocyte growth factor/scatter
factor, IGFBP-2, and vascular endothelial growth factor
(VEGF) D were elevated. Unfortunately, no obvious candidate
for the strong proliferative support from pHPL could be identified: basic fibroblast growth factor (bFGF), GITR, macrophage migration inhibitory factor (MIF), macrophage inflammatory protein-1b (MIP-1b), MSP-a, regulated on activation,
normal T-cell expressed, and secreted (RANTES; CCL-5),
and VEGF were differentially regulated in pHPL/pHPL-CM
compared with HS/HS-CM and tPRP/tPRP-CM.
Bieback, Hecker, Kocaömer et al.
Figure 7. Selection of differentially regulated growth factors evaluated by human cytokine array. Medium supplemented with either
10% human serum (HS), pooled thrombin-activated platelet-richplasma (tPRP), or pooled human platelet lysate (pHPL), and in addition 3 days of conditioned medium of fetal bovine serum, HS, tPRP,
and pHPL (each n ¼ 1) were analysed. Depicted cytokines from a list
of 174 (supporting information Table 1) have been selected based on
noticeable differences in the signal intensities indicating different concentrations in medium and conditioned medium. Abbreviations: FBS,
fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet
lysate.
DISCUSSION
Currently, the ex vivo expansion of MSCs seems to be inevitably to get the common therapeutic dose of >2 106/kg
body weight for infusion (e.g., in treatment of graft vs. host
disease). Also, for other indications, there exists a need to
study the applicability of MSCs with dose escalation, indicating the need to propagate MSCs in sufficient quantity.
In a recent concise review in this Journal, Manello and
Tonti underlined that elaboration of a culture medium for the
production of MSCs for clinical application still remains a
crucial matter [12]. We and others [2-4, 14-24, 27, 33-38]
have since developed various protocols for the clinical scale
propagation of human MSCs. Most of these protocols actually
avoid the use of animal serum and some get rid off antibiotics
and density gradient separation of the culture initiating cells.
The major limitation of these studies relates to the fact that
they compare FBS-based media to only selected FBS-free culture conditions (only HS or only pHPL). There are some data
www.StemCells.com
2339
indicating that autologous serum in general supports greater
amplification of MSCs than FBS [14, 39]. Limited availability
and high variability regarding MSC growth clearly hamper
the clinical applicability of autologous serum for large-scale
MSCs production. Pooled preparations of allogeneic human
serum can be produced in large amounts for pharmaceutical
manufacturing and are easily controlled for quality according
to blood banking standards (one batch ¼ 25 blood donors;
produced in the Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tübingen, Tübingen,
Germany). However, studies investigating the effects of allogeneic serum on BM-MSCs are contradictory [24, 28, 36, 40].
Alternatively, tPRP requires a complicated manufacturing
process [27]. pHPL, in contrast, can be produced by simple
freeze thaw cycles from the standard blood product buffy
coat-derived pooled platelet concentrates. A further advantage
is the possible use of platelet concentrates after their expiry
period of 4-5 days. The freeze-thaw process furthermore
allows for quarantine storage, potentially leading to a larger
batch representing 40 donors. Based on a previously developed GMP-compliant large-scale protocol, a volume of 200
ml pHPL would be sufficient for one clinical scale BM-MSC
expansion [4]. Thus, one batch of pHPL may be sufficient to
expand MSCs from 10-20 patients.
Based on these results, we performed a comprehensive
comparison of four standardized culture protocols together
with three initial MSC enrichment modalities to define optimized clinical scale MSC culture conditions. We selected one
commercially available, pretested FBS batch, analyzed as
being superior to other batches.
Our results showed for the first time that MSC population
doublings and expansion kinetics were significantly enhanced
in pHPL-supplemented BM-MSC cultures compared with cultures supplemented with selected FBS, HS, or tPRP. Using
pooled HS (or tPRP) exerted comparable expansion kinetics
in early passage BM-MSCs like the pretested FBS batch.
Clinically relevant numbers of MSCs could be obtained
within a maximum of three passages with HS or tPRP, equivalent to FBS cultures. These numbers could certainly be
obtained within the first to second passage in pHPL-supplemented cultures. Compared with AT-MSCs, pHPL, but not
HS or tPRP, consistently surpassed FBS in expanding BMMSCs. Cell yields in terms of CFU were maintained.
No change in the cellular quality and potency was
obvious. No lot-to-lot variability of pHPL and no variability
between two manufacturing sites, Graz and Mannheim, were
observed. This is in contrast to findings with FBS, where only
selected lots are appropriate for MSC expansion [19]. Interestingly, in AT-MSCs, pHPL at a concentration of 10% did not
allow the expansion of AT-derived cells beyond p1 [22, 27].
Currently, it is not known why pHPL has a stronger mitogenic effect than HS and tPRP on BM-MSCs. Activating platelets by thrombin or clotting induces secretion of more than 300
proteins and small molecules [41]. Platelet a granules are heterogeneous and contain either pro- or antiangiogenic factors.
Depending on the mode of activation, release of these granule
contents can be differentially induced [42]. Thus far, it cannot
be excluded that various agonists used for platelet activation
select for a certain platelet growth factor composition. We
detected only a limited number of cytokines as differentially
concentrated in feeding or conditioned medium containing
pHPL compared with HS and tPRP. These include bFGF,
GITR, IGFBP-3, latency associated peptide of TGFb, MIF,
MIP-1b, MSP-a, RANTES, VEGF, and all different isoforms
of PDGF. PDGF either as homodimer of the A or B chain or
the AB heterodimer showed the highest concentration in pHPL
(supporting information Table 1). PDGF and bFGF are well-
Human Alternatives to FBS for BM-MSC Expansion
2340
described growth factors for MSCs [36, 43]. In a recent study,
the combination of PDGF, bFGF, and transforming growth factor b was sufficient to expand MSCs in a serum-free medium
under laboratory scale conditions [44]. MIP-1b has recently
been attributed to the promotion of fibrosis [45]. Besides stimulatory activities, inhibitory activities might be promoted by
the growth factors present. Also the variety of extracellular
matrix components including fibrin, fibronectin, vitronectin,
and osteonectin may play pivotal roles [46]. In this context, the
modified expression of the fibronectin receptor CD29 (lowest
positivity in tPRP and highest intensity in HS) will be elucidated in further studies. Because of its complexity, multivariate
designs are planned to identify the most relevant components
[47] .
HS, tPRP, and pHPL allowed the isolation of BM-MSCs
with comparable immune phenotype, in vitro functionality
regarding T-cell suppression, and a differentiation potential
like FBS. Focusing on the intended therapeutic application,
additional tests for genomic stability and in vivo differentiation potential will be necessary [4]. Preliminary data by us
and others suggest that autologous serum may even favor
genomic stability compared with FBS [28, 4]. Despite rare
spontaneous transformation events in FBS-cultured MSCs [18,
48, 49], recent data have shown localized genomic instabilities in human BM-MSCs at clinically relevant passages irrespective of the serum source used [37, 50]. However, cells in
autologous serum displayed a preserved methylated and
unmethylated state compared with FBS [37]. Related to this,
a recent study suggested that allogeneic AB serum may select
for a more immature MSC phenotype, called mesodermal progenitor cells, which can be induced to differentiate into MSCs
by switching the culture to FBS [38].
Unlike AT-MSCs, hematopoietic contamination was only
detectable in the primary culture of BM-MSCs. In contrast to
previous reports of selecting highly proliferative cells by
enrichment of MSCs by RosetteSep or CD271 sorting [51], in
our study, both strategies were not advantageous in any of the
culture conditions tested. Admittedly, we used standardized
and not method-optimized culture conditions. The addition of
growth factors has been suggested for highly purified precursor
cells that do not get trophic support from other cell types [52].
Culturing cells under different conditions may affect the
secretion of trophic mediators. With the cytokine array
applied, we cannot directly compare medium supplemented
with FBS to the human supplements because of the antihuman specificity of most antibodies. The response of MSCs
cultured in FBS shows certain differences as described in
detail within the Results. Currently the cytokine array represents only a preliminary insight into the complex secretome
of MSCs. We have therefore already initiated further studies
to evaluate cytokines that may function as markers to ensure
a quality control of supplement batches and to further monitor
MSC potency and therapeutic efficacy.
Although human components can easily be prepared
according to blood banking standards, there remains the risk
REFERENCES
1
2
3
Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair---current views. Stem Cells 2007;25:2896–2902.
Reinisch A, Bartmann C, Rohde E et al. Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for
clinical application. Regen Med 2007;2:371–382.
Schallmoser K, Bartmann C, Rohde E et al. Human platelet lysate can
replace fetal bovine serum for clinical-scale expansion of functional
mesenchymal stromal cells. Transfusion 2007;47:1436–1446.
of sensitization by blood group substances or by adventitious
agents not covered by routine blood donor testing. Implementation of further procedures such as quarantine storage or
pathogen inactivation into a large-scale, GMP-compliant
pHPL manufacturing may ensure the highest possible quality
standards [53].
Our approach is currently limited by the in vitro comparison of MSC qualities. Further studies focusing on genomic
stability or lack of transformation and in vivo differentiation
potential, as well as homing capacities, are currently underway to show that MSCs isolated and expanded by using
pHPL share properties of FBS-cultured MSCs. The data published thus far support maintenance of differentiation and biologic safety, even in vivo [3, 4, 17, 21]. Presently, the first
application of pHPL expanded BM-MSCs has been performed
to treat refractory graft versus host disease [54].
CONCLUSION
Our data based on a paired analysis of 14 bone marrow donor
MSCs using three different human alternative supplements
compared with FBS for MSC isolation and expansion indicate
that all tested human supplements support the isolation and
expansion of BM-MSCs comparably to FBS. Human platelet
lysate, however, seems to be the optimal component, assuring
enriched cell numbers, maintained viability, cell identity, purity, sterility, and potency of BM-MSCs. pHPL favors not
only very rapid but also long-term expansion while maintaining the immune phenotype, differentiation, and immunomodulatory capacities. The combined fast, profound, and extended
expansion suggests that the progenitor compartment in pHPLsupplemented cultures is best preserved.
ACKNOWLEDGMENTS
We thank Angela Lenzen (H.L.) and Claudia Url (K.S. and D.S.)
for excellent technical assistance, Monica Farrell and Daniele
Griffiths for proofreading the manuscript, and our colleagues
from the German Red Cross Blood Donor Service, especially
from the production unit, for support. This work was supported
by a research fund of the German Federal Ministry of Education
and Research (O1GN O531; K.B., H.L., and H.K.); ‘‘OsteoCord’’ (LSHB-CT-2005-O18999), a project commissioned by
the European Community (K.B.); and Austrian Research Foundation Grant N211-NAN (D.S.).
DISCLOSURE
OF
OF
POTENTIAL CONFLICTS
INTEREST
The authors indicate no potential conflicts of interest.
4
5
6
7
Schallmoser K, Rohde E, Reinisch A et al. Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone
marrow without animal serum. Tissue Eng Part C Methods 2008;14:
185–196.
Honn KV, Singley JA, Chavin W. Fetal bovine serum: A multivariate
standard. Proc Soc Exp Biol Med 1975;149:344–347.
Heiskanen A, Satomaa T, Tiitinen S et al. N-glycolylneuraminic acid
xenoantigen contamination of human embryonic and mesenchymal
stem cells is substantially reversible. Stem Cells 2007;25:197–202.
Sundin M, Ringden O, Sundberg B et al. No alloantibodies against
mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell
recipients. Haematologica 2007;92:1208–1215.
Bieback, Hecker, Kocaömer et al.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Note for guidance on the use of bovine serum in the manufacture of
human medicinal products. EMEA CPMP/BWP/1793/02; 2003.
Note for the guidance on minimising risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal
products EMEA/410/01 rev. 2; 2004.
Halme DG, Kessler DA. FDA regulation of stem-cell-based therapies.
N Engl J Med 2006;355:1730–1735.
Horwitz EM, Gordon PL, Koo WK et al. Isolated allogeneic bone
marrow-derived mesenchymal cells engraft and stimulate growth in
children with osteogenesis imperfecta: Implications for cell therapy of
bone. Proc Natl Acad Sci U S A 2002;99:8932–8937.
Mannello F, Tonti GA. Concise review: No breakthroughs for human
mesenchymal and embryonic stem cell culture: conditioned medium,
feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold!Stem
Cells 2007;25:1603–1609.
Guideline on human cell-based medicinal products. EMEA/CHMP/
410869/2006; 2008.
Stute N, Holtz K, Bubenheim M et al. Autologous serum for isolation
and expansion of human mesenchymal stem cells for clinical use. Exp
Hematol 2004;32:1212–1225.
Lin HT, Tarng YW, Chen YC et al. Using human plasma supplemented medium to cultivate human bone marrow-derived mesenchymal stem cell and evaluation of its multiple-lineage potential.
Transplant Proc 2005;37:4504–4505.
Phadnis SM, Joglekar MV, Venkateshan V et al. Human umbilical
cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells. In Vitro Cell
Dev Biol Anim 2006;42:283–286.
Doucet C, Ernou I, Zhang Y et al. Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cellbased therapy applications. J Cell Physiol 2005;205:228–236.
Bernardo ME, Avanzini MA, Perotti C et al. Optimization of in vitro
expansion of human multipotent mesenchymal stromal cells for celltherapy approaches: Further insights in the search for a fetal calf serum substitute. J Cell Physiol 2007;211:121–130.
Capelli C, Domenghini M, Borleri G et al. Human platelet lysate
allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplant 2007;40:785–791.
Gregory CA, Reyes E, Whitney MJ et al. Enhanced engraftment of
mesenchymal stem cells in a cutaneous wound model by culture in
allogenic species-specific serum and administration in fibrin constructs. Stem Cells 2006;24:2232–2243.
Lange C, Cakiroglu F, Spiess AN et al. Accelerated and safe expansion of
human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cell Physiol 2007;213:18–26.
Davenport M, Verrier S, Droeser R et al. Platelet lysate as a serum substitute for 2D static and 3D perfusion culture of stromal vascular fraction
cells from human adipose tissue. Tissue Eng Part A 2009;15:869–875.
Müller I, Kordowich S, Holzwarth C et al. Animal serum-free culture
conditions for isolation and expansion of multipotent mesenchymal
stromal cells from human BM. Cytotherapy 2006;8:437–444.
Oreffo ROC, Virdi AS, Triffitt JT. Modulation of osteogenesis and
adipogenesis by human serum in human bone marrow cultures. Eur J
Cell Biol 1997;74:251–261.
Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev 1993;7:
52–62.
Janetzko K, Kluter H, van Waeg G et al. Fully automated processing
of buffy-coat-derived pooled platelet concentrates. Transfusion 2004;
44:1052–1058.
Kocaoemer A, Kern S, Kluter H et al. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf
serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 2007;25:1270–1278.
Shahdadfar A, Fronsdal K, Haug T et al. In vitro expansion of human
mesenchymal stem cells: Choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability.
Stem Cells 2005;23:1357–1366.
Lannert H, Able T, Becker S et al. Optimizing BM harvesting from
normal adult donors. Bone Marrow Transplant 2008;42:443–447.
Kern S, Eichler H, Stoeve J et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose
tissue. Stem Cells 2006;24:1294–1301.
2341
31 Bieback K, Kern S, Kluter H et al. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells
2004;22:625–634.
32 Nguyen XD, Eichler H, Dugrillon A et al. Flow cytometric analysis of
T cell proliferation in a mixed lymphocyte reaction with dendritic
cells. J Immunol Methods 2003;275:57–68.
33 Bartmann C, Rohde E, Schallmoser K et al. Two steps to functional
mesenchymal stromal cells for clinical application. Transfusion 2007;
47:1426–1435.
34 Bieback K, Schallmoser K, Klüter H, et al. Clinical protocols for the
isolation and expansion of mesenchymal stromal cells. Transfusion
Med Hemother. 2008;35:286–295.
35 Carrancio S, Lopez-Holgado N, Sanchez-Guijo FM et al. Optimization
of mesenchymal stem cell expansion procedures by cell separation
and culture conditions modification. Exp Hematol 2008;36:1014–1021.
36 Yamaguchi M, Hirayama F, Wakamoto S et al. Bone marrow stromal
cells prepared using AB serum and bFGF for hematopoietic stem cells
expansion. Transfusion 2002;42:921–927.
37 Dahl JA, Duggal S, Coulston N et al. Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. Int J Dev Biol 2008;52:
1033–1042.
38 Trombi L, Pacini S, Montali M et al. Selective culture of mesodermal
progenitor cells (MPCs). Stem Cells Dev 2009 [Epub ahead of print].
39 Kobayashi T, Watanabe H, Yanagawa T et al. Motility and growth of
human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum. J Bone Joint Surg Br 2005;87:1426–1433.
40 Kuznetsov SA, Mankani MH, Robey PG. Effect of serum on human
bone marrow stromal cells: Ex vivo expansion and in vivo bone formation. Transplantation 2000;70:1780–1787.
41 Coppinger JA, Cagney G, Toomey S et al. Characterization of the
proteins released from activated platelets leads to localization of novel
platelet proteins in human atherosclerotic lesions. Blood 2004;103:
2096–2104.
42 Italiano JE Jr, Richardson JL, Patel-Hett S et al. Angiogenesis is regulated by a novel mechanism: Pro- and antiangiogenic proteins are
organized into separate platelet alpha granules and differentially
released. Blood 2008;111:1227–1233.
43 Levy O, Dvir T, Tsur-Gang O et al. Signal transducer and activator of
transcription 3-A key molecular switch for human mesenchymal stem
cell proliferation. Int J Biochem Cell Biol 2008;40:2606–2618.
44 Ng F, Boucher S, Koh S et al. PDGF, TGF-beta, and Fgf signaling is
important for differentiation and growth of mesenchymal stem cells
(MSCs): Transcriptional profiling can identify markers and signaling
pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood 2008;112:295–307.
45 Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol
2008;214:199–210.
46 Gronthos S, Simmons PJ, Graves SE et al. Integrin-mediated interactions between human bone marrow stromal precursor cells and the
extracellular matrix. Bone 2001;28:174–181.
47 Thomas RJ, Hourd PC, Williams DJ. Application of process quality
engineering techniques to improve the understanding of the in vitro
processing of stem cells for therapeutic use. J Biotechnol 2008;136:
148–155.
48 Rubio D, Garcia-Castro J, Martin MC et al. Spontaneous human adult
stem cell transformation. Cancer Res 2005;65:3035–3039.
49 Zhang ZX, Guan LX, Zhang K et al. Cytogenetic analysis of human
bone marrow-derived mesenchymal stem cells passaged in vitro. Cell
Biol Int 2007;31:645–648.
50 Meza-Zepeda LA, Noer A, Dahl JA et al. High-resolution analysis of
genetic stability of human adipose tissue stem cells cultured to senescence. J Cell Mol Med 2008;12:553–563.
51 Poloni A, Maurizi G, Rosini V et al. Selection of CD271(þ) cells and
human AB serum allows a large expansion of mesenchymal stromal
cells from human bone marrow. Cytotherapy 2009;11:153–162.
52 Quirici N, Soligo D, Bossolasco P et al. Isolation of bone marrow
mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002;30:783–791.
53 Janetzko K, Cazenave JP, Kluter H et al. Therapeutic efficacy and
safety of photochemically treated apheresis platelets processed with an
optimized integrated set. Transfusion 2005;45:1443–1452.
54 von Bonin M, Stolzel F, Goedecke A et al. Treatment of refractory
acute GVHD with third-party MSC expanded in platelet lysate-containing medium. Bone Marrow Transplant 2009;43:245–251.
See www.StemCells.com for supporting information available online.