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Article
Human Platelet Lysate Can Replace Fetal Calf Serum
as a Protein Source to Promote Expansion and
Osteogenic Differentiation of Human
Bone-Marrow-Derived Mesenchymal Stromal Cells
Maria Karadjian, Anne-Sophie Senger, Christopher Essers, Sebastian Wilkesmann,
Raban Heller , Joerg Fellenberg , Rolf Simon and Fabian Westhauser *
Center of Orthopedics, Traumatology, and Spinal Cord Injury, Heidelberg University Hospital,
Schlierbacher Landstraße 200a, 69118 Heidelberg, Germany; karadjian.maria@gmail.com (M.K.);
annesophie.senger@gmail.com (A.-S.S.); c.essers@stud.uni-heidelberg.de (C.E.);
sebastianwilkesmann@web.de (S.W.); raban.heller@outlook.com (R.H.);
Joerg.Fellenberg@med.uni-heidelberg.de (J.F.); dr.rolf.simon@web.de (R.S.)
*Correspondence: Fabian.Westhauser@med.uni-heidelberg.de; Tel.: +49-6221-56-25000
Received: 16 February 2020; Accepted: 6 April 2020; Published: 9 April 2020
Abstract:
Fetal calf serum (FCS) is frequently used as a growth factor and protein source in
bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic
product presenting multiple disadvantages including but not limited to ethical concerns. A promising
alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates
and happens to be a stable and reliable protein source. In this study, we investigated the influence
of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on
the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed
population doublings during cell expansion, performed alizarin red staining to evaluate the calcium
content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as
osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented
with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the
calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented
ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a
protein supplier in cell culture media and does not negatively affect the osteogenic differentiation
capacity of BMSC.
Keywords:
osteogenic differentiation; population doublings; human mesenchymal stromal cells; fetal
calf serum; human platelet lysate
1. Introduction
Despite some limitations, fetal calf serum (FCS) is still the most common source of proteins
and growth factors in cell culture media [
1
]. However, the composition of different batches of FCS
varies crucially [
2
,
3
], making it necessary to evaluate different batches of FCS before ordering a new
batch to cope with the batch-to-batch variability [
1
,
4
]; this evaluation takes time and resources [
1
].
The influence of FCS as a xenogenic compound of cell culture media on human cells is still not
completely understood [
1
,
5
–
7
] as its composition differs substantially from the very complex human
serum [
8
–
11
]. In addition, the conditions of FCS production regarding animal welfare have to be
viewed critically, especially since production circumstances often remain unclear and information
gathering is reported to be complicated [
12
]. This raises the question whether an alternative protein
source standard in cell culture media is required.
Cells 2020,9, 918; doi:10.3390/cells9040918 www.mdpi.com/journal/cells
Cells 2020,9, 918 2 of 12
The most common alternative to FCS is human platelet lysate (hPL) [
1
], which is produced
from human platelets and contains proteins and growth factors necessary for, inter alia, in-vitro
human bone-marrow-derived mesenchymal stromal cell (BMSC) expansion [
13
–
18
] and osteogenic
differentiation [
15
,
16
,
19
]. hPL products can have smaller batch-to-batch variabilities by pooling multiple
batches of platelet concentrates, hence extensive pre-testing becomes obsolete [
17
,
20
]. It has already been
shown that hPL-supplemented expansion medium (ESM) augments the proliferation rates of BMSC [
11
,
16
–
18
,
21
–
29
], and does not affect BMSC properties as part of ESM [
21
]. Several studies evaluated
BMSC differentiation potential after cell expansion in hPL-containing medium, while the differentiation
medium itself contained only FCS as a supplement [
17
,
18
,
22
,
23
,
28
–
30
]. There have also been multiple
studies reporting that hPL does not affect osteogenic differentiation [
16
,
17
,
21
,
22
,
24
,
27
–
29
,
31
] or even
increases the osteogenic differentiation potential of BMSC [
22
,
23
,
25
,
26
]. However, it remains unclear
how hPL as a part of an osteogenic differentiation medium (ODM) influences the kinetics of the
osteogenic differentiation of BMSC. Furthermore, the necessary concentrations of hPL reported differ
from 2% to 10% [
18
,
20
,
25
,
26
,
29
–
34
], pointing out that there is no established standard protocol for hPL
supplementation in ODM for BMSC yet, although the concentration of serum supplement in media
resulting in different concentrations of growth factors and other components of the serum influences
cells in culture [1].
The aim of this study was to compare the performance regarding the proliferation rate and
osteogenic differentiation of BMSC cultured in ESM and ODM containing different concentrations of
hPL or FCS and to evaluate the impact of the protein supplement’s concentration on the osteogenic
differentiation of BMSC. As most available studies on hPL assess its effects on mesenchymal stromal
cells (MSC) during the expansion period, we focused on the effects of hPL as part of ODM on the
kinetics of BMSC osteogenic differentiation by evaluating alkaline phosphatase (ALP) activity and
calcium deposition as osteogenic differentiation markers at multiple time points.
2. Materials and Methods
The aims of this study were (i) to compare the impact of either FCS or hPL on the growth of
BMSC under expansion conditions and (ii) to evaluate the osteogenic differentiation potential of BMSC
cultivated in either FCS or hPL containing ODM. In order to compare the proliferation potential,
population doublings of BMSC incubated in ESM with either 10% FCS or 10% hPL were calculated.
For osteogenic differentiation experiments, the cells were incubated in ODM (containing either 1% or
10% of FCS or hPL, respectively) and measurement of ALP activity as well as alizarin red staining as
markers for osteogenic differentiation were performed after incubation periods of 1, 7, 14 and 21 days
(D1, D7, D14, D21). Twelve identical samples were analyzed for each assay at each time point.
BMSC were harvested from the bone marrow washouts of n =10 patients undergoing surgery at
the proximal femur at Heidelberg University Hospital. Prior to bone marrow harvesting, every patient
gave written informed consent. The local ethics committee approved the study (S-443/2015) that was
conducted according to the declaration of Helsinki in its present form. Cells derived from six male
and four female patients with an average age of 50 years (range 29 to 73 years, median 46 years) were
included in the study.
BMSC were isolated from bone marrow, cultivated according to previously published protocols
and pooled in order to reduce inter-individual variability as published previously [
35
,
36
]. During
cultivation and expansion, ESM containing either 10% FCS (Thermo Fisher Scientific, Dreieich, Germany;
lot 42G2082K) according to our standard expansion protocol or 10% of commercially available hPL
(PL BioScience, Aachen, Germany) according to the manufacturer’s instructions for high proliferation
rates was used. The ESM was composed of 25 mM DMEM high-glucose (Thermo Fisher Scientific,
Dreieich, Germany), Penicillin/Streptomycin 100 mg/L (Merck, Darmstadt, Germany), L-Glutamine
200 mM, MEM non-essential amino acids solution, 2-Mercaptoethanol 50 mM (all Thermo Fisher,
Dreieich, Germany) and 4 µg/L fibroblast growth factor two (Abcam, Cambridge, United Kingdom).
Cells 2020,9, 918 3 of 12
BMSC were split after 72 hours of expansion and cultured for a further 96 hours before they were
introduced to osteogenic differentiation conditions. Cells were then stained with trypan blue and
counted in a Neubauer’s cell chamber. Population doublings within a total incubation period of seven
days of expansion as a parameter for the proliferation rate were calculated using Formula (1).
population doublings =
log N −log N0
log 2 (1)
where N =cell number at the end of the expansion period and N0 =cell number at time point zero.
Population doublings of the passages were added in order to obtain cumulative population doublings.
For osteogenic differentiation, 35,000 BMSC were seeded in a well of a 24-well plate (Thermo Fisher
Scientific, Dreieich, Germany) and incubated in ODM. The ODM was composed of 25 mM DMEM
high-glucose with L-Glutamine (Thermo Fisher Scientific, Dreieich, Germany), Penicillin/Streptomycin
100 mg/L (Merck, Darmstadt, Germany), dexamethasone 0.1
µ
M (Sigma Aldrich, Steinheim, Germany),
ascorbic acid-2-phosphate 2.5 mg/L (Sigma Aldrich, Steinheim, Germany) and
β
-glycerophosphate
10 mM (Merck, Darmstadt, Germany). The growth factor sources in ODM were either FCS or hPL,
added in concentrations of either 1% (according to hPL’s manufacturer’s advice for differentiation
culture) or 10% according to the well-known FCS standard concentration (Table 1). In the ODM
containing hPL, 2 IU/mL heparin (PL BioScience, Aachen, Germany) were added according to the
manufacturer’s instructions to avoid gel formation of the medium. The cells were incubated for up to
21 days at 37
◦
C, 5% CO
2
following well-established protocols [
36
–
38
]. Media were changed twice
per week.
Table 1. Overview over the experimental groups.
Group Protein Source and
Concentration in ESM
Protein Source and
Concentration in ODM
F1 FCS 10% FCS 1%
F10 FCS 10% FCS 10%
H1 hPL 10% hPL 1%
H10 hPL 10% hPL 10%
ALP correlates with osteogenic differentiation of BMSC since it is produced during differentiation
towards osteoblasts [
37
,
38
]. The cells were washed with 1x PBS (Thermo Fisher Scientific, Dreieich,
Germany), then lysed in 1% Triton buffer (Sigma Aldrich, Steinheim, Germany). The lysates were freeze
stored at
−
80
◦
C until the ALP activity measurement was conducted; after thawing at room temperature
and short centrifugation, the lysates were added to a para-nitrophenylphosphat (p-NPP) (Sigma Aldrich,
Steinheim, Germany) solution for 90 minutes. ALP hydrolyzes p-NPP to para-nitrophenol (p-NP),
causing a change of color to yellow. The extinction of p-NP which corresponds to the ALP activity was
measured photometrically at 405 nm with a reference wavelength of 490 nm using a MRX Microplate
Reader (Dynatech Laboratories, Stuttgart, Germany). ALP activity was normalized to the total protein
content as correlate for the cell number of the samples, determined using the Micro BCA Protein Assay
Kit (Thermo Fisher Scientific, Dreieich, Germany) according to manufacturer’s instructions.
Alizarin red stains the calcium deposits in the extracellular matrix formed by the osteoblastic
(precursor) cells [
38
]. The cells were washed with 1x PBS and then fixed in 70% ethanol (Carl Roth,
Karlsruhe, Germany). After fixation, the ethanol was removed, the cells were washed with distilled
water (Thermo Fisher Scientific, Dreieich, Germany), then stained with alizarin red (Waldeck, Münster,
Germany) and washed again. Finally, the cells were incubated in 10% hexadecylpyridinium (Merck,
Darmstadt, Germany) to dissolve the stained calcium. The extinction of the solution corresponds to
the calcium content of the sample and was measured photometrically at 570 nm.
Cells 2020,9, 918 4 of 12
The experiments were performed on 12 experimental replicates per study group and two technical
replicates per experimental sample.
Statistical analyses were conducted with IBM SPSS Statistics (Version 25; IBM, Mannheim,
Germany) and graphs were created using GraphPad Prism (Version 7, GraphPad Software, La Jolla,
USA). Before further analysis, values were tested for normal distribution by a Shapiro-Wilk test.
Normally distributed samples were tested with the unpaired T-Test; not normally distributed samples
were tested using the Wilcoxon signed-rank test. Results were described as statistically significant for
p<0.05. Unless otherwise stated, differences mentioned in the text are non-significant. Values are
shown as rounded means with standard deviation (SD) where applicable.
3. Results
3.1. Population Doublings
Cells cultured in 10% hPL-supplemented ESM had significantly (p<0.01) more population
doublings, with an average of 4.46 cumulative population doublings, than cells cultured in 10%
FCS-supplemented ESM with an average of 2.22 cumulative population doublings (Figure 1).
Cells 2020, 9, x FOR PEER REVIEW 4 of 12
Statistical analyses were conducted with IBM SPSS Statistics (Version 25; IBM, Mannheim,
Germany) and graphs were created using GraphPad Prism (Version 7, GraphPad Software, La Jolla,
USA). Before further analysis, values were tested for normal distribution by a Shapiro-Wilk test.
Normally distributed samples were tested with the unpaired T-Test; not normally distributed
samples were tested using the Wilcoxon signed-rank test. Results were described as statistically
significant for p < 0.05. Unless otherwise stated, differences mentioned in the text are non-significant.
Values are shown as rounded means with standard deviation (SD) where applicable.
3. Results
3.1. Population Doublings
Cells cultured in 10% hPL-supplemented ESM had significantly (p < 0.01) more population
doublings, with an average of 4.46 cumulative population doublings, than cells cultured in 10%
FCS-supplemented ESM with an average of 2.22 cumulative population doublings (Figure 1).
Figure 1. Population doublings of BMSC incubated in ESM. Values are presented as means with SD,
* marks significant differences.
3.2. Alkaline Phosphatase Activity
ALP activity kinetics differed among the four groups during the incubation period (Figure 2a).
In the F1 group, ALP activity increased significantly from D1 to its maximum on D14, then remained
on a stable level until D21, showing no significant differences between D14 and D21. The H1 group
increased almost tenfold from D1 to D7, then decreased to D14 to further decrease until D21. The F10
group showed similar kinetics to the H1 group, but presented significantly different values to all
time points. H10 showed its maximum ALP activity on D7, then significantly decreased to D14 to
re-increase until D21. Differences between the F1 and H1 group were significant in the beginning of
differentiation culture on D1 and D7, but showed no significant differences at D14 and D21.
Differences between the H1 and the F10 group were significant at any time. Differences between the
F10 and H10 group were significant on D7 and D21. In both hPL and FCS groups, cells incubated in
the 10% supplemented media showed significantly higher ALP activity than the 1% groups on D7 to
D21, but not on D1 (Figure 2b,c).
Figure 1.
Population doublings of BMSC incubated in ESM. Values are presented as means with SD,
* marks significant differences.
3.2. Alkaline Phosphatase Activity
ALP activity kinetics differed among the four groups during the incubation period (Figure 2a).
In the F1 group, ALP activity increased significantly from D1 to its maximum on D14, then remained
on a stable level until D21, showing no significant differences between D14 and D21. The H1 group
increased almost tenfold from D1 to D7, then decreased to D14 to further decrease until D21. The F10
group showed similar kinetics to the H1 group, but presented significantly different values to all time
points. H10 showed its maximum ALP activity on D7, then significantly decreased to D14 to re-increase
until D21. Differences between the F1 and H1 group were significant in the beginning of differentiation
culture on D1 and D7, but showed no significant differences at D14 and D21. Differences between the
H1 and the F10 group were significant at any time. Differences between the F10 and H10 group were
significant on D7 and D21. In both hPL and FCS groups, cells incubated in the 10% supplemented
media showed significantly higher ALP activity than the 1% groups on D7 to D21, but not on D1
(Figure 2b,c).
Cells 2020,9, 918 5 of 12
Cells 2020, 9, x FOR PEER REVIEW 5 of 12
Figure 2. (a) Alkaline phosphatase activity during time of incubation in ODM in IU/mL of all groups.
(b) ALP activity of FCS groups. (c) ALP activity of hPL groups. Values are shown as means with SD.
* mark significant differences. D = day.
3.3. Alizarin Red Staining
Calcium content increased in all groups over time of incubation. Calcium content in the hPL
groups peaked on day 14, and the FCS groups showed maximum calcium content on day 21 (Figure
3a). During the whole differentiation period, the H1 group showed significantly higher calcium
levels compared to the F1 group. H10 showed significantly higher calcium values at D1, D7 and D14
compared to F10, but lower values at D21 – however, the differences on D21 remained
non-significant. When comparing H1 and F10, H1 showed significantly higher values from D1 to
D14 and lower values on D21. When comparing the hPL groups (Figure 3c), H1 showed significantly
higher calcium deposition than H10 on D1 and D7; this relation changed on D14 and D21 where H10
presented significantly higher values than H1. F1 presented the lowest values of all four groups on
days seven to 21, significantly lower than the F10 group (Figure 3b).
Figure 2.
(
a
) Alkaline phosphatase activity during time of incubation in ODM in IU/mL of all groups.
(
b
) ALP activity of FCS groups. (
c
) ALP activity of hPL groups. Values are shown as means with SD.
* mark significant differences. D =day.
3.3. Alizarin Red Staining
Calcium content increased in all groups over time of incubation. Calcium content in the hPL
groups peaked on day 14, and the FCS groups showed maximum calcium content on day 21 (Figure 3a).
During the whole differentiation period, the H1 group showed significantly higher calcium levels
compared to the F1 group. H10 showed significantly higher calcium values at D1, D7 and D14
compared to F10, but lower values at D21 – however, the differences on D21 remained non-significant.
When comparing H1 and F10, H1 showed significantly higher values from D1 to D14 and lower
values on D21. When comparing the hPL groups (Figure 3c), H1 showed significantly higher calcium
deposition than H10 on D1 and D7; this relation changed on D14 and D21 where H10 presented
significantly higher values than H1. F1 presented the lowest values of all four groups on days seven to
21, significantly lower than the F10 group (Figure 3b).
Cells 2020,9, 918 6 of 12
Cells 2020, 9, x FOR PEER REVIEW 6 of 12
Figure 3. (a) Calcium content after alizarin red staining during time of incubation in ODM in mg/mL
of all groups. (b) Calcium content of FCS groups. (c) Calcium content of hPL groups. (d–g) Cell layer
in 24-well plate after alizarin red staining on D7. (d) F1. (e) F10. (f) H1. (g) H10. Values are shown as
means with SD. * mark significant differences.
4. Discussion
The aim of the study was to investigate whether hPL is a valid substitute for FCS in cell culture
media for BMSC, especially in ODM, since FCS shows some limitations. For example, FCS
composition differs essentially from human serum: Shanskii et al. [11] showed that hPL has
significantly higher amounts of growth factors like IGF-1 (insulin-like growth factor 1) and PDGF
(platelet-derived growth factor) than FCS. Despite the long-term use of FCS in cell culture, the effects
of this xenogenic compound on human cells in culture remain unclear, also due to the fact that a
majority of research on FCS dates from the past century [2,3,8]. Apart from the mentioned scientific
concerns, also an ethical component has to be taken into account when discussing further usage of
FCS: the European Food Safety Authority (EFSA) published a scientific opinion to, amongst others,
answer the central question how to treat unborn livestock during slaughter, which is relevant for the
procedure of FCS harvesting [39]. FCS is produced out of the blood collected by puncturing the heart
of unborn, generally non-anesthetized cattle [20,40]. According to data obtained from Jochems et al.
[12], cardiac puncture of the fetus begins between five and 30 minutes after the death of the dam and
the bleeding procedure takes between two and five minutes. Another concern when it comes to
ethical questions is the fact that FCS is mostly produced outside of the European Union; specifically,
the exact origin of commercially available FCS often remains vague (e.g., origin “South America”)
[12,39–41], making it almost impossible to trace specific FCS products.
hPL is generally produced out of a varying number of pooled human platelet concentrates
[13,18,42–45] after platelet lysis by freezing—thawing cycles [20,43–45] or activation by thrombin
[13,42] to liberate the substances required for cell culture that are stored in platelet granules [14,15].
However, there is no good manufacturing practice statement concerning the platelet lysis method in
hPL production [13]. The hPL available for scientific purposes can be produced out of outdated
platelet concentrates having no clinical use whilst being still suitable for (in-vitro research) cell
culture [13,20,43]. Furthermore, the hPL composition creates a milieu closer to the physiological
milieu of the human body than FCS does [8–11]. Therefore, hPL has favorable properties in terms of
Figure 3.
(
a
) Calcium content after alizarin red staining during time of incubation in ODM in mg/mL of
all groups. (
b
) Calcium content of FCS groups. (
c
) Calcium content of hPL groups. (
d
–
g
) Cell layer in
24-well plate after alizarin red staining on D7. (
d
) F1. (
e
) F10. (
f
) H1. (
g
) H10. Values are shown as
means with SD. * mark significant differences.
4. Discussion
The aim of the study was to investigate whether hPL is a valid substitute for FCS in cell culture
media for BMSC, especially in ODM, since FCS shows some limitations. For example, FCS composition
differs essentially from human serum: Shanskii et al. [
11
] showed that hPL has significantly higher
amounts of growth factors like IGF-1 (insulin-like growth factor 1) and PDGF (platelet-derived growth
factor) than FCS. Despite the long-term use of FCS in cell culture, the effects of this xenogenic compound
on human cells in culture remain unclear, also due to the fact that a majority of research on FCS dates
from the past century [
2
,
3
,
8
]. Apart from the mentioned scientific concerns, also an ethical component
has to be taken into account when discussing further usage of FCS: the European Food Safety Authority
(EFSA) published a scientific opinion to, amongst others, answer the central question how to treat
unborn livestock during slaughter, which is relevant for the procedure of FCS harvesting [
39
]. FCS is
produced out of the blood collected by puncturing the heart of unborn, generally non-anesthetized
cattle [
20
,
40
]. According to data obtained from Jochems et al. [
12
], cardiac puncture of the fetus begins
between five and 30 minutes after the death of the dam and the bleeding procedure takes between two
and five minutes. Another concern when it comes to ethical questions is the fact that FCS is mostly
produced outside of the European Union; specifically, the exact origin of commercially available FCS
often remains vague (e.g., origin “South America”) [
12
,
39
–
41
], making it almost impossible to trace
specific FCS products.
hPL is generally produced out of a varying number of pooled human platelet
concentrates [
13
,
18
,
42
–
45
] after platelet lysis by freezing—thawing cycles [
20
,
43
–
45
] or activation
by thrombin [
13
,
42
] to liberate the substances required for cell culture that are stored in platelet
granules [
14
,
15
]. However, there is no good manufacturing practice statement concerning the platelet
lysis method in hPL production [
13
]. The hPL available for scientific purposes can be produced out of
outdated platelet concentrates having no clinical use whilst being still suitable for (in-vitro research)
cell culture [
13
,
20
,
43
]. Furthermore, the hPL composition creates a milieu closer to the physiological
milieu of the human body than FCS does [
8
–
11
]. Therefore, hPL has favorable properties in terms
Cells 2020,9, 918 7 of 12
of ethics and scientific practice and should seriously be considered as a new standard cell culture
medium substitute.
To evaluate the impact of hPL on cell proliferation, we compared the expansion of BMSC in
ESM supplemented with either 10% FCS or 10% hPL. We could show that BMSC expanded in
hPL-supplemented ESM had more population doublings than their FCS-supplemented counterparts.
However, the approach for evaluating cell proliferation in our study is a basic approach.
For a more detailed evaluation, assays such as the 3H-thymidine incorporation assay or the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay might provide a more
detailed and standardized quantification of cell proliferation. The promoting effects of hPL on BMSC
proliferation have been described before multiple times, in studies using the aforementioned more
detailed assays; it has been shown that an enhanced proliferation of MSC is one of the main advantages
in hPL supplementation of ESM [
11
,
17
,
21
,
23
,
25
,
26
]. Since our main focus was to assess the influence
of hPL on the osteogenic differentiation of BMSC, we did not look into proliferation in greater detail.
However, based on the assays performed, the results presented in this study confirm the results
of previous studies that can be explained by the higher amount of growth factors in hPL, favoring
cell proliferation and leading to a higher absolute growth factor amount in cell culture media when
supplemented in the same concentration as FCS [11].
Many preliminary studies evaluated the effects of hPL-supplemented ESM on MSC in order
to assess whether hPL changes MSC characteristics during expansion. Most of these studies
evaluated the immunophenotype of the cells after expansion by analyzing cell surface markers
with fluorescence-activated cell sorting (FACS): stem cell defining surface markers [
46
] were analyzed
in almost any study; the consistent results were that expansion of MSC in hPL-supplemented ESM
does not alter the expression of stem cell defining surface markers in comparison to MSC expanded in
FCS-supplemented ESM [
16
–
18
,
22
,
23
,
26
,
28
,
29
,
43
,
44
,
47
]. Viau et al. [
18
] and Reis et al. [
47
] assessed the
expression of further surface markers, revealing that the expression of some surface markers differs
significantly between MSC expanded in hPL or FCS, but the majority of the markers are only mildly
influenced by the source of protein supplement in ESM. Some studies additionally performed gene
expression analyses of expanded MSC and revealed that, along with the results of protein expression
of the investigated markers, the ESM’s protein supplement only mildly influences the gene expression
pattern of MSC [
29
,
43
,
44
]. Fernandez-Rebollo et al. [
44
] performed DNA methylation analysis and
revealed no significant differences between hPL- and FCS-expanded MSC on an epigenetic level.
Viau et al. [
18
] evaluated BMSC morphology by immunofluorescence and FACS after expansion in
hPL-containing ESM and revealed that these cells were smaller and less granular but more homogenous
than their counterparts expanded in the FCS-supplemented ESM. Given the amount of studies that
evaluated MSC characteristics after expansion in hPL-supplemented ESM and the extensiveness of
this aspect, we did not perform any experiments regarding MSC characteristics after expansion in
this study.
In order to evaluate hPL as a substitute for FCS in ODM, we compared the osteogenic differentiation
of BMSC cultured in ODM supplemented with 1% hPL, 10% hPL, 1% FCS and 10% FCS, respectively.
Alizarin red staining and ALP activity measurement were performed at four time points that cover all
relevant steps of osteogenic differentiation in the used setting [
48
]. Osteogenic differentiation increased
over time in all groups. The ALP activity of the BMSC in the hPL groups was higher than or as high
as it was in the matching FCS groups. In addition, BMSC incubated in hPL-supplemented media
presented a calcium content higher than or equal to the FCS groups at any time during cell culture.
It has been shown previously that hPL can have favorable effects on the osteogenic differentiation of
BMSC [
22
,
23
,
26
], providing every component required from serum necessary for cell culture [
13
–
19
]
while respecting human serum composition [
8
–
10
]. However, our study is the first to evaluate the
osteogenic parameters’ evolution under the influence of hPL over the differentiation period in two
different concentrations, making it possible to track the influence of hPL on commonly used time
points during osteogenic differentiation. This culture setting has been used regularly and covers
Cells 2020,9, 918 8 of 12
the major steps of osteogenic differentiation by using the specified evaluation time points between
one and 21 days of culture [
36
–
38
,
49
]: as described previously, the early phases in the differentiation
of BMSC towards osteoblasts takes place from day five to 14 in culture and is characterized by
ALP expression [
48
,
50
]. The calcification of the extracellular matrix produced during osteoblastic
differentiation occurs from day 14 to 28 in culture [48,51,52].
The ALP activity of the hPL groups as well as the F10 group reach their maxima on day seven
and decrease afterwards, so the kinetics of cells incubated in hPL- and FCS-supplemented ESM are
similar. Only the F1 group increases until its maximum on day 14 and remains stable until day 21.
Birmingham et al. [
48
] showed that osteoblastic cells show higher ALP activity than riper stages of the
osteoblastic lineage. Considering the fact that H1, H10 and F10 groups reach their maxima earlier, it
could be stated that osteogenic differentiation under those circumstances happens faster than under
1% FCS supplementation. There are no preliminary data concerning ALP kinetics of BMSC under
hPL supplementation, as photometrical ALP activity measurement of hPL-supplemented BMSC was
hardly performed before. Doucet et al. [
21
] described that ALP activity of BMSC in hPL-supplemented
ODM was comparable to BMSC in FCS-supplemented ODM after 21 days, but no statement regarding
ALP activity kinetics is available. Chevallier et al. [
27
] revealed that hPL can significantly up-regulate
osteogenic genes like ALP in undifferentiated BMSC without exogenous osteogenic stimulation, whilst
FCS-incubated BMSC reach the same level of gene expression only after one week of culture in ODM
containing
β
-glycerophosphate and ascorbic acid. This finding could explain the superior ALP activity
of the hPL groups, especially at the beginning of cell culture.
Focusing on the kinetics of extracellular calcium content, it is remarkable that both FCS groups
increase to their maxima on day 21, whereas the hPL groups reach their maxima earlier, on day 14.
Hoemann et al. [
51
] described that in-vitro mineralization normally occurs after two or three weeks of
differentiation culture, hence both FCS and hPL groups show adequate kinetics, whilst hPL-incubated
BMSC seem to differentiate faster than their FCS incubated counterparts. It is remarkable that no
significant differences can be found between the H10 and the F10 group on day 21, implying that the
final calcium content is similar in both groups while only the kinetics differ. Alizarin red staining
is probably the most investigated parameter for osteogenic differentiation in hPL research, hence
there is a lot of data available [
16
,
22
,
23
,
25
–
27
,
29
]. However, most studies performed a single time
measurement and did not quantify the calcium content photometrically [
16
,
26
,
27
,
29
]. The studies that
performed quantification of calcium content revealed that hPL-incubated BMSC show higher amounts
of calcium than their FCS-incubated counterparts [22,23], which was confirmed in this study.
Comparing FCS concentrations, it is remarkable that the commonly used concentration of 10% [
1
]
seems to be necessary for adequate osteogenic differentiation as both ALP activity and calcium content
remain higher (mostly to a significant extent) compared to 1% FCS-supplemented ODM. The hPL
manufacturer recommended 1% hPL supplementation for BMSC differentiation, which is sufficient for
a higher or comparable calcium content compared to 10% FCS (and 10% hPL). However, ALP activity
in the H1 group remains beneath F10 and H10 groups at any time but D1. Interestingly, most studies
determining hPL concentration in the context of osteogenic differentiation only determined calcium
content [
16
,
22
,
23
,
25
–
27
,
29
,
33
,
53
]; only a few studies assessed ALP activity [
18
,
21
] and, therefore, did not
detect the described discrepancy in ALP activity and calcium deposition. Only 10% hPL-supplemented
ODM showed a constantly better or equal osteogenic differentiation compared to FCS. This discrepancy
persists when comparing the absolute results of alizarin red staining and ALP activity measurement:
in alizarin red staining, the hPL groups showed calcium content higher than or as high as the FCS
groups while ALP activity varies more, especially when comparing H1 and F10 groups, where the
F10 group presents higher ALP activity throughout differentiation from day seven onwards. For the
pairs of H1 vs. F1 and H10 vs. F10, the previously described statement of superiority or equality of the
hPL [16,17,21–24,26–28,31] is still valid.
According to our results and the data reported in the literature, hPL can be used as a supplement
in ESM and ODM for BMSC, providing higher proliferation rates and higher or equal osteogenic
Cells 2020,9, 918 9 of 12
differentiation potential. Our study was the first to evaluate the dynamics of ALP activity under the
influence of two different concentrations of hPL in ODM, providing additional information about
the kinetics of osteogenic differentiation under the influence of hPL. Furthermore, we reported a
discrepancy of the impact of hPL in ODM on BMSC between two osteogenic differentiation parameters
on a cellular/protein level, as there are hardly any studies that have evaluated two quantitative
osteogenic differentiation parameters before. As we did not perform further assays evaluating
osteogenic differentiation of BMSC on a genetic level, future studies should also evaluate osteogenic
gene expression in order to obtain a more detailed impression of the impact of hPL in comparison to
FCS on osteogenic differentiation in one and the same setting.
The concentration of supplementation matters; in our case, only 10% hPL-supplemented ODM
guaranteed an equal or higher osteogenic differentiation than the common approach using 10% FCS.
However, we were the first study directly comparing two different concentrations of hPL in ODM,
therefore further studies should follow in order to approach an optimum hPL concentration in ODM
for BMSC. Considering the higher costs of hPL compared to FCS as well as the fact that the differences
between the H10 and H1 groups were only detectable in the ALP activity assay, the use of hPL in
smaller concentrations seems to be reasonable in order to avoid a significant increase of the budget
necessary for cell culture experiments [
54
]. Based on the findings of this study, hPL should at least be
considered as a potential alternative to FCS when analyzing osteogenic differentiation.
5. Conclusions
The main aim of this study was to evaluate the influence of hPL supplementation in different
concentrations in an osteogenic differentiation medium on the osteogenic differentiation of BMSC,
evaluated by alizarin red staining and ALP activity measurements. As already described in the
literature before, we could show a positive impact of hPL supplementation in an expansion medium
on BMSC population doublings, presenting a proliferation rate almost twice as fast as under FCS
supplementation. Furthermore, we showed that osteogenic differentiation is not compromised, yet,
favored by a hPL-supplementation in ODM in a concentration-dependent manner; the positive effect is
most visible when hPL concentration is 10% and at the beginning of the differentiation period, implying
an accelerated, but absolutely comparable osteogenic differentiation potential of human BMSC.
Author Contributions:
Conceptualization, M.K. and F.W.; Data curation, M.K.; Formal analysis, M.K. and
R.H.; Investigation, M.K., C.E. and S.W.; Methodology, M.K. and A.-S.S.; Project administration, M.K. and
F.W.; Resources, R.S. and F.W.; Supervision, F.W.; Visualization, M.K.; Writing—original draft, M.K. and F.W.;
Writing—review & editing, M.K., A.-S.S., C.E., S.W., R.H., J.F., R.S. and F.W. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
The human platelet lysate used in this study was provided by PL BioScience GmbH, Aachen,
Germany. We acknowledge financial support for open access publishing by the Baden-Württemberg Ministry of
Science, Research and the Arts and by Ruprecht-Karls-Universität Heidelberg.
Conflicts of Interest: The authors declare no conflict of interest.
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