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EFFECTS OF HUMAN PLATELET LYSATE PREPARATIONS
ON THE PROLIFERATION OF BONE MARROW DERIVED
MICE MESENCHYMAL STEM CELLS
Majid Hameed Mohammed
Faculty os science, Center of Scientific Research,University of
Dohouk,Kurdistan,Iraq
(Received31June 2016, Accepted 10 august2016)
Keywords: Stromal cells, Platelet lysate, lysate.
ABSTRACT
Human platelet lysates (HPL) have been described as appropriate cell culture
supplement for cultures of mesenchymal stem cells (MSCs). The increase in demand
for harmless and animal-free cultures of MSCs is related to the potential application
of MSCs grow in human source supplements
In this study, human platelet lysates are derived from fresh and expired blood
donor platelet concentrates. Mouse bone marrow-derived mesenchymal stem cells
were cultured with one of three culture supplements; fetal bovine serum, lysates from
fresh and expired prepared human platelet concentrates. The impacts of these platelet
derived culture supplements on basic mesenchymal stem cell characteristics were
evaluated. All cultures preserved the mesenchymal stem cells surface marker
expression, growth carves and viability test. The result demonstrated that
mesenchymal stem cells complemented with platelet lysates proliferated faster than
conventional FBS cultured cells and increased the expression of the cells. In
conclusion, the use of fresh and expired platelet units from blood banks to prepare
platelet lysates for the culture of MSCs is appropriate as well as keep cells phenotype
characteristics and also shortens culture time by increasing their growth.
INTRODUCTION
Mesenchymal stem cells considered great potential cellular tools used in
regenerative medicine due to their remarkable ability to self-renew and differentiate
into multiple tissues. They have been isolated from different sources including, but
not limited to, bone marrow, adipose tissue, umbilical cord, skin, skeletal muscle,
dental pulp and other dental tissues such as the apical papilla [1, 2, 3 and 4]. Because
MSCs can be found, albeit in small numbers and necessitates there in vitro expansion,
should they be used for therapeutic purposes [5, 6 and 7]. In this regard, the cells are
cultured, under conventional conditions, in the presence of fetal bovine serum (FBS)
should be replacement with other supplement new media
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Until now fetal animal sera still use as medium supplements is widespread,
despite the many disadvantages associated with that [8]. A major problem
encountered is the risk of possible contamination with viruses, prions, bacteria, and
mycoplasma. Additional problem in this culture supplement is cross species
contamination, leading to the expansion of antibodies against FBS as noted in patients
which were infused with MSCs cultured in the presence of FBS [9]. Additional
scientific complications related with the use of FBS include batch-to-batch variability,
fluctuating availability, unexpected cell growth characteristics, cytotoxicity of
uncharacterized factors in the serum, and so forth [10]. The determination of
appropriate culture conditions for optimal growth and suitable functional abilities of
MSCs still remains a critical problem.
Numerous serum-free media have been used in experimental applications for
the in vitro expansion of MSCs [11]. Human platelet lysate (HPL) containing media
were recently described as possible substitutes for FBS-containing media for the
expansion of MSCs for clinical use [12]. Indeed, HPL was described to stimulate
MSC proliferation rate and maintain their differentiation potential and
immunophenotypic characteristics [6-13]. Nevertheless, different scientist has used
different approaches for platelet lysate preparation, leading to great variability
amongst published work regarding which concentrations should be utilized for
optimum results. In this study, the objectives were to describe and standardize
method for the preparation and use of human fresh and expired platelets (FPL and
EPL) in cultures, as well as describe the in vitro effects of FPL and EPL containing
media on MSCs derived from mouse bone marrow.
MATERIALS AND METHODS
1- Preparation of human platelet lysates
Fresh and expired human donor platelets (10 bags, 5 per each of them) were
provided by the blood bank of the Duhok-Kurdistan, Azadi Medical University
Hospital. Platelet donor bags (one bag per donor) were moved under sterile situations
into 250 ml centrifugation cups and centrifuged at 7319 rpm for 20 mins in order to
remove platelet additive solution (PAS) The supernatant was aspirated and the
platelets were washed with 0.9% NaCl. Platelets were resuspended in 15 ml 0.9%
NaCl with a final cell count of ~1.5 x 1010 platelets/ml. The suspension was stored at -
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20°C before lysate preparation by three freeze/thawing cycles. Aliquots of the platelet
lysate were again stored at -20°C for use within 4 weeks. Before addition to serumfree
culture media, aliquots were thawed and centrifuged at 8452 rpm for 10 mins
and supernatants were taken. To identify the most efficient platelet activation method
in order to achieve a maximum yield of growth factors [14].
2- Isolation of Mice Bone Marrow Stem Cells (MBSCs)
Mouse bone marrow stem cells (MBSCs) were obtained and processed as
previously described [15]. MSCs were harvested from the bone marrow (BM) of the
femurs and tibiae, Apply pools of 4-6 mice were sacrificed by cervical dislocation.
Femurs and tibiae were dissected from the surrounding tissues. The extra growth
plates were removed and the BM was collected by flushing with syringe through a 25
gauge needle containing 1 ml of growth medium MEM (US-Biological-USA)
containing 100U/ml penicillin, 100μg/ml streptomycin and 10%FBS. Suspension
cultures were grown in MEM medium supplemented with 10% FBS and antibiotics.
After one passage the MSCs were harvested and transferred to expansion in
mesenchymal stem cell screened FBS (MSCs-FBS), human fresh platelet rich
concentrates (FPL) or expired
platelet rich concentrates (EPL). After two passages in expansion the MSCs were
harvested and seeded in in 25 cm2 sterile disposable polystyrene cell culture flask
(Nunc Easyflasks, Sigma) for experiments. Morphology was evaluated after staining
the MSCs with crystal violet and imaging them. Proliferation analysis was done by
continuing the expansion in each media type and counting the cells after each passage.
3-Subculturing and harvesting of cells
When seeded cells had reached 80%-90% confluence, they were subcultured and
cells were seeded for expansion in the three different expansion media (10% MSCs-
FBS, 10% FPL or 10% EPL). Basal medium was prepared by adding 4 IU/ml of
heparin (LEO Pharma A/S, Ballerup, Denmark) and 1% penicillin/streptomycin
(Gibco, Grand Island, NY, USA) into MEM growth medium (US-Biological-USA)
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4- Growth dynamics
Cells were seeded on 24-well microplates at the density of about 1.5×105 cells
per well, cultivated for 7 days and calculated every day (3 wells each time)
subsequently. The mean cell counts at each time point were then used to plot a growth
curve, based on which the population doubling time (PDT) was calculated, with the
following formula. PDT = 1 / [3.32 (log N– log N1) / (t2– t1)] where NH is the
number of harvested cells at the end of the growth period, N1 is the number of seeded
cells, t1 is the time at seeding, and t2 is the time elapsed between t1 and cell
harvesting [16].
5-Cells Cryopreservation
After three passages, the growth cells were suspended in three different media
containing 40% MEM, (50% MSC-FBS, 50% FPL and 50% EPL) respectively and
10% dimethyl sulfoxide (DMSO). Cells in logarithmic growth phase were counted
with a hemocytometer and adjusted to a density of 3×106 viable cells per mL [17].
Aliquots of the cell suspension were transferred into sterile cryovials and labeled. The
vials were sealed and kept at 4°C for 20-30 min to equilibrate DMSO, put into -30°C
for one h, then into -80°C overnight, and finally transferred to liquid nitrogen for
long-term storage.6 Vials taken from the liquid nitrogen were thawed in a 37°C water
bath, then transferred to flasks with MEM containing (10% FBS, 10% FPL and 10%
EPL) respectively and cultured at 37°C with 5% CO2. The medium was renewed after
24 h.
6- Immunocytochemistry analysis of BM-MSCs
The Immunocytochemistry was done according to the method of Ooi et al [18].
growth cells were tested at passage two mouse MSCs were seeded at a density 1x106
cells per coverslip and fixed with cold methanol: acetone (50:50 volum/volum) for 5
mins. The glass slides were then immersed in 1% hydrogen peroxide in absolute
methanol for 30 mins. The phosphate puffer salain was then added to the glass slide
for 15 mins. The glass slides were then air dried. The primary antibody of the
following CD markers (CD44 and CD90,) were added, and then incubated for 30
mins. The cells were washed three time with PBS for 5 mins. Biotinylated secondary
antibody approximately 1-3 drops was added to the coverslip for 30 mins. (Mouse,
Rat, and Goat secondary antibodies added to specific primary antibody). Then the
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cells were washed three time with PBS for 2 mins each. The mixture of streptavidin
conjugated to horseradish peroxidase was incubated at room temperature for (30)
mins before use, then added to cover the cells, and then the cells were washed with
PBS three times. Each plate was received 1-2 drops of DAB solution and this step was
made in darkness and then left for 10-20 mins. The cells were washed one time with
distilled water. Two drops of Harris Haematoxylin stain were added and left for 5-10
seconds, and then washed with distilled water. Then left for five mins to dry. Drops of
DPX were added to the slide, coverslip were adhered carefully, and finally the slide
were inspected by inverted microscope for detection of MSCs.
7- Statistical analysis
GraphPad® version 5 and Microsoft Office Excel 2007 were used for all data
analysis. Two-way ANOVA was used where applicable and Student’s t-test was used
to confirm statistical significance. P<0.05 was considered statistically significant
RESULTS
1- Morphology
Morphology was examined visually and imaged in an inverted microscope at
10 x magnifications during expansion in growth medium supplemented with MSCs-
FBS, FPL or EPL at 10% concentration respectively. After 1 weeks of culture, an
adherent and stable cell layer was obtained from BM-derived MSCs with all media. In
fact, all the cells shown same morphological changes initially from time 0 were found
to be round and glistening. After 7 days the cells were changed to spindle-shaped cells
like fibroblast (Figure 1)
2- Population doubling assay
MSCs cultured in either FPL or EPL supplemented media showed a higher
growth rate than MSCs cultured in MSC-FBS (n=2 for each expansion media at each
passage). The maximum difference was observed at P4-P5 with mean difference of
3.221 ± 0.6212 CPD (p<0.05, Figure 2) between cells expanded in MSC-FBS or HPL
containing media.
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3- Indirect Immunoperoxidase characterization of MSCs
The morphological appearance of the monolayer cultures at the second passage
by immunocytochemistry staining analysis, for the MSCs isolated in both FBS and
HPL. Immunophenotypic evaluation demonstrated that mouse BM-MSCs uniformly
positive for CD44 and CD90 (Figure 3), All immunoreactivity staining cells were
performed with marker stained strongly brown.
4- Cryopreservation
MSCs in three different media recovered from storage in liquid nitrogen one
month and after 6 months. Cells grew to confluence within 7 days post one month
freezing while no confluence noticed post 6 months freezing (Table 1).
Table 1: Comparison of the viability and confluence of MSCs fibroblast cells
before freezing and after
Freezing time
Cell viability and
confluence
No. of days to reach
confluent
Before freezing
+++
5
1 month
+
7-10
6 months
+/-
_
(+++): Excellent growth, <106 viable cells, <80% confluence
(+): Medium growth, 103 viable cell, 20-40% confluence
(+/-): Little growth, 10-102 viable cells
(-): No growth, no confluence
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Figure: 1 Images of MSCs after day 0 (A-C), 3 (D-F) and 7 days (G-I) in culture with
MSC-FBS, FPL or EPL supplemented media. Cells grown in FPL or EPL exhibit
spherical growth patterns (E-F) and faster growth compared to MSC-FBS grown cells
(H-I). Images were taken in an inverted microscope at 10x magnification. Images are
representative of three experiments.
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Figure 2: Cumulative population doublings of MSCs after culture in FPL,EPL
and FBS
Population doubling assay was performed at the end of every passage for a total of six
passages (P1–P6). MSC cultured in either FPL or EPL consistently had higher
numbers of population doublings at the end of every passage compared to MSC
cultured in FBS.
Figure 3: Immunophenotypic analysis of mouse BM-MSCs at the second passage of
culturing on coverslips revealed by light microscope (X10). (A and B): the most of
adherent MSCs were positive response for CD44 andCD90 marker respectively were
stained with brown color stain DAB.
DISCUSSION
In this study, the results of three different expansion media (10% MSC-FBS,
10% FPL and 10% EPL) on MSC basic appearances were compared. The
characteristics analyzed were proliferation and immunohistochemistry. Mesenchymal
stem cells from three mice were expanded for six passages in each media and then
used in experiments evaluating the basic characteristics.
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After growth in platelet lysates and FBS supplemented media, all cells displayed the
same characteristic morphology of MSCs, long, spindle-shaped cells resembling
fibroblasts and doesn't exhibit any different in morphology and characteristics of
expansion cells throughout the study, This result might have been different with [19]
where noted that the spherical growth pattern of HPL supplemented cultures was
accompanied by circular areas free of cell growth before cultures reached confluence.
We found that culturing MSCs in both FPL and EPL-supplemented medium
significantly increased proliferation rate by 2-fold compared with FBS-cultured MSC
in passage six because platelet lysates are extremely rich in various growth factors
necessary for MSC growth, with even higher concentrations than in FBS[19].
Scientists noted that the mature cells in HPL supplemented media proliferate faster
and reach the limit of population doublings earlier, there is the possibility that these
cells will cease growth sooner than cells grown in media supplemented with FBS
[20]. Moreover, the actual proliferation rate after long term culture in FBS media
compared to HPL supplemented media can be expected to be very similar since a
plateau phase is observed after a few passages in cultures with platelet lysates.
Our study clearly showed that the presence of FPL or EPL is necessary for MSC
growth to substitute the standard expansion medium containing the FBS. The addition
of FPL or EPL in expansion media did not modify the immunophenotype of MSCs,
irrespectively of the amounts used and of the presence or absence of FBS. These cells
display a typical feature of MSCs bearing CD44 and CD90 [21].
Cryopreservation, a method to preserve cells, is as important as cell culture method.
The application of cryopreserved mouse MSCs in cell therapy also requires
preservation of their differentiation and proliferation ability. Thus, it is essential to
investigate whether the freezing process influenced or not by using three different
media FBS, FPL and EPL and their proliferation ability . The study showed excellent
cell adhesion rate, proliferation capability, with appreciable recovery after thawing of
up to 90%. Furthermore, these cells did not lose their differentiation capacity after
cryopreservation. After storage for 1 month, cryopreserved cells had a fairly high
degree of viability. Most of the thawed cells can attach surface of culture flasks,
similar to primary cultured cells. In addition to characteristics described above,
another defining feature that thawed MSCs still kept was their differentiation potential
or their ability into fibroblast when compared with that of primary cultured cells.
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These observations clearly showed cryopreserved MSCs could be stored and
maintained high degrees of viability and differentiation potential.
CONCLUSION
The possibility to use of HPL in supplemented media may be offered some
benefits like faster growth of MSC and improved differentiation as compared to the
traditional use of FBS. Meanwhile no difference between expired and fresh HPL
could be observed, expired platelet units at blood banks and transfusion centers are an
attractive choice for MSC culture, instead of being discarded as is the general practice
today
تاثیر استخدام الصفائح الدمویة البشریة المحضره على نمو وانتشار الخلایا الجذعیة المعزولھ
من نخاع العظام فی الفئران
ماجد حمید محمد
کلیة العلوم، مرکز الابحاث العلمیھ، جامعة دھوک کردستان-العراق
الخلاصھ
لقد وصفت الصفائح الدمویھ البشریھ على انھا وسط مغذی ومکمل لنمو الخلابا الجذعیھ ونتیجة الى زیادة
الطلب عاى الخلایا الجذعیھ بدء التفکیر فی استخدام مصادر مغذیھ غیر حیوانیھ وغیر ضاره ومعزولھ من البشر
یمکن ان تستخدم کوسط مغذی یساعد فی نمو الخلایا الجذعیھ. فی ھذه الدراسھ تم استخدام ثلاث اوساط زرعیھ
مغذیھ وھی الصفائح الدمویھ المشتقھ من دم طازج ودم قدیم وکذلک المصل الجنینی للابقار وملاحظة تاثیرھا فی
نمو وتضاعف الخلایا الجذعیھ المشتقھ من نخاع العظم للفئران. قیم التاثیر اعتمادا فی خصائص الخلایا الجذعیھ،
ومنحنی النمو ومستقبلات الخلایا الجذعیھ على الغشاء. اظھرت النتائج ان الخلایا التی نمت فی وسط الصفائح
الدمویھ المشتقھ دم طازج ودم قدیم قد نمت اسرع من وسط المصل الجنینی للابقار کما لم یظھر ھناک فرق فی
النمو بین الصفائح الدمویھ المشتقھ من الدم الطازج والدم القدیم
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