HUMAN PLASMA AND HUMAN PLATELET-RICH PLASMA AS A SUBSTITUTE FOR FETAL CALF SERUM DURING LONG-TERM CULTIVATION OF MESENCHYMAL DENTAL PULP STEM CELLS

Aims: Our aims were to isolate and cultivate mesenchymal dental pulp stem cells (DPSC) in various media enriched with human blood components, and subsequently to investigate their basic biological properties. Methods: DPSC were cultivated in five different media based on α MEM containing different concentrations of human plasma (HP), platelet-rich plasma (PRP), or fetal calf serum (FCS). The DPSC biological properties were examined periodically. Results: We cultivated DPSC in the various cultivation media over 15 population doublings except for the medium supplemented with 10% HP. Our results showed that DPSC cultivated in medium supplemented with 10% PRP showed the shortest average population doubling time (DT) (28.6 ± 4.6 hours), in contrast to DPSC cultivated in 10% HP which indicated the longest DT (156.2 ± 17.8 hours); hence this part of the experiment had been cancelled in the 6th passage. DPSC cultivated in media with 2% FCS+ITS (DT 47.3 ± 10.4 hours), 2% PRP (DT 40.1 ± 5.7 hours) and 2% HP (DT 49.0 ± 15.2 hours) showed almost the same proliferative activity. DPSC’s viability in the 9th passage was over 90% except for the DPSC cultivated in the 10% HP media. Conclusions: We proved that human blood components are suitable substitution for FCS in cultivation media for long-term DPSC cultivation.


Introduction
Stem cells (SC) constitute one of the first cell populations in the nascent organism.Their main roles are to produce specialized cell types and simultaneously to maintain the SC population into adulthood.For this reason, SC are endowed with special properties that distinguish them from all the other cells in the body; such as high proliferative activity, differentiation potential, and the ability of self-renewal.Altogether SC play the key role in development and regeneration of damaged tissues.The growing body of knowledge about their behavior and properties suggests that SC have a great potential as a tool for tissue engineering and cell therapy.
Dental pulp stem cells (DPSC) are typically isolated by enzymatic dissociation of dental pulp tissue, obtained most often from non-erupted lower third molars.They exhibit typical fibroblast-like morphology and retain high proliferation ability, even during prolonged cultivation (1).DPSC express the cell surface mesenchymal stem cells markers and also some markers of bone marrow stem cells (STRO-1 and CD-146) as well as embryonic stem cells markers (Oct4) (2).DPSC cultivation in osteogenic, chondrogenic, dentinogenic, adipogenic, neurogenic and myogenic medium demonstrated their wide differential potential (2)(3)(4).Dental pulp is easily accessible and ethically acceptable source of SC.
Fetal calf serum (FCS, FBS) became, due to its high content of growth factors and conversely a low content of inhibitory factors, the most widely used growth supplement in cultivation media.Due to the origin of the serum, it is not possible to maintain stable and unchanging composition of individual batches (different amount of protein, other bioactive substances, endocrine parameters etc.) (5,6).Many current clinical trials use cultivation media for stem cells enriched with FCS, even though FCS is a potentially dangerous xenogeneic component.Particularly bovine serum proteins can be internalized by stem cells and stimulate immunogenicity (7,8) in this context many problems may also arise due to viral and prion transmission (9,10), or immunological reactions as responses to antigenic substrate (11).Although these "side effects" are well realized, in literature they are often overlooked; an example would be a treatment of myocardial infarction by myoblasts cultured in FCS, which resulted in the formation of malignant ventricular arrhythmias and death of the patient (12); treatment or lack of adenosine deaminase with autologous T cells cultured in medium with FCS supplemented leading to the development of IgG antibodies against FCS (13).Bovine serum contains many polypeptides which can induce metabolic and morphological changes (14,15).Therefore, the interest of the scientific community turns to human blood components or serum-free media containing artificial supplements (16,17).Although, it isn't possible to standardize the human blood components, their usage avoids internalization of the animal substances, thus excluding the risk of zoonosis and immunogenicity (7,9,10).As the cultivation supplement we can used manufactory produced approved AB-serum or umbilical cord blood, which is very hard to obtain.Stojkovic proved that embryonic stem cells cultivated in medium with human serum retain all the characteristics of embryonic cells during long-term cultivation and have unlimited proliferation capacity while maintaining a stable karyotype (18) and Bo Chen, Ui-Lyong Lee and Vijayendram (19,20,21) proved that DPSC can be cultivated in presence of PRP without losing their SC characteristics and differentiation capacity.The effect of platelet rich plasma (PRP) and plasma (HP) derived from human blood is based on degranulation α granules from activated platelets, which contain growth factors.These growth factors are platelet derived growth factor (PDGF), transforming growth factors beta 1 and beta 2 (TGF β1, TGF β2 ), vascular endothelial growth factor (VEGF), platelet derived endothelial cell growth factor, interleukin-1 (IL-1), basic fibroblast growth factor (bFGF), and platelet activating factor-4 (PAF-4 ) (22,23).The human blood serum is obtained by centrifugation of clotted blood (24).Platelet concentrates are defined as components which have 3 to 5 fold higher concentration of platelets in the plasma compared to whole blood (25).It was proved that stem cells cultivated in a media enriched with PRP maintain differentiation capacity, demonstrate higher proliferative activity (19)(20)(21)(26)(27)(28)(29)(30), while it was also shown that human serum alone does not fully support the growth of human mesenchymal stem cells in vitro (31).Another option for replacing the FCS is artificially mixed supplement, but due to the complexity and not completely known composition of FCS it will be very expensive and hard to obtain it.
The aim of this study is to determine the effect of different human blood components on cultivated dental pulp stem cells and if these supplements could be used instead of FCS.

Material and Methods
The dental pulp (DP) used in the experiment was obtained from the twenty years old male patient, who undergone extraction of an impacted third molar due to an orthodontic indication.Prior to donation the patient was informed of the stem cell theory, the planned experiment and has signed an informed consent.The isolation of the DPSC for the experi-mental purposes, the informed consent and a text containing information for the patient were approved by the Ethical Committee of the University Hospital Hradec Králové ref.
200712 S01P; the obtaining and use of blood products in the culture medium was also approved by the Ethical Committee of the University Hospital Hradec Králové ref. 201011 S14P.Blood components were prepared in collaboration with the Transfusion Department.
The isolation of the DP and the DPSC from the tooth took place under sterile conditions in a tissue culture laboratory at the Department of Histology and Embryology LFHK by enzymatic dissociation (DP was cut into pieces of about 1 mm 3 , enzymaticaly dissociated for 50 minutes at temperature of 37.0 °C in a solution of enzyme collagenase type I (Sevapharma, CR), dispase (Invitrogen, USA), PBS (Invitrogen, USA) and HBSS (Invitrogen, USA) in the 1:1:1:1 ratio).The obtained cell suspension was centrifuged for 5 minutes at 2000 rev/min (600 g).The resulting cell pellet was resuspended and inoculated into culture vessels, Cell + (Sarstedt, USA).
The experiment used pooled HP and PRP obtained from 5 donors according to the protocols of Transfusion Department FNHK.

Statistical Analysis
Data are presented as arithmetic mean ± SD.All statistical tests were performed using Microsoft Office Excel 2010 (Microsoft, Redmond, WA).

Results
The DPSC were spindle-shaped with elongated processes.DPSC cultivated in media 1 were more rounded

L-ascorbic acid
1 ml 1 ml 1 ml 1 ml 1 ml (Sigma, USA) (10 mg) (10 mg) (10mg) (10 mg) (10 mg) Glutamin 1.9 ml 1.9 ml 1.9 ml 1.9 ml 1.9 ml (Invitrogen, USA) PEN/STM 0.6 ml 0.6 ml 0.6 ml 0.6 ml 0.6 ml (Invitrogen, USA) GEN 0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.  in compare to those cultivated in media 2, 4, 5. DPSC cultivated in media 2 and 4 share similar morphology and those cultivated in media 5 were more prolonged and less adhered.In media 3 the DPSC were more adhered to the surface of the culture vessel.All the tested media with PRP (2%, 10%) contained microscopic inhomogeneous particles.These were most probably the aggregated residues of the platelets membranes.We assume so because these particles were not found in any other tested media and the presence of platelets was the only difference; moreover in the media 5 (10% PRP) we found substantially more of these (Fig. 1, A-E).DPSC cultured in media 5 exhibited the highest proliferative activity and the associated the shortest population doubling time.DPSC cultivated in all media showed a very high viability, usually in excess of 90%.The viability of DPSC was measured in the 9th passage.Only in the media 3, we were forced to measure the viability already in the 6th passage because of the low proliferative activity and termination of the testing media.Viability in the media 1 was 96.2%, 96.6% in the media 2, 88.1% in the media 3, 96.3% in the media 4, and 91.7% in the media 5.
DPSC cultured in the media 2 showed, compared to the control group cultured in the standard media with FCS, the same expression of tested markers CD 29, CD 73, CD 105 and CD 146, while the positivity for CD 44 increased to 64.78% compared to 41.7%.The DPSC cultivated in the media 4 and 5 showed the same positivity for the CD 29, but compared to standard media significantly lower positivity for other tested markers.Due to the low proliferative activity it was impossible to do the phenotypic analysis of the DPSC cultured in the media 3.The positivity for each marker in media order 1,2,4,5 were for CD 29

Discussion
Despite the stem cells research is already quite advanced, the usage of cell therapy (32,33,34) is not possible in every day human medicine.One of the major reason is the usage of xenogeneic supplements during the SC cultivation (35).
Even though the xenogeneic components represent cheap and easily accessible source of growth factors and substances important for the stem cells proliferation, their usage can cause internalisation of xenogeneic proteins into the cell membrane and transmission of zoonoses, viral or prion infection.The internalized xenogeneic proteins, which are arduous to detect, can later lead to immunological reaction in patients (36,37).
In our study we compared 2 different human blood components in different concentrations and successfully re-placed the FCS in cultivation media.To obtain comparable data we decided to test all 5 different media on one lineage.We are fully aware that DPSC were cultivated in a medium containing 2% FCS during first three passages and this may affect the results of the experiment, but on the other hand through this procedure we have been able to clearly define and demonstrate the changes in phenotype, proliferation ability and viability of the tested DPSC.To investigate the differences among the properties of hMSC cultivated in various media supplemented with FCS, or human blood components (49,50,51,52,19,20,21); however neither of them provides statistically relevant results on which we could build.
The hMSC cultured in media of different compositions resulted in similar morphological variances as previously described for hMSC (38,42,19,20,21).The fastest colony formation and proliferation was observed in the media containing 10% platelet rich plasma.These results confirm that PRP enhances MSC proliferation as described in some earlier studies (50, 51).We have not used any higher concentration of FCS and human blood components than 10%.In our earlier study (unpublished data) we had already proven higher concentrations are cytotoxic and led to senescence of DPSC lineage.DPSC cultivated in media containing 10% PRP proliferate faster, on the other hand DPSC cultivated in media containing 10% HP did not proliferate at all.Media containing 2% HP or 2% PRP enriched by ITS, EGF and PDGF showed similar proliferation activity, which was slower than in case in media of 10% PRP.We conclude that those three supplements support the cells enough to keep proliferation, but still there are more important growth factors which enhanced the proliferation rate.The DPSC cultivated in 10% PRP grew even faster than the ones cultivated in 2% FCS.In our previous studies we had proven that the DPSC cultivated in a media containing 2% FCS proliferate faster than those cultivated in media containing 10% FCS (43), thus we conclude, that DPSC cultivated in 10% PRP media proliferate faster than DPSC cultivated in 10% FCS and therefore this medium seems to be optimal.We didn't observe any significant visible differences between the DPSC cultured in media with 2% FCS, 2% HP and 2% PRP.We suggest that the cause of this result is the comparable amount of growth factors in the Tab.2: The positivity of the DPSC's CD cultivated in tested media.We can see decreasing positivity for CD44, CD73, CD90, CD105, CD146 in DPSC cultivated in media supplemented with PRP. .We can see decreasing positivity for CD44, CD73, CD90, CD105, CD146 in DPSC cultivated in media supplemented with PRP.The decreasing positivity is connected with increasing concentration of PRP in cultivation media.media; either added or released from blood platelets.Our results correlate with the results of studies made on DPSC cultivated in the presence of PRP (19,20,21).The differences among the expression of individual epitopes on the cell surface were very significant between the group of cells cultured in the media containing 2% FCS and 2% HP and the cells cultured in the media containing 2% and 10% PRP.Compared to the cells cultured in the 2% FCS and 2% HP media the expression of the markers CD44, CD73, CD90 and the CD105 was lower on the DPSC cultivated in 2% PRP media and the lowest positivity showed DPSC cultivated in 10% PRP.Conversely the CD29 and CD146 markers expression were analogously high in all the media, with exclusion of the CD146 marker expression in PRP cells, where it was moderate.These findings do not correlate with the studies made by Govindasamy, who did not observe any significant changes.The reason for this disparity can be the fact that we cultivated the DPSC in the PRP medium for 5 passages before phenotypic analysis, in the study made by Govindasamy the analysis was done in passage No. 2 (21).

Conclusions
PRP in concentration of 10% highly increased the proliferation activity, but on the other hand PRP affected the phenotype of the cultivated DPSC.Further study of this effect is necessary, mostly wider panel of tested CD has to be compared and the differentiation capacity has to be proved.HP which was not substitute by the growth factors did not support DPSC enough and the lineage was slowly lost.The experiment has succesfully proven that DPSC can be cultivated in cultivation media where the FCS was replaced by human blood components and thus this xenogeneic component can be substitute by allogeneic or better autologous blood components.

Fig. 1 :
Fig.1: A) Stem cells cultured in the media 1, the image was captured in the 9th passage, 3 days after seeding into the culture vessel.B) Stem cells cultured in the media 2, the picture taken in the 9th passage, 2 days after seeding into the culture vessel.C) Stem cells cultured in the media 3, the picture was taken in the 5th passage 3 days after seeding into the culture vessel.DPSC were more adhrent to the surface of the culture vessel than in other media, therefore they seem to be bigger.D) Stem cells cultured in the media 4, the picture taken in the 9th passage, 3 days after seeding into the culture vessel.E) Stem cells cultured in the media 5, the picture was taken in the 9th passage, 3 days after deployment into the culture vessel.In theFigures A, B, D, Ewe can see the typical DPSC morphology.In the FiguresD, and E, we can see tiny particles that float freely in the culture media.A-E pictures were taken with a phase contrast microscope, original magnification 200×.
Composition of the test media and the quantities of supplements per 100 ml of media.