THE EFFECT OF FETAL CALF SERUM ON HUMAN DENTAL PULP STEM CELLS

Summary: Aims: Authors studied potential side effects of fetal calf serum (FCS) in cultivation media on human dental pulp stem cells (DPSC) during long term cultivation. Methods: Two lines of DPSC obtained healthy donors (male 22 years, female 23 years) were used. Both lines were cultivated under standard cultivation conditions in four different media containing 10% or 2% FCS and substituted with growth factors. During long term cultivation proliferation ability, karyotype and phenotype of DPSC were measured. Results: Both lines of DPSC cultivated in a media containing 2% FCS and ITS supplement showed the highest number of population doublings. On the other hand the proliferation rate of DPSC cultivated in a media with 2% FCS without ITS supplement was slowest. Proliferation rate of DPSC cultivated in 10% FCS media with or without FGF-2 was comparable. DPSC cultivated in a media with 10% FCS showed a significantly higher amount of chromosomal aberrations. These chromosomal aberrations do not seem to be clonal but sur-prisingly we found large amounts of tetraploid cells in the 9th passage in both media containing 10% FCS. Conclusions: Our study proved that cultivation of DPSC in media containing higher concentration of FCS has critical side effects on cell chromosomal stability.


Introduction
Multipotent mesenchymal stromal stem cells (MSC) are defined as cells which are able to selfrenew or differentiate into other mature cell types (at last into adipocytes, chondrocytes and ostoblasts). These cells are moreover able to adhere to plastic surface of the cultivation flasks, are supposed to be positive at least for CD105, CD73, CD90 and have imunosupressive abilities and can undergo gene modification. MSC can be found in many different tissues in the multicellular organism during the whole lifetime (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Their main function in the body is to create specialized cell types and maintain an unchanged line for future repair processes, which is why the stem cells can undergo asymmetric division. In contrast with embryonic stem cells, isolation of MSC does not provoke any ethical issues. For these reasons, these cells promised to be a good source for tissue engineering and gene therapy.
We can find many different articles describing properties of MSC, but the main question is: How does the cultivation medium affect the MSC during the long term cultivation?
Nowadays many scientists are focused on finding optimal cultivation media for MSC. Their main goal is to find animal free component media, which will not force MSC to differentiate or to change any of their properties like phenotype or differentiation potential and keeps high proliferation ability (16). There is no question that different types of cells need different approaches during the cultivation. For example, using fetal calf serum (FCS) can enhance the activity of glycerolphosphate dehydrogenase in adipocyte precursors and cause their differentiation (17). Expansion and proliferation of neural progenitors is reduced when cultured in FCS with N2 and without cytokines (18). On the other hand, FCS contains high levels of growth stimulatory factors and keeps lines in high proliferation activity without adding supplementary growth factors (19).
Since the year 2000 where the dental pulp stem cells (DPSC) were isolated from the dental pulp (9), these cells begin to be intensively studied. Their main advantage is relatively easy accessible source and the simplicity of isolation.
In the last 3 years some new approaches were established for isolation and cultivation of human dental pulp stem cells (DPSC). Recently described the way how to isolate the dental pulp from non-extracted tooth (20) and the protocol of imunomagnetic separation during DPSC isolation was established (21). Scientists have started to work on animal component free medium for DPSC (22) because using of animal components during cultivation prohibits clinical trials of SC in human medicine.
The most widely used animal component during the long term cultivation is FCS. Its amount in cultivation media varied from 2-20%. The problem is that FCS is not ORIGINAL ARTICLES a standardized supplement and the composition of each batch can vary slightly. Moreover it can be the source of different contamination which can cause allergic reactions or infectious diseases like bovine disease transmitted by prions and other zoonosis.
In this study we have focused on finding how the FCS can affect DPSC (e.g. karyotype, phenotype, proliferation ability) and which supplements could be used in the case of decreasing amount of FCS.

Isolation
We isolated two different lines of DPSC, both from semierupted third molars. The first line marked DPSC 1 was obtained from a 23 year old female, the second line DPSC 2 was isolated from the third molar of a 22 year old male. The isolation procedure and usage of DPSC was approved by the Ethical Committee of the Faculty hospital Hradec Králové and both donors signed an informed consent. Both lines were isolated according to the procedures described previously (23). Briefly: Third molars were extracted under sterile conditions and transported to a tissue culture laboratory in 4 °C cold Hank's balanced salt solution (Gibco, UK). Roots of the third molars were broken using Luer's forceps, vital pulp was removed using extirpation needle or tweezers through the root canal. Then the pulp tissue was exposed to enzymatic dissociation collagenase (Sevapharma, Czech Republic) and dispase (Gibco, UK) for 70 minutes. Followed by a centrifugation (600 g, 5 min.) we have obtained a cell pellet.

Tested media composition
DPSC were cryopreserved in the third passage. After it was approved that both lines can bypass Hayflick's limit and undergo differentiation, cryopreserved cells were thawed and seeded into four different media. In these media the cells were cultivated from 3rd to 9th passage. Media A composed of alfa-MEM, 2% FCS, 10 ng/ml EGF, 10 ng/ml PDGF, 0.2 mM L-ascorbic acid, 2% glutamine, 100 U/ml penicilin/ 100 µg/ml streptomycin (Gibco, UK), 20 µg/ml gentamycin, 50 nM dexamethasone (Sigma, USA) and 10 µl/ml ITS (catalog number I3146, Sigma, USA) is our standard cultivation media and DPSC cultivated in this media were used as control group.

Statistics
The descriptive statistics of the results were calculated and the charts were made in either Microsoft Offi ce Excel 2010 (Microsoft, Redmond, WA). In this study, all the values were expressed as arithmetic means with SD.

Tab. 4:
Phenotypic analysis of DPSC 1 and DPSC 2 cultivated in tested media in passage No. 9. The value is a percentage expression of the CD.

Discussion
Fetal calf serum is a widely used media supplement which can highly affect cultivated cells and the results of the experiments (19). FCS is so widely used because it contains high amount of growth stimulatory factors and it is cheap. On the other hand, because it is not manufactured but extracted from calves and their natural products, its composition varied not only between producers, but even between each batches from the same producer. The scientist cannot be sure how much their results will be influenced and repeatable (24).
In our studies we are focused on decreasing amount, or better discarding the FCS from cultivation media since 2005. In the previous studies we were able to reduce the FCS to 2%, but still we were not able to discard it totally. In this study we tried to compare the effect of higher concentration of FCS to the DPSC. It is well known, that high amount (about 40%) is even toxic for the cultivated cells. So surely there has to be some negative side effect of FCS on the cells. In this study we tried to find some of these side effects by comparing the DPSC cultivated in 4 different media containing 2% or 10% of FCS and substituted with growth factors. We proved that the FCS can have critical side effect at least on chromosomal stability of the cultivated cells and affect the phenotype of cultivated cells.
We were analyzing proliferation activity, karyotype and phenotype of 2 lines of DPSC cultured in media with different amount of FCS and substituted with different growth factors. To acquire comparative results we cultivated DPSC from two donors and after reaching a sufficient amount of DPSC we split each line into 4 cultivation flasks with different media. Unlike other investigators (25,26) we cultured both lines over 40 population doublings in the tested media A, C, D (about 20 PD from initial 3 passages + at least 22 passages in tested media). We proved, that using 10% FCS medium can highly affect proliferation activity of DPSC (results from samples C + D) in compare to 2% FCS medium (samples B), but when adding ITS in to 2% FCS medium (samples A) proliferation activity will be even faster than in media containing 10% FCS media. Some authors reported that usage of FGF-2 can lead to higher proliferation ability of MSC and does not affect their differentiation ability (27,28). In contrast with another study by Sotiropoulou (29) in which FGF-2 was used in different concentrations and mitogenic activity was proven, we found that proliferation activity did not improve significantly when adding FGF-2 to cultivated in media. The main difference was the positivity for CD105, where DPSC cultivated in media without FGF-2 had higher positivity. The difference between our findings and the findings of the other mentioned author can be explained by different stem cells population. All cited authors used bone marrow stem cells, and we did not find any article describing the effect of FGF-2 on DPSC.
Analysis of karyotype showed that DPSC cultivated in media containing 10% of FCS had significantly more chromosomal aberrations than those cultivated in medium containing only 2% FCS and ITS. Chromosamal aberration during in vitro expansion did not represent high risk for the researchers. Those cells probably undergo apoptosis and will not effect the results, but the it surely represents unacceptable risks for the patients when used in human medicine. After 6 passages in cultivation media containing 10% of FCS the chromosomal aberration occurred in 8/12 of checked mitosis in DPSC 1 line and in 5/29 of checked mitosis in DPSC 2 line, compare to 2 aberration of 11 checked mitosis in DPSC 1 line and in 1 aberration of 25 checked mitosis in DPSC 2 line cultivated in media containing 2% of FCS. The most frequently observed aberrations in DPSC lines cultivated in 10% FCS media were tetraploid. In compare to structural aberrations observed in DPSC lines cultivated in 2% FCS, numerical aberrations are more severe. We did not find any article that described this side effect, but in these days more authors worldwide warns again using FCS as the supplement for cultivation media (30) and offers possible ways of how the FCS can be replaced by other substituent (e.g. human blood serum) (31). Even that these aberrations seems not to be clonal because they did not occur repeatedly we are supposed to be wary when some stem cells cultivated in medium containing FCS are used in human medicine.
Because of low proliferation activity we were not able to compare the results with medium containing only 2% FCS without ITS. This results evokes a new question; does the FCS in higher concentration cause more chromosomal aberrations, or does the usage of FCS result in chromosomal aberrations while the ITS supplement protects the cells? We have to continue with new experiments to answer this question.
Our phenotypic analysis supports our previous findings (32). DPSC showed high or moderate positivity (in the case ITS was added into cultivation media) for mesenchymal stem cell markers (CD29, CD44, CD90 and for CD166 which is also known as ALCAM [Activated leukocyte cell adhesion molecule]) which is a cell surface sensor for cell density and regulates cell migration. Adding ITS supplement into the cultivation media leads to higher expression of CD45, CD71, CD105 and CD133. The higher expression of CD45 (recognize leukocyte common antigen, monocytes and T-cell subset), CD105 (Endoglin, part of the TGF-be-ta1 receptor komplex) which plays a role in the binding of TGF-beta1, TGF-beta3, activin-A, BMP-2 and BMP-7 and CD133 (hematopoietic stem cells, endothelial progenitor cells, glioblastoma, neuronal and glial stem cells) supports the idea that ITS supplement keeps the DPSC less differentiated and prepared for wider differentiation into mature cells lines. Higher expression of CD71 (receptor for transferrin) in a medium with ITS supplement can be explained by the presence of transferrin in cultivation media. This receptor helps the cells import the transferrin-iron complex into the cells through endocytosis. We found that decreasing concentrations of FCS leads to decreasing expression of CD90 (in medium B falls even into low positivity for DPSC 1 and into moderate positivity on media A, B for DPSC 2 ). We are not able to explain this side effect.

Conclusion
Even though some animal component free media are known and already tested, we are far from using FCS free media during in vitro MSC experiments. Even when some clinical trials reported usage of FCS in patient treatment without any side effects, there is no doubt that usage of FCS should be avoided in future human medicine. In our study we proved that fetal calf serum in cultivation media can affect the phenotype profile of cultivated dental pulp stem cells, but moreover it cause chromosomal aberration. These side effects of cultivation can lead into a false result or even more when used in human medicine. The risk of transmission of zoonosis or inducing immunization of the recipients is well known, but the risk of malignant transformation is relatively new problem. If the clinicians will count on the preclinical studies results it can lead not just to the failure of the clinical treatment but also to the threat of patient health. Research with MSC should continue but scientists should also be aware about the results acquired from cells cultivated in media containing high concentration of FCS because it surely affects the basic biological properties of DPSC.