Eliminating the need of serum testing using low serum culture conditions for human bone marrow-derived mesenchymal stromal cell expansion
© Wappler et al.; licensee BioMed Central Ltd. 2013
Received: 5 December 2012
Accepted: 17 February 2013
Published: 20 February 2013
The conventional expansion of human mesenchymal stromal cells (hMSC) for tissue engineering or (pre-) clinical investigation includes the use of 10% fetal bovine serum (FBS). However, there exists immense lot-to-lot variability in FBS samples and time consuming as well as cost intensive lot pre-testing is essential to guarantee optimal hMSC proliferation and stem cells characteristics maintenance. Furthermore, lot-to-lot variability impedes the long-term consistency of research and comparability between research groups. Therefore, we investigated the use of defined, invariable, non-synthetic FBS in low serum culture conditions for isolation and expansion of hMSC.
hMSC were isolated from bone marrow in Panserin 401 supplemented with growth factors and 2% MSC-tested or non-tested, defined, invariable, non-synthetic FBS and further cultivated in vitro. The surface marker expression, differentiation capacity as well as cell proliferation and cytotoxicity was analyzed and compared between serum samples.
Cells isolated and cultivated with low concentrations of MSC-tested or non-tested FBS demonstrated no differences in surface marker expression or differentiation capacity. Proliferation of hMSC was equal in medium supplemented with either serum with no indication of cell death.
The low serum concentration in Panserin 401 supplemented with growth factors enables the use of defined, invariable, non-synthetic FBS for the isolation and expansion of hMSC. The required hMSC characteristics like surface marker expression and differentiation capacity are maintained. Importantly, no differences in the cell proliferation could be detected. Therefore, using these low-serum culture conditions, the need for lot-to-lot pre-testing of FBS usually needed for optimal hMSC expansion is abolished leading to long-term consistency and comparability of results.
KeywordsCytotoxicity Mesenchymal stromal cells MSC-tested serum Proliferation Serum-reduced medium
The therapeutic application of human mesenchymal stromal cells (hMSC) is of great interest in a variety of diseases including graft-versus-host disease (GvHD) [1, 2], osteogenesis imperfecta [3, 4], acute myocardial infarction [5, 6], spinal cord injury [7, 8], multiple sclerosis [9, 10], and critical limb ischemia [11, 12]. Although hMSC can differentiate into adipocytes, osteocytes and chondrocytes , the beneficial effect in the above mentioned diseases is a paracrine mechanism . hMSC are capable of expressing and releasing a variety of different growth factors (basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), stromal-derived growth factor 1α (SDF-1 α), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF)) and cytokines (interleukin 1 β (IL-1β), IL-6, IL-8, tumor necrosis factor α (TNFα)) in a donor-dependent manner [15–19].
For tissue engineering purposes as well as for the clinical application of hMSC, adequate numbers of viable, transplantable (autologous or allogenic) cells need to be generated. Since hMSC represent only a small proportion (0.01-0.02%) of bone marrow cells, the expansion to high cell numbers is crucial . The conventional applied medium for such expansion procedures is Dulbecco’s modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) . FBS has, however, the disadvantage that there exists immense lot-to-lot variability and unexpected cell growth characteristics . To overcome these difficulties, time-consuming lot pre-testing is required to guarantee optimal cell growth and maintenance of hMSC characteristics. Alternatively, the use of commercial hMSC-tested serum can be applied. However, these pre-tested sera are highly expensive and lot-to-lot variability still exists. In a previous study, we were able to develop serum-reduced culture conditions for even better hMSC proliferation than conventional DMEM with 10% FBS . This serum-reduced medium consists of Panserin 401 supplemented with only 2% FBS and growth factors. The aim of the current study was to evaluate if this low serum concentration can be replaced by defined, invariable, non-synthetic FBS which is available at around 10% of the costs of commercially pre-tested sera and guarantees the consistency and comparability of research results.
Cancellous bone fragments were obtained during operation procedures from four fully anonymous patients (with informed consent according to local ethical board approval of the University Hospital, Aachen) and selected by their plastic adherence as demonstrated previously [15, 17, 23]. Briefly, bone fragments were washed with Panserin 401 media (Pan Biotech GmbH, Aidenbach, Germany) supplemented with 2% FBS (hMSC-tested serum: PromoCell, Heidelberg, Germany; non-tested serum: FBS Gold, PAA Laboratories GmbH, Pasching, Austria) and growth factors (10 ng/ml EGF, 1 ng/ml bFGF, 1 ng/ml PDGF-BB; all PeproTech GmbH, Hamburg, Germany) and 10 nM dexamathasone (Sigma, Steinheim, Germany). The collected media was centrifuged with 500 g for 5 min and the cell pellet was resuspended in 10 ml fresh media, transferred to a T75 flask (Greiner Bio-One, Frickenhausen, Germany) and maintained in a humidified cell culture incubator at 37°C and 5% CO2. Non-adherent cells were removed by media exchange on the next day. Medium exchange was performed every 3–4 days. Pictures were taken using a Leica DMI4000 B microscope (Leica, Wetzlar, Germany) and Diskus software (Carl H. Hilgers Technisches Büro, Königswinter, Germany). When the cells reached nearly confluence, they were detached with trypsin/ethylene diamine tetra acetic acid (EDTA) (PAA) and re-seeded with a density of 4000 cells/cm2. All experiments were performed with cells of passage 1.
Surface marker profiles and multipotent differentiation capacities of isolated hMSC of those four donors were analyzed according to the guidelines set by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy as demonstrated previously [23, 24].
To detect specific surface antigens, cells were detached and fixed with 4% paraformaldehyde for 20 min. After washing with 0.1 M phosphate-buffered saline (PBS), cells were incubated in blocking solution (20% FBS in PBS) for 20 min. After washing with PBS, the cell pellets (300,000 per antibody) were resuspended in 100 μl PBS, 2 μl primary antibody was added and incubated for 30 min. Monoclonal primary antibodies recognizing surface markers CD11b (Invitrogen, Carlsbad, USA), CD19, CD34, CD45, CD73, CD90 (Becton Dickinson, San Jose, USA), CD105 (Invitrogen) and HLA-DR (Abcam, Cambridge, UK) were used. After three washing steps with PBS, 2 μl of the secondary antibody (Alexa-488 conjugated goat anti-mouse, Invitrogen) was incubated in 100 μl PBS for 30 min in the dark. After two final washing steps, the cells were re-suspended in 400 μl PBS and analyzed using a FACSCalibur and FACSCalibur software (Becton Dickinson, Heidelberg, Germany).
For adipogenic differentiation, hMSC were cultivated alternately in adipogenic induction medium and adipogenic maintenance medium for 3–4 days for 3 weeks. Adipogenic induction medium consisted of DMEM (Lonza, Verviers, Belgium) containing 10% FBS, 0.01 mg/ml insulin, 0.2 μM indomethacin, and 0.5 mM isobutyl-methyl-xanthine (all Sigma). Adipogenic maintanance medium consisted of DMEM supplemented with 10% FBS and 0.01 mg/ml insulin. Oil Red O staining (Sigma) was used to visualize lipid droplets formation in differentiated adipocytes. Osteogenic differentiation was induced by cultivation of hMSC in DMEM containing 10% FCS, 10 nM sodium β-glycerophosphate, 100 nM dexamethasone, and 0.05 mM 1-ascorbic acid 2-phosphate (all Sigma). Medium was changed every 3 days for 3 weeks. Osteogenic differentiation was visualized by Alizarin Red S (Sigma) staining to demonstrate matrix mineralization associated with osteoblast differentiation.
Proliferation and cytotoxicity
Cell proliferation and cytotoxicity were measured using the CellTiter-Blue Cell Viability Assay and CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega, Madison, USA) according to the manufacturer’s protocol twice a week for two weeks without passaging of cells . Therefore, 5 × 104 cells of four donors were seeded into 12-wells and grown in culture media supplemented with the different sera. For cytotoxicity measurement, 100 μl of the supernatant were transferred into a black 96-well plate, and incubated with 100 μl CytoTox-ONE for 10 min at 37°C. To measure proliferation, CellTiter-Blue was diluted 1:5 in medium and 1 ml added to each well for 1 h at 37°C and 5% CO2. Subsequently, 200 μl of the supernatant were transferred into a black 96-well plate. For both assays fluorescence intensity was measured using the fluorometer FLUOstar OPTIMA (BMG Labtech, Jena, Germany) with an excitation at 560 nm and emission at 590 nm. After washing with medium, 1 ml fresh medium was added to the cells and incubated until the following measurement.
All data are presented as the mean value ± SD. Statistical differences were determined by one-way ANOVA followed by Bonferroni post hoc testing. A p value of less than 0.05 was considered to be significant. All the statistical analyses and the evaluation of data were performed using GraphPad Prism version 4.03 (GraphPad Software, San Diego, USA).
To evaluate if serum-reduced culture conditions for expansion of bone marrow-derived hMSC to relevant cell numbers for tissue engineering purposes or direct clinical application might be performed with defined, invariable, non-synthetic FBS rather than MSC-tested FBS, hMSC of four different donors were isolated in Panserin 401 supplemented with growth factors and 2% MSC- or non-tested serum. Cultivated hMSC were characterized according to the Mesenchymal Stem Cell Committee of the International Society for Cellular Therapy by three criteria: (I) adherence to tissue culture plastic, (II) specific surface marker expression, and (III) multipotent differentiation capacity.
The use of pre-tested sera is essential to guarantee optimal hMSC proliferation and maintenance of hMSC characteristics. The aim of the current study was to investigate if in low serum culture conditions MSC-tested serum might be exchanged by defined, invariable, non-synthetic FBS. Therefore, hMSC of four different donors were isolated and expanded in both sera and compared regarding their surface marker expression, differentiation capacity and cell proliferation. Analysis of the surface marker expression demonstrated the existence of CD73, CD90 and CD105 as well as the absence of CD11b, CD19, CD34, CD45 and HLA-DR of hMSC expanded with either serum. Furthermore, the differentiation capacity into adipocytes and osteocytes was not affected by the different sera. According to the Mesenchymal Stem Cell Committee of the International Society for Cellular Therapy , all cells isolated by media supplemented with MSC-tested or non-tested serum were proved to be hMSC.
Important for tissue engineering purposes as well as for the clinical application of isolated hMSC is, however, the generation of relevant cell numbers. In a previous study, we were able to demonstrate higher cell proliferation using low serum culture conditions based on Panserin 401 than the conventional applied DMEM with 10% FBS . Comparison of the hMSC proliferation in Panserin 401 supplemented with growth factors and 2% of MSC-tested or non-tested serum revealed no significant differences. Interestingly, lower cell attachment could be detected in cultures that were cultivated with non-tested serum. However, this difference was not significant and could be explained by discrepancies in attachment factor concentration which might also exist between different MSC-tested lots. Since hMSC cultivated with non-tested serum reached equal cell numbers already after 7 days in culture, this lower attachment capacity can be disregarded. In conclusion, hMSC isolated and cultivated in Panserin 401 supplemented with growth factors and 2% non-tested FBS maintain their cell-specific characteristics and proliferative capacity.
The defined, invariable, non-synthetic FBS used in this study is chromatographically purified and fractionated. Afterwards, the individual constituents are recombined to guarantee the defined composition (http://www.paa.com). The advantage of the use of this FBS is, besides the lower price, that the necessity of serum lot testing is abolished. Furthermore, research groups from all over the world could work with the same FBS which might lead to more comparable results as well as an efficient transfer of results to the clinic.
In an attempt to eliminate FBS in hMSC culture conditions, Lohmann et al.  investigated the use of human platelet lysate (HPL). Primary isolation and differentiation by using 10% HPL could be achieved; however, the HPL donor age immensely affected hMSC proliferation rate.
Main cultivation protocols of human bone marrow-derived MSC (grey: serum-free protocol)
Primary isolation demonstrated
DMEM 10% FBS
DMEM/F12 20% FBS or 20% human autologous serum or 20% human allogenic serum
Shahdadfar et al. 
DMEM 10% HPLs
Lohmann et al. 
Panserin 401 2% FBS and GF
Montzka et al. 
ESF basal medium and supplements
Mimura et. al 
Stem Pro MSC SFM (Invitrogen)
Chase et al. 
In conclusion, the current study demonstrated the isolation, expansion and maintenance of hMSC characteristics by the use of low serum concentrations using a defined, invariable, non-synthetic FBS. This media composition enables the generation of consistent research results over long periods and importantly, simplifies the collaboration and comparability within different research groups.
Basic fibroblast growth factor
Dulbecco’s modified eagle medium
Ethylene diamine tetra acetic acid
Epidermal growth factor
Fetal bovine serum
US food and drug administration
Hepatocyte growth factor
Human mesenchymal stromal cells
Human platelet lysate
Platelet-derived growth factor
Transforming growth factor
Tumor necrosis factor
Vascular endothelial growth factor.
This work was supported by the START-program from the RWTH Aachen University.
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