TY - JOUR
AU - Klüter, Harald
AB - Abstract Mesenchymal stromal cells (MSCs) are promising candidates for novel cell therapeutic applications. For clinical scale manufacturing, human factors from serum or platelets have been suggested as alternatives to fetal bovine serum (FBS). We have previously shown that pooled human serum (HS) and thrombin-activated platelet releasate in plasma (tPRP) support the expansion of adipose tissue-derived MSCs. Contradictory results with bone marrow (BM)-derived MSCs have initiated a comprehensive comparison of HS, tPRP, and pooled human platelet lysate (pHPL) and FBS in terms of their impact on MSC isolation, expansion, differentiation, and immunomodulatory activity. In addition to conventional Ficoll density gradient centrifugation, depletion of lineage marker expressing cells (RosetteSep) and CD271+ sorting were used for BM-MSC enrichment. Cells were cultured in medium containing either 10% FBS, HS, tPRP, or pHPL. Colony-forming units and cumulative population doublings were determined, and MSCs were maximally expanded. Although both HS and tPRP comparable to FBS supported isolation and expansion, pHPL significantly accelerated BM-MSC proliferation to yield clinically relevant numbers within the first two passages. MSC quality and functionality including cell surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar in MSCs from all culture conditions. Importantly, spontaneous cell transformation was not observed in any of the culture conditions. Telomerase activity was not detected in any of the cultures at any passage. In contrast to previous data from adipose tissue-derived MSCs, pHPL was found to be the most suitable FBS substitute in clinical scale BM-MSC expansion. Disclosure of potential conflicts of interest is found at the end of this article. Mesenchymal stromal cells, Fetal bovine serum, Platelet-derived factors, Pooled platelet lysate, Human serum, Bone marrow Introduction Bone marrow (BM) is a complex tissue harboring hematopoietic stem and progenitor cells, endothelial cells, adipocytes, osteocytes, and fibroblastoid stromal cells. On cell culture expansion, BM can yield a multipotent precursor population. These mesenchymal stromal cells (MSCs) have been assessed in a variety of preclinical and clinical settings ranging from regenerative medicine to immunological or hematopoietic support [1]. With MSCs becoming established in the clinical setting, issues have been raised regarding how to expand these cells in large-scale good-manufacturing practice (GMP)-compliant protocols [2–4]. Most expansion protocols use a medium supplemented with fetal bovine serum (FBS). Serum supplementation is practical because it provides the cells with vital nutrients, attachment factors, and growth factors. However, the use of xenogenic serum is complicated because of high lot-to-lot variability and is associated with a risk of transmitting infectious agents and immunizing effects [5–7]. Regulatory guidelines aiming to minimize the use of FBS have further reinforced an intensive search for possible alternatives [8–10]. Most current clinical data have been accomplished with MSCs having been expanded in FBS supplemented media without the appearance of major side effects. In some cases, however, immunological reactions and anti-FBS antibodies have been observed and considered as having possibly affected the therapeutic outcome [7, 11]. A chemically defined standardized, xenogeneic antigen- and serum-free media composition would be the preferential solution for pharmaceutical scale manufacturing. Such a formulation allowing for both isolation and expansion has not been achieved thus far [12]. Based on extensive demand, FBS may also become scarce and expensive. In the development of a cell-based medicinal product, any change in the manufacturing process that impacts final product quality must show comparability or superiority [13]. Human blood products are already considered to represent drugs and are produced accordingly, thus offering certain advantages as potential FBS substitutes. Accordingly, a variety of human supplements have been postulated as alternatives to FBS to provide nutrients, attachment factors, and especially growth factors. These include autologous or allogeneic human serum, human plasma, cord blood serum, human platelet derivatives including platelet lysate, and platelet released factors [3, 4, 14-24]. Analysis of platelet releasates, lysates, and subcellular fractions has shown that numerous bioactive molecules are stored within distinct platelet organelles including adhesive proteins, coagulation factors, mitogens, protease inhibitors, and proteoglycans [25]. Compared with serum, buffy coat-derived platelet preparations are of particular interest because they do not compete with erythrocyte and plasma preparation for the limited available blood donations [26]. In a previous study, we evaluated a variety of platelet activation protocols to obtain biologically active proteins to isolate and expand MSCs. Thrombin-activated platelet releasate in plasma (tPRP) and human blood type AB serum (HS) were found to be superior adjuvants in isolating and expanding human adipose tissue-derived MSCs (AT-MSCs) [27]. The efficiency of both HS and tPRP, but not of pooled human platelet lysate (pHPL), in expanding AT-MSCs was notable in contrast to previous reports on BM-MSCs [20, 28]. Consequently, in a recent study we compared the effects of these three human alternatives on the isolation, expansion, differentiation, and immunomodulatory capacities, as well as the immunophenotype of BM-MSCs using FBS as the standard substitute. The experimental setup was expanded by additional analysis of product purity, because our previous observations showed reduced depletion of contaminating hematopoietic cells in AT-MSCs cultured in human supplements. In this new study, the standard Ficoll gradient density centrifugation method was therefore compared with the enrichment of MSCs by either depleting mature hematopoietic cells or by purifying MSCs expressing CD271 (low affinity nerve growth factor receptor [LNGFR]) and cultivating the obtained mononuclear cells in the four different supplements. Materials and Methods Media and Supplements Dulbecco modified Eagle's medium low glucose (Lonza Group Ltd., Basel, Switzerland, http://www.lonza.com), supplemented with 4 mM L-glutamine (PAA, Coelbe, Germany, http://www.paa.at), 50,000 units (U) penicillin/50,000 μg streptomycin (PAA) served as basal medium in all instances. It was completed with (a) 10% FBS (MSCGM Single Quots; Lonza Group Ltd.), (b) 10% HS, (c) 10% tPRP, or (d) 10% pHPL. Human AB Serum HS was derived from whole blood donations of prescreened AB blood group-typed donors. From each donor, whole blood was drained into blood bags without anticoagulants and allowed to clot overnight at 4°C. The serum was aliquoted and separated by centrifugation at 2,000g for 15 minutes. Subsequently, the supernatant was aliquoted into 15-ml sterile tubes (Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com/en) and frozen at –30°C. After thawing aliquots from at least five donors, HS was pooled and sterilely filtered through 0.2-μm pore filters (Nalgene filtration device; Nalgene Nunc International, Rochester, NY, USA, http://www.nuncbrand.com). HS-supplemented medium was pretested to maintain its mitogenic capacity over a period of at least 4 weeks. Thus, HS medium was not freshly made for each individual use. At least 10 different pools were checked to verify reproducibility. Thrombin-Activated Platelet Releasate Plasma Four whole blood donations of AB or O blood group-typed donors were used to prepare one pooled platelet concentrate derived from buffy coats. Instead of using an additive solution like T-Sol, the pooled platelet concentrate was suspended in AB plasma of one donor. Platelet counts ranged between 20 × 1011 and 30 × 1011 platelets per liter determined by CellDyn 3,200 (Abbott, Wiesbaden, Germany, http://www.abbott.de). Subsequently, the platelet concentrate was activated by 1 U of human thrombin (Sigma Aldrich, Hamburg, Germany, http://www.sigmaaldrich.com) [27]. The released factors were separated from the cellular debris by centrifugation at 3,000g, followed by filtration through 0.2-μm pores. By pooling two pooled platelet concentrates, tPRP finally represented eight donors. Five-milliliter aliquots were stored at –80°C. After thawing, the aliquot was centrifuged again for 5 minutes at 1,500g to remove any developing clots. To prevent in vitro gel formation, 2 U of heparin (Heparin-Natrium-5000-ratiopharm; Ratiopharm, Ulm, Germany, http://www.ratiopharm.de)/ml of medium was added before the tPRP. tPRP was shown to rapidly lose mitogenic activity. A storage time exceeding 48 hours resulted in extensive loss of mitogenic activity; thus, the medium was prepared freshly for each individual use. To verify reproducibility, at least 11 different pools were applied. Pooled Human Platelet Lysate pHPL was prepared in Graz as previously described [3]. Briefly, four buffy coat units of blood group O-typed donors were pooled in AB plasma and centrifuged (340g, 6 minutes, 22°C). The platelet rich plasma (PRP) was leukocyte depleted by inline filtration and was frozen at –30°C. After thawing at 37°C, at least 10 units of freeze-thaw lysed human platelets were further pooled resulting in approximately 40-50 donations per batch to minimize donor variations. pHPL was aliquoted and stored at –30°C. Before use in cell culture, pHPL was thawed and centrifuged at 4,000g for 15 minutes, whereas only the supernatant was added to the culture medium containing 2 U/ml of preservative-free heparin. Reproducibility of pHPL effects was verified by using at least seven different batches of pHPL identically prepared in Mannheim by pooling platelet concentrates from eight donors. Where specified, tPRP and pHPL were prepared from one platelet concentrate split in two halves to directly compare both. Isolation and Culture of BM-Derived MSCs BM aspirates were harvested using an optimized bone marrow harvesting technique [29]. Illiac crest bone marrow aspirates were derived from 14 young healthy donors (median age 22) after having received informed consent. Mononuclear cells (MNCs) were isolated from all heparinized BM aspirates by density gradient centrifugation (Ficoll Paque, GE Healthcare, Uppsala, Sweden, http://www.gehealthcare.com) as described elsewhere [30]. Independent of the cell number, the MNCs were split into equal subfractions and cultured within the respective basal medium supplemented with either FBS (n = 14), HS (n = 12), tPRP (n = 12), or pHPL (n = 6) (Fig. 1B). 1 Open in new tabDownload slide Morphology of bone marrow (BM)-mesenchymal stromal cells (MSCs) and BM-MSC allocation to specific tests. (A): Photomicrographs of one representative donor at primary culture at day 10 for fetal bovine serum human serum, thrombin-activated platelet releasate in plasma, and pooled human platelet lysate are shown in rows. Columns reflect cells either isolated using Ficoll density centrifugation, depletion of lineage positive cells by RosetteSep, or CD271 selection followed by plastic adhesion. Magnification, ×100. (B): The scheme shows the total number BM samples and those used for the respective parallel tests. Numbers of BM samples were reduced in subsequent passages because of replicative senescence-induced growth retardation. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. 1 Open in new tabDownload slide Morphology of bone marrow (BM)-mesenchymal stromal cells (MSCs) and BM-MSC allocation to specific tests. (A): Photomicrographs of one representative donor at primary culture at day 10 for fetal bovine serum human serum, thrombin-activated platelet releasate in plasma, and pooled human platelet lysate are shown in rows. Columns reflect cells either isolated using Ficoll density centrifugation, depletion of lineage positive cells by RosetteSep, or CD271 selection followed by plastic adhesion. Magnification, ×100. (B): The scheme shows the total number BM samples and those used for the respective parallel tests. Numbers of BM samples were reduced in subsequent passages because of replicative senescence-induced growth retardation. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. In n = 6 BM samples, MSC enrichment using RosetteSep (StemCell Technologies Inc, St. Katharinen, Germany, http://www.cellsystems.de) was compared with Ficoll-only isolation. The RosetteSep antibody cocktail (CD3, CD11b, CD14, CD16, CD19, CD56, CD66b, and glycophorin A) crosslinks undesirable cells and forms immunorosettes with red blood cells. These are pelleted after Ficoll gradient centrifugation. In this case, the BM aspirate was split into two equal aliquots before MNC isolation. Resulting cells were cultured in media supplemented with FBS, HS, or tPRP. On four other samples, CD271 (LNGFR) enrichment was performed in one half of the BM sample, whereas the other half was split to perform Ficoll and RosetteSep separation. CD271 sorting involved a magnetic bead-assisted preselection (AutoMACS device; program “Possel D” [2 columns] and “Possel S” [sensitive]) using CD271 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Because purity reached, at best, 80%, a flow cytometric sorting followed the enrichment (BD FACS Vantage TM SE: sorter used for flow cytometric cell sorting). This yielded a >99% CD271+ cell population as assured by flow cytometric analysis (CD 271-FITC and CD 271-PE; Miltenyi Biotec GmbH). All cell cultures were incubated with the respective supplements at 37°C, 5% CO2 in a humidified atmosphere. In a standardized fashion, all nonadherent cells were removed 24 hours after initial plating by media changes. The cells were cultured with media changed twice weekly until reaching confluence of 70-80%. At this time, cells were passaged using 1× trypsin-EDTA (PAA). At each passage (p), cells were replated at a standard density of 200 cells per cm2 at any subsequent passage. Proliferation Kinetics Cells were passaged and counted once they reached a subconfluence of 70-80%. The population doubling (PD) rate was determined using the following formula [31]: NH is the harvested cell number and N1 is the plated cell number. The PD for each passage was calculated and added to the PD of the previous passages to generate data for cumulative population doublings (CPD). In addition, the generation time (average time between two cells doublings) of four BM within all media conditions was calculated at passage 1 (p1) and p4 using the following formula: The effects of heparin and thrombin, which are present in tPRP and pHPL, were checked separately. BM-MSCs of two donors were cultured in (a) 10% FBS, (b) 10% FBS + 2 U heparin/ml, or (c) 10% FBS + 1 U thrombin/ml for three passages. No impact on MSC growth kinetics was observed within the three passages. Colony-Forming Unit-Fibroblast Assays The colony-forming unit-fibroblast (CFU-F) assay in primary culture was determined for six donor BM, and colonies were counted after 10 days. Freshly isolated BM-MNCs derived from the three different isolation methods and cultured within the four different media were plated in duplicate in 6-well plates at densities of 1 × 104, 5 × 104, and 1 × 105 per well. On day 10, the cell layer was fixed with methanol and stained with Giemsa solution (Merck, Darmstadt, Germany, http://www.merck.de). Individual colonies composed of at least 50 cells were counted. CFU-F frequency was calculated based on the respective input cell number as CFU-F per 1 × 104 MNCs. In Vitro Differentiation Potential The adipogenic and osteogenic differentiation capacity of MSCs was assessed at p2/p3 for all BM donors and for all culture conditions [27]. To detect the osteogenic differentiation, cells were stained for calcium deposition using von Kossa stain. Adipogenic differentiation was indicated by the morphological appearance of lipid droplets stained with Oil Red O. Flow Cytometry Analysis Immunophenotypic analyses were performed on three BM-MSC batches for all supplements and selected MSC samples derived from the different isolation methods at p3. The following mouse anti-human antibodies were used in multiplexed flow cytometric analysis: CD105-FITC (clone 8E11; Chemicon/Millipore, Schwalbach/TS, Germany, http://www.millipore.com), CD144-PE (TEA1/31; Beckman Coulter GmbH, Krefeld, Germany, http://www.beckman.com), CD90-APC (5E10; Becton Dickinson GmbH, Heidelberg, Germany, http://www.bdeurope.com), CD106-FITC (51-10C9; Becton Dickinson GmbH), CD146-PE (TEA-1/34; Beckman Coulter), CD34-PerCP-Cy5.5 (8G12; Becton Dickinson GmbH), CD133/1-APC (AC133; Miltenyi), CD44-APC-Alexa750 (IM7; NatuTec, Frankfurt/Main, Germany, http://www.natutec.de), CD15-FITC (HI98; Becton Dickinson GmbH), CD45-FITC (HI30; Becton Dickinson GmbH), CD3-FITC (UCHT1; Becton Dickinson GmbH), CD235a-FITC (GA-R2; Becton Dickinson GmbH), CD14-FITC (M5E2; Becton Dickinson GmbH), CD19-FITC (AE1; Diatec/Dianova Hamburg, Germany, http://www.dianova.de), CD117-PE (104D2; Becton Dickinson GmbH), CD33-PerCP-Cy5.5 (P67.6; Becton Dickinson GmbH), CD31-APC (WM59; NatuTec), CD29-APC-Cy7 (TS2/16 BioLegend/Biozol Eching b, München, Germany, http://www.biozol.de), CD73-PE (AD2; Becton Dickinson GmbH), HLA-ABC-APC (G46-2.6; Becton Dickinson GmbH), HLA-DR-PE-Cy7 (L243; Becton Dickinson GmbH), and 7-AAD (Beckman Coulter) for dead cell exclusion. The samples were analyzed using the BD FACS-Canto II and DIVA software. Comparative analysis was performed with FlowJo Version 7.2.5 (Tree Star, Inc., Ashland, OR, USA, http://www.treestar.com). Inhibition of Phytohemagglutin-Induced T-Cell Proliferation To test the allostimulatory or inhibitory effect of MSCs on T-cell proliferation, we co-cultured phytohemagglutin (PHA)-stimulated MNCs derived from healthy human buffy coat preparations with decreasing numbers of MSCs. Specifically, 1 × 104, 5 × 103, and 2.5 × 103 MSCs were preseeded as quadruplicates into wells of a flat-bottom 96-well plate in Roswell Park Memorial Institute 1640 (PAA) medium containing 10% FBS. On the following day, the medium was discarded, and 1× 105 MNCs were added to each well in RPMI medium containing 10% FBS and interleukin-2 (IL-2; 20 U/ml = 0.01 μg/ml; Roche Applied Science, Mannheim, Germany, http://www.roche-applied-science.com). One half of the wells were stimulated with PHA (2.5 μg/ml PHA-L; Roche Applied Science) to induce T-cell proliferation. Controls included nonstimulated, co-cultured MNCs, nonstimulated MNCs, and stimulated MNCs without MSC co-culture. To simultaneously quantify the cell number, viability, phenotype, and activation level of T-cell subsets, we used a modified method established by Nguyen et al. [32]. CD3, CD4, and CD8 antibodies were used to distinguish T-cell subsets, whereas T-cell activation was measured by the assessment of CD71 expression. For T-cell quantification, fluorescent microparticles with defined concentrations were used. The absolute count of target cells was calculated on the basis of the known bead concentrations using the following equation: After 5 days, the MNCs were harvested and stained with a cocktail of the following reagents: flow-count fluorospheres (Beckmann Coulter) to directly determine absolute cell counts, anti-CD3-PE-Cy7 (Becton Dickinson GmbH), anti-CD4-PE (Becton Dickinson GmbH), anti-CD8-FITC (Becton Dickinson GmbH), anti-CD71-APC (Becton Dickinson GmbH), and 7-AAD (Beckmann Coulter) to exclude dead cells. The samples were analyzed using the FACS-Canto II and DIVA software. The percentage of inhibition of T cells was calculated by comparing control cultures stimulated with PHA in the absence of MSCs (=0% inhibition) to those in the presence of MSCs. We used BM-MSC samples from three donors cultured in the differing supplements. Because of the fact that, in two HS-MSC cultures, T cells showed reduced viability (<70%) in unstimulated co-cultures because of unknown reasons, data for HS-MSCs were not statistically evaluated. Detection of Telomerase Activity To detect potential telomerase activity, we used the Telo TAGGG Telomerase PCR ELISA (Roche Applied Science) following the manufacturer's instructions. Samples of BM aspirates and BM-MSCs at different passages cultured in the various supplements were analyzed. Samples were regarded as telomerase negative if the difference in absorbance after subtraction of the negative control was <0.2. Human Cytokine Expression Profile The cytokine profile of culture medium supplemented with 10% FBS, HS, tPRP, or pHPL and that of 3-day MSC-conditioned medium was analyzed with a semiquantitative human cytokine antibody array that can detect 174 cytokines per experiment (RayBio Human Cytokine Antibody Array G series 2000; Tebu-bio GmbH, Berlin, Germany, http://www.tebu-bio.com). To minimize variances, tPRP and pHPL were derived from the same initial platelet concentrates. Despite the human specificity of the array, we also tested FBS, but finally interpreted only the conditioned medium. All sample measurements were performed in duplicate according to the manufacturer's instructions. The signals were detected using a laser scanner (GMS 418 array scanner; Affymetrix, Santa Clara, CA, USA, http://www.affymetrix.com) and analyzed with array vision version 7 (Imaging Research, Inc., St. Catharines, Canada, http://www.imagingresearch.com). Signals were normalized using positive, negative and internal controls included on the array. For analysis, the internal negative controls were used to determine the cut-off rate for a positive signal as 2 × SD. Thus, signal intensity values of >2,000 were regarded as positive. Statistical Analysis Statistical tests were performed using SPSS 12.0 (SPSS, Inc., Chicago, IL, USA, http://www.spss.com) or SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA, USA, http://www.systat.com) statistical software. Data are represented as arithmetic mean ± SD. Data were tested for normality and equal variance before analysis. Statistical differences were calculated using analysis of variance (ANOVA; or ANOVA on ranks if equal variance testing failed) and t test (paired t test where applicable). Differences were considered significant at ∗︁, p < .05 or ∗︁∗︁, p < .01. Results MSCs Isolation and Expansion Effect of Isolation Strategies. Because our previous experiments with AT-MSCs resulted in a transient contamination with hematopoietic cells in human supplement cultures, we applied two MSC enrichment strategies. First, we depleted mature lineage marker expressing cells by rosetting to erythrocytes (RosetteSep). Second, we used magnetic combined with flow cytometric sorting of CD271-expressing cells to enrich MSCs. Both enrichment strategies reduced contaminating round and loosely adherent cells in the primary passage (Fig. 1). Ficoll gradient-derived cultures supplemented with FBS had experientially few contaminating cells indicated by the presence of small loosely adherent round cells reactive with anti-CD45 (data not shown). These cells were easily depleted by repetitive media changes that occurred in the primary culture. RosetteSep very efficiently depleted the round contaminating cells in all culture conditions and yielded MSCs to be passaged 11.2 ± 1.48 days after seeding compared with 15.42 ± 4.46 days after seeding for Ficoll/FBS, respectively (p = .01; Fig. 2). A more rapid proliferation was observed by p3. However, this did not correlate with higher cumulative population doublings. Up to p3 expansion kinetics of RosetteSep-enriched cells showed a higher proliferation. Interestingly, immunodepleted cells from some donors showed an earlier onset of replicative senescence compared with Ficoll-isolated cells from p4 on, indicated by reduced proliferation and morphologic changes. We observed differences in the effects of HS and tPRP on RosetteSep-enriched cultures. The cell increment in HS exceeded that of ficolled cells up to p4 (Fig. 2). Proliferation rates were accelerated in HS, with a significant increase only in p1. 2 Open in new tabDownload slide Effect of isolation strategies. (Top) Mean cumulative population doublings of bone marrow-mesenchymal stromal cells isolated using Ficoll gradient centrifugation, RosetteSep, or CD271 sorting followed by plastic adhesion in medium supplemented with fetal bovine serum, human serum, or pooled thrombin-activated platelet-rich-plasma. (Bottom) Cumulative days needed for each MSC culture to be passaged. Low numerical values indicate high proliferative activity (for initial n, see Fig. 1B). ∗︁In comparison to Ficoll, p < .05 using analysis of variance and paired t test. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma. 2 Open in new tabDownload slide Effect of isolation strategies. (Top) Mean cumulative population doublings of bone marrow-mesenchymal stromal cells isolated using Ficoll gradient centrifugation, RosetteSep, or CD271 sorting followed by plastic adhesion in medium supplemented with fetal bovine serum, human serum, or pooled thrombin-activated platelet-rich-plasma. (Bottom) Cumulative days needed for each MSC culture to be passaged. Low numerical values indicate high proliferative activity (for initial n, see Fig. 1B). ∗︁In comparison to Ficoll, p < .05 using analysis of variance and paired t test. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma. FBS-supplemented cultures sorted for CD271+ cells showed bacterial contamination in three of four cases. This necessitated the abandonment of said cultures. Despite the addition of the same concentration of penicillin/streptomycin, parallel cultures using the human alternatives displayed no bacterial outgrowth. This may suggest that human FBS alternatives might have intrinsic antibacterial components. In summary, the experiments with CD271+ cells cultured in FBS, HS, or tPRP showed that CD271+ cells tended to grow in colonies during the entire culture period, never forming confluent monolayers. Expansion kinetics were delayed in FBS-driven cultures of CD271+ cells after p3 (Fig. 2). This corresponded to the low cell numbers yielded within each passage. Cells stopped proliferation in p5 yielding maximum 24.91 CPD for FBS (n = 1; 17.47 for HS, p4, n = 1 and 21.92 for tPRP, p5, n = 1). Effect of Supplements. As indicated above, the culturing of MNCs after density gradient centrifugation in FBS-supplemented medium yielded few contaminating hematopoietic cells. In contrast, supplementing MNCs with HS or tPRP resulted in variably high numbers of hematopoietic cells. Interestingly, and unlike HS and tPRP, pHPL-supplemented cultures were devoid of contaminating cells (Fig. 1). Calculating the number of CFU-Fs showed a precursor frequency of 1:25,000 MSCs/MNCs, which was not affected by the use of different culture substitutes. However, the number of cells composing the colonies was larger in all of the human supplements. Colonies in pHPL were densely packed with very small spindle-shaped cells compared with only loosely connected cells in FBS cultures (Fig. 3). 3 Open in new tabDownload slide Colony-forming unit-fibroblast of bone marrow (BM)-mesenchymal stromal cells (MSCs). Photomicrographs represent BM-MSCs from one donor assessed after 10 days cultivated in fetal bovine serum, pooled human serum, pooled thrombin-activated platelet-rich-plasma, or pooled human platelet lysate (magnification, ×100). Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. 3 Open in new tabDownload slide Colony-forming unit-fibroblast of bone marrow (BM)-mesenchymal stromal cells (MSCs). Photomicrographs represent BM-MSCs from one donor assessed after 10 days cultivated in fetal bovine serum, pooled human serum, pooled thrombin-activated platelet-rich-plasma, or pooled human platelet lysate (magnification, ×100). Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. The comparison of the expansion rates of MSCs in FBS to either HS- or tPRP-supplemented culture conditions showed no significant differences (Fig. 4). However, cells cultured in HS and tPRP decelerated proliferation from p4, reaching 14.46 ± 3.46 (HS, n = 3) and 18.47 ± 2.92 CPD (tPRP, n = 7) compared with 18.73 ± 1.96 CPD (FBS, n = 13). This correlated well with an increased population doubling time. Beginning in p1, the generation time of ficolled MSCs cultured in HS was significantly prolonged with 3.41 ± 1.23 days compared with 2.51 ± 0.87 days in the FBS cultures. In p4, both HS and tPRP showed an extended generation time (12.53 ± 6.54 days for HS and 12.72 ± 7.39 days for tPRP) compared with FBS (3.72 ± 0.44 days). Cells from one donor (HS) or two donors (tPRP) expanded until p5. In contrast, 11 samples from a total of 14 donors cultured in FBS reached p5. 4 Open in new tabDownload slide Effect of supplements. (Top) Mean cumulative population doublings of bone marrow-mesenchymal stromal cells (MSCs) isolated using Ficoll gradient centrifugation and cultivation either in fetal bovine serum (FBS), pooled human serum, pooled thrombin-activated platelet-rich-plasma, or pooled human platelet lysate. (Middle) Cumulative days needed for each MSC culture to be passaged (for initial n, see Fig. 1B). (Bottom) Generation time of MSCs at p1 (black) and p4 (white) (n = 4). ∗︁p < .05 and ∗︁∗︁p < .01 in comparison to FBS using analysis of variance (ANOVA) or ANOVA on the ranks, respectively. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate; CPD, cumulative population doublings. 4 Open in new tabDownload slide Effect of supplements. (Top) Mean cumulative population doublings of bone marrow-mesenchymal stromal cells (MSCs) isolated using Ficoll gradient centrifugation and cultivation either in fetal bovine serum (FBS), pooled human serum, pooled thrombin-activated platelet-rich-plasma, or pooled human platelet lysate. (Middle) Cumulative days needed for each MSC culture to be passaged (for initial n, see Fig. 1B). (Bottom) Generation time of MSCs at p1 (black) and p4 (white) (n = 4). ∗︁p < .05 and ∗︁∗︁p < .01 in comparison to FBS using analysis of variance (ANOVA) or ANOVA on the ranks, respectively. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate; CPD, cumulative population doublings. Consistently, cultures supplemented with pHPL yielded significantly higher expansion rates than cells in FBS, reaching a maximum of 52.82 CPD (in p8; n = 1 from initially six donors) compared with 31.43 ± 3.13 in FBS (in p7; n = 8 from initially 14 donors, p = .004). Calculating the generation time at p1 and p4 yielded, in both cases, significantly reduced population doubling times: 1.27 ± 0.23 days in p1 and 1.9 ± 0.32 days in p4 compared with FBS (2.51 ± 0.87 days in p1 and 3.72 ± 0.44 days in p4; p = .012 and p = .004, respectively). MSC Quality and Functionality Immune Phenotype. Typical CD44, CD73, CD90, CD105, CD146, and HLA-ABC surface marker expression was detected in all MSC cultures at p3 despite a measurable donor variance. CD29 was expressed on 44.46 ± 9.49% of BM-MSCs cultured in tPRP, whereas the other supplement yielded significantly higher positivity; for example, in HS, 98.12 ± 0.76%. CD29 mean fluorescence intensity was significantly higher in HS (FBS, 967.38 ± 476.55; HS, 2,176.07 ± 416.98; tPRP, 134.57 ± 19.18; pHPL, 438.88 ± 306.06). CD15, CD33, lineage (CD45, CD3, CD235a, CD14, and CD19), CD117, CD144, and HLA-DR showed less than 5% positivity. Selected antigens representing one donor are depicted in Figure 5. No further statistically significant differences between FBS and the other supplements were detected. 5 Open in new tabDownload slide Flow cytometric characterization of bone marrow (BM)-mesenchymal stromal cells (MSCs). Comparison of the expression of surface proteins of ficolled BM-MSCs cultured in fetal bovine serum, pooled human serum, pooled thrombin-activated platelet-rich-plasma, or pooled human platelet lysate analyzed by flow cytometry. One representative donor and typical MSC marker expression are depicted in the overlay to the unstained/control. For statistical analyses, n = 3 BM-MSC donors were paired assessed at passage 3. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. 5 Open in new tabDownload slide Flow cytometric characterization of bone marrow (BM)-mesenchymal stromal cells (MSCs). Comparison of the expression of surface proteins of ficolled BM-MSCs cultured in fetal bovine serum, pooled human serum, pooled thrombin-activated platelet-rich-plasma, or pooled human platelet lysate analyzed by flow cytometry. One representative donor and typical MSC marker expression are depicted in the overlay to the unstained/control. For statistical analyses, n = 3 BM-MSC donors were paired assessed at passage 3. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. Other markers showed donor-dependent variable reactivity perhaps influenced by the supplement used and/or the degree of hematopoietic cell contamination: CD31 (FBS, 0.95 ± 0.74; HS, 0.71 ± 0.31; tPRP, 5.5 ± 4.48; pHPL, 19.12 ± 10.78), CD133 (FBS, 6.45 ± 11.23; HS, 5.14 ± 7.21; tPRP, 6.04 ± 5.94; pHPL, 0.77 ± 0. 89), and CD106 (FBS, 6.18 ± 7.91; HS, 8.35 ± 9.7; tPRP, 13.54 ± 14.34; pHPL, 18.28 ± 9.41). Differentiation Potential. MSCs derived from all conditions demonstrated differentiation toward the osteogenic and adipogenic lineage as assessed by von Kossa and Oil Red O staining (supporting information Fig. 1). Inhibition of PHA-Induced T-Cell Proliferation We used a flow cytometric method to simultaneously quantify mitogen-driven T-cell proliferation, subtypes, activation level, and viability. T-cell stimulation by PHA led to strong proliferation and activation. To study the impact of culture conditions on MSC inhibitory activity, the same donor MNCs were used for all MSC samples. In co-culture controls without adding PHA, MSCs did not induce an alloreaction of the T cells but rather a loss of T cells in the range of 10-20% compared with the control. All MSCs independent of the culture conditions inhibited the PHA-driven T-cell proliferation and activation dose dependently (Fig. 6). Both CD4+ and CD8+ T-cell subsets were similarly affected. MSCs cultured in the human platelet-derived substitutes showed a tendency toward aggravated inhibitory activity at ratios of 1:10 and 1:20 that was not statistically significant. 6 Open in new tabDownload slide Immunomodulatory capacity of bone marrow (BM)-mesenchymal stromal cells (MSCs). BM-MSCs, irrespective of the supplement, mediated a dose-dependent inhibition of phytohemagglutin-induced T-cell/CD3 stimulation. Proliferation of the CD4 (T-helper) and CD8 (cytotoxic) subsets were similarly affected. Simultaneously, we quantified the proportion of activated T cells by means of CD71 expression. Like T-cell proliferation, T-cell activation was inhibited dose dependently. The overlay depicts CD71 expression of CD3+ cells. The same buffy coat mononuclear cells were used for all experiments. Three MSC batches were paired assessed at passage 3. Dose dependent differences were observed compared to ratio 1:10 with p < .05 (* = fetal bovine serum (FBS), # = thrombin-activated platelet releasate in plasma (+PRP) and $ = pooled human platelety lysate (pHPL)). tPRP- and pHPL MSCs-induced CD3 (plus CD4 for tPRP MSCs and CD8 for pHPL MSCs) inhibition did not differ significantly from that at 1:10. Statistically reduced inhibition was found at the ratio 1:40. A paired t test was used to compare dose dependency and analysis of variance to compare culture supplements. 6 Open in new tabDownload slide Immunomodulatory capacity of bone marrow (BM)-mesenchymal stromal cells (MSCs). BM-MSCs, irrespective of the supplement, mediated a dose-dependent inhibition of phytohemagglutin-induced T-cell/CD3 stimulation. Proliferation of the CD4 (T-helper) and CD8 (cytotoxic) subsets were similarly affected. Simultaneously, we quantified the proportion of activated T cells by means of CD71 expression. Like T-cell proliferation, T-cell activation was inhibited dose dependently. The overlay depicts CD71 expression of CD3+ cells. The same buffy coat mononuclear cells were used for all experiments. Three MSC batches were paired assessed at passage 3. Dose dependent differences were observed compared to ratio 1:10 with p < .05 (* = fetal bovine serum (FBS), # = thrombin-activated platelet releasate in plasma (+PRP) and $ = pooled human platelety lysate (pHPL)). tPRP- and pHPL MSCs-induced CD3 (plus CD4 for tPRP MSCs and CD8 for pHPL MSCs) inhibition did not differ significantly from that at 1:10. Statistically reduced inhibition was found at the ratio 1:40. A paired t test was used to compare dose dependency and analysis of variance to compare culture supplements. Telomerase Activity Telomerase activity was analyzed in MSCs at different passages to control the onset of spontaneous immortalization. We never detected telomerase activity except for the primary BM. Here we could attribute the low telomerase activity to the CD34+ proportion (data not shown). Cytokine Content in Supplements and Conditioned Medium The cytokine content in the 10% supplemented media and the conditioned media (CM) after 3 days of culture was evaluated. For these analyses, tPRP and pHPL were derived from the same pools to eliminate donor-specific differences. Because the cytokine array is human specific, data for the FBS-containing medium were not interpreted (Fig. 7; supporting information Table 1). 7 Open in new tabDownload slide Selection of differentially regulated growth factors evaluated by human cytokine array. Medium supplemented with either 10% human serum (HS), pooled thrombin-activated platelet-rich-plasma (tPRP), or pooled human platelet lysate (pHPL), and in addition 3 days of conditioned medium of fetal bovine serum, HS, tPRP, and pHPL (each n = 1) were analysed. Depicted cytokines from a list of 174 (supporting information Table 1) have been selected based on noticeable differences in the signal intensities indicating different concentrations in medium and conditioned medium. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. 7 Open in new tabDownload slide Selection of differentially regulated growth factors evaluated by human cytokine array. Medium supplemented with either 10% human serum (HS), pooled thrombin-activated platelet-rich-plasma (tPRP), or pooled human platelet lysate (pHPL), and in addition 3 days of conditioned medium of fetal bovine serum, HS, tPRP, and pHPL (each n = 1) were analysed. Depicted cytokines from a list of 174 (supporting information Table 1) have been selected based on noticeable differences in the signal intensities indicating different concentrations in medium and conditioned medium. Abbreviations: FBS, fetal bovine serum; HS, pooled human serum; tPRP, pooled thrombin-activated platelet-rich-plasma; pHPL, pooled human platelet lysate. Overall growth factor levels in FBS-CM were lower than in any of the human supplemented cultures, indicating that detectable levels are continuously present in the human supplements and remain unchanged including Acrp30 (adiponectin), angiogenin, CD14, glucocorticoid-induced tumor necrosis factor receptor (GITR), platelet-derived growth factor (PDGF) AB, and sgp130 (soluble gp130). Other cytokine levels dropped during culture (because of consumption or degradation) such as epidermal growth factor, macrophage-derived chemokine/CCL22, pulmonary and activation-regulated chemokine, PDGF-AA, and PDGF-BB. Insulin-like growth factor binding protein (IGFBP)-3, interleukin 6, monocyte chemoattractant protein-1, macrophage stimulating protein (MSP)α, osteoprotegerin, thrombopoietin, and tissue inhibitor of metalloproteinases-1 and -2 levels increased during culture, presumably because of production by MSCs. tPRP differed from HS and pHPL with regard to a variety of cytokines. In tPRP-CM, hepatocyte growth factor/scatter factor, IGFBP-2, and vascular endothelial growth factor (VEGF) D were elevated. Unfortunately, no obvious candidate for the strong proliferative support from pHPL could be identified: basic fibroblast growth factor (bFGF), GITR, macrophage migration inhibitory factor (MIF), macrophage inflammatory protein-1β (MIP-1β), MSP-α, regulated on activation, normal T-cell expressed, and secreted (RANTES; CCL-5), and VEGF were differentially regulated in pHPL/pHPL-CM compared with HS/HS-CM and tPRP/tPRP-CM. Discussion Currently, the ex vivo expansion of MSCs seems to be inevitably to get the common therapeutic dose of >2 × 106/kg body weight for infusion (e.g., in treatment of graft vs. host disease). Also, for other indications, there exists a need to study the applicability of MSCs with dose escalation, indicating the need to propagate MSCs in sufficient quantity. In a recent concise review in this Journal, Manello and Tonti underlined that elaboration of a culture medium for the production of MSCs for clinical application still remains a crucial matter [12]. We and others [2-4, 14-24, 27, 33-38] have since developed various protocols for the clinical scale propagation of human MSCs. Most of these protocols actually avoid the use of animal serum and some get rid off antibiotics and density gradient separation of the culture initiating cells. The major limitation of these studies relates to the fact that they compare FBS-based media to only selected FBS-free culture conditions (only HS or only pHPL). There are some data indicating that autologous serum in general supports greater amplification of MSCs than FBS [14, 39]. Limited availability and high variability regarding MSC growth clearly hamper the clinical applicability of autologous serum for large-scale MSCs production. Pooled preparations of allogeneic human serum can be produced in large amounts for pharmaceutical manufacturing and are easily controlled for quality according to blood banking standards (one batch = 25 blood donors; produced in the Institute of Clinical and Experimental Transfusion Medicine, University Hospital Tübingen, Tübingen, Germany). However, studies investigating the effects of allogeneic serum on BM-MSCs are contradictory [24, 28, 36, 40]. Alternatively, tPRP requires a complicated manufacturing process [27]. pHPL, in contrast, can be produced by simple freeze thaw cycles from the standard blood product buffy coat-derived pooled platelet concentrates. A further advantage is the possible use of platelet concentrates after their expiry period of 4-5 days. The freeze-thaw process furthermore allows for quarantine storage, potentially leading to a larger batch representing 40 donors. Based on a previously developed GMP-compliant large-scale protocol, a volume of 200 ml pHPL would be sufficient for one clinical scale BM-MSC expansion [4]. Thus, one batch of pHPL may be sufficient to expand MSCs from 10-20 patients. Based on these results, we performed a comprehensive comparison of four standardized culture protocols together with three initial MSC enrichment modalities to define optimized clinical scale MSC culture conditions. We selected one commercially available, pretested FBS batch, analyzed as being superior to other batches. Our results showed for the first time that MSC population doublings and expansion kinetics were significantly enhanced in pHPL-supplemented BM-MSC cultures compared with cultures supplemented with selected FBS, HS, or tPRP. Using pooled HS (or tPRP) exerted comparable expansion kinetics in early passage BM-MSCs like the pretested FBS batch. Clinically relevant numbers of MSCs could be obtained within a maximum of three passages with HS or tPRP, equivalent to FBS cultures. These numbers could certainly be obtained within the first to second passage in pHPL-supplemented cultures. Compared with AT-MSCs, pHPL, but not HS or tPRP, consistently surpassed FBS in expanding BM-MSCs. Cell yields in terms of CFU were maintained. No change in the cellular quality and potency was obvious. No lot-to-lot variability of pHPL and no variability between two manufacturing sites, Graz and Mannheim, were observed. This is in contrast to findings with FBS, where only selected lots are appropriate for MSC expansion [19]. Interestingly, in AT-MSCs, pHPL at a concentration of 10% did not allow the expansion of AT-derived cells beyond p1 [22, 27]. Currently, it is not known why pHPL has a stronger mitogenic effect than HS and tPRP on BM-MSCs. Activating platelets by thrombin or clotting induces secretion of more than 300 proteins and small molecules [41]. Platelet α granules are heterogeneous and contain either pro- or antiangiogenic factors. Depending on the mode of activation, release of these granule contents can be differentially induced [42]. Thus far, it cannot be excluded that various agonists used for platelet activation select for a certain platelet growth factor composition. We detected only a limited number of cytokines as differentially concentrated in feeding or conditioned medium containing pHPL compared with HS and tPRP. These include bFGF, GITR, IGFBP-3, latency associated peptide of TGFβ, MIF, MIP-1β, MSP-α, RANTES, VEGF, and all different isoforms of PDGF. PDGF either as homodimer of the A or B chain or the AB heterodimer showed the highest concentration in pHPL (supporting information Table 1). PDGF and bFGF are well-described growth factors for MSCs [36, 43]. In a recent study, the combination of PDGF, bFGF, and transforming growth factor β was sufficient to expand MSCs in a serum-free medium under laboratory scale conditions [44]. MIP-1β has recently been attributed to the promotion of fibrosis [45]. Besides stimulatory activities, inhibitory activities might be promoted by the growth factors present. Also the variety of extracellular matrix components including fibrin, fibronectin, vitronectin, and osteonectin may play pivotal roles [46]. In this context, the modified expression of the fibronectin receptor CD29 (lowest positivity in tPRP and highest intensity in HS) will be elucidated in further studies. Because of its complexity, multivariate designs are planned to identify the most relevant components [47] . HS, tPRP, and pHPL allowed the isolation of BM-MSCs with comparable immune phenotype, in vitro functionality regarding T-cell suppression, and a differentiation potential like FBS. Focusing on the intended therapeutic application, additional tests for genomic stability and in vivo differentiation potential will be necessary [4]. Preliminary data by us and others suggest that autologous serum may even favor genomic stability compared with FBS [28, 4]. Despite rare spontaneous transformation events in FBS-cultured MSCs [18, 48, 49], recent data have shown localized genomic instabilities in human BM-MSCs at clinically relevant passages irrespective of the serum source used [37, 50]. However, cells in autologous serum displayed a preserved methylated and unmethylated state compared with FBS [37]. Related to this, a recent study suggested that allogeneic AB serum may select for a more immature MSC phenotype, called mesodermal progenitor cells, which can be induced to differentiate into MSCs by switching the culture to FBS [38]. Unlike AT-MSCs, hematopoietic contamination was only detectable in the primary culture of BM-MSCs. In contrast to previous reports of selecting highly proliferative cells by enrichment of MSCs by RosetteSep or CD271 sorting [51], in our study, both strategies were not advantageous in any of the culture conditions tested. Admittedly, we used standardized and not method-optimized culture conditions. The addition of growth factors has been suggested for highly purified precursor cells that do not get trophic support from other cell types [52]. Culturing cells under different conditions may affect the secretion of trophic mediators. With the cytokine array applied, we cannot directly compare medium supplemented with FBS to the human supplements because of the anti-human specificity of most antibodies. The response of MSCs cultured in FBS shows certain differences as described in detail within the Results. Currently the cytokine array represents only a preliminary insight into the complex secretome of MSCs. We have therefore already initiated further studies to evaluate cytokines that may function as markers to ensure a quality control of supplement batches and to further monitor MSC potency and therapeutic efficacy. Although human components can easily be prepared according to blood banking standards, there remains the risk of sensitization by blood group substances or by adventitious agents not covered by routine blood donor testing. Implementation of further procedures such as quarantine storage or pathogen inactivation into a large-scale, GMP-compliant pHPL manufacturing may ensure the highest possible quality standards [53]. Our approach is currently limited by the in vitro comparison of MSC qualities. Further studies focusing on genomic stability or lack of transformation and in vivo differentiation potential, as well as homing capacities, are currently underway to show that MSCs isolated and expanded by using pHPL share properties of FBS-cultured MSCs. The data published thus far support maintenance of differentiation and biologic safety, even in vivo [3, 4, 17, 21]. Presently, the first application of pHPL expanded BM-MSCs has been performed to treat refractory graft versus host disease [54]. Conclusion Our data based on a paired analysis of 14 bone marrow donor MSCs using three different human alternative supplements compared with FBS for MSC isolation and expansion indicate that all tested human supplements support the isolation and expansion of BM-MSCs comparably to FBS. Human platelet lysate, however, seems to be the optimal component, assuring enriched cell numbers, maintained viability, cell identity, purity, sterility, and potency of BM-MSCs. pHPL favors not only very rapid but also long-term expansion while maintaining the immune phenotype, differentiation, and immunomodulatory capacities. The combined fast, profound, and extended expansion suggests that the progenitor compartment in pHPL-supplemented cultures is best preserved. Acknowledgements We thank Angela Lenzen (H.L.) and Claudia Url (K.S. and D.S.) for excellent technical assistance, Monica Farrell and Daniele Griffiths for proofreading the manuscript, and our colleagues from the German Red Cross Blood Donor Service, especially from the production unit, for support. This work was supported by a research fund of the German Federal Ministry of Education and Research (O1GN O531; K.B., H.L., and H.K.); “OsteoCord” (LSHB-CT-2005-O18999), a project commissioned by the European Community (K.B.); and Austrian Research Foundation Grant N211-NAN (D.S.). Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. References 1 Phinney DG , Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair---current views . Stem Cells 2007 ; 25 : 2896 – 2902 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Reinisch A , Bartmann C, Rohde E et al. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: K.B.: Conception and design, financial support, administrative support, collection and assembly of data, data analysis, manuscript writing; A.H.: Conception and design, collection and assembly of data, data analysis, manuscript writing, final approval of the manuscript; A.K.: Conception and design, provision of study material; H.L.: provision of study material, collection of data; K.S.: provision of study material, manuscript writing; D.S.: financial support, data interpretation, manuscript editing; H.K.: financial and administrative support, final approval of the manuscript. K.B. and A.H. contributed equally to this work. First published online in Stem Cells Express June 4, 2009. Telephone: 49-621-383-9720; Fax: 49-621-383-9720; Copyright © 2009 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow
JF - Stem Cells
DO - 10.1002/stem.139
DA - 2009-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/human-alternatives-to-fetal-bovine-serum-for-the-expansion-of-DJBaDcZJjO
SP - 2331
EP - 2341
VL - 27
IS - 9
DP - DeepDyve
ER -