Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells
2016; Elsevier BV; Volume: 44; Issue: 6 Linguagem: Inglês
10.1016/j.exphem.2016.02.004
ISSN1873-2399
AutoresPatrick Wuchter, Marcel Vetter, Rainer Saffrich, Anke Diehlmann, Karen Bieback, Anthony D. Ho, Patrick Horn,
Tópico(s)3D Printing in Biomedical Research
Resumo•Xeno-free human platelet lysate-based cell expansion of human bone marrow-derived mesenchymal stromal cells (MSCs) was comprehensively analyzed and directly compared with two commercially available media: StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only).•Quantitative and qualitative analysis of the resulting cell populations included assessment of proliferation kinetics, differentiation capacity, cell morphology, and immunophenotype.•A blueprint for an MSC-expansion strategy on a clinically relevant scale is presented.•Cost analyses for MSC expansion strategies with all three media were assessed. Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media—StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)—in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media—StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)—in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. In the last decade, numerous therapeutic strategies based on human mesenchymal stem/stromal cells (MSCs) have been evaluated. They addressed various diseases, such as osteogenesis imperfecta [1Horwitz E.M. Gordon P.L. Koo W.K.K. et al.Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone.Proc Natl Acad Sci USA. 2002; 99: 8932-8937Crossref PubMed Scopus (1386) Google Scholar], myocardial infarction [2Chen S.L. Fang W.W. Ye F. et al.Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction.Am J Cardiol. 2004; 94: 92-95Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar], regeneration of cartilage [3Nejadnik H. Hui J.H. Feng Choong E.P. Tai B.C. Lee E.H. Autologous bone marrow-derived mesenchymal stem cells versus autologous chondrocyte implantation: An observational cohort study.Am J Sports Med. 2010; 38: 1110-1116Crossref PubMed Scopus (422) Google Scholar], graft-versus-host disease (GvHD) [4Le Blanc K. Frassoni F. Ball L. et al.Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: A phase II study.Lancet. 2008; 371: 1579-1586Abstract Full Text Full Text PDF PubMed Scopus (2116) Google Scholar], and improvement of hematopoietic stem cell engraftment [5Ball L.M. Bernardo M.E. Roelofs H. et al.Cotransplantation of ex vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem-cell transplantation.Blood. 2007; 110: 2764-2767Crossref PubMed Scopus (431) Google Scholar]. However, the comparison of data among different research groups is severely limited by the lack of specific markers for MSCs and the fact that a vast number of different isolation and expansion protocols exist [6Bieback K. Wuchter P. Besser D. et al.Mesenchymal stromal cells (MSCs): Science and f(r)iction.J Mol Med. 2012; 90: 773-782Crossref PubMed Scopus (49) Google Scholar, 7Ikebe C. Suzuki K. Mesenchymal stem cells for regenerative therapy: Optimization of cell preparation protocols.Biomed Res Int. 2014; 2014: 951512Crossref PubMed Scopus (134) Google Scholar]. Criteria proposed by the International Society of Cellular Therapy (ISCT) [8Dominici M. Le Blanc K. Mueller I. et al.Minimal criteria for defining multipotent mesenchymal stromal cells: The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (11642) Google Scholar] still comprise human MSC populations with considerable intrinsic heterogeneity [9Wagner W. Feldmann R.E.J. Seckinger A. et al.The heterogeneity of human mesenchymal stem cell preparations: Evidence from simultaneous analysis of proteomes and transcriptomes.Exp Hematol. 2006; 34: 536-548Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 10Wagner W. Ho A.D. Mesenchymal stem cell preparations: Comparing apples and oranges.Stem Cell Rev. 2007; 3: 239-248Crossref PubMed Scopus (222) Google Scholar, 11Wuchter P. Boda-Heggemann J. Straub B.K. et al.Processus and recessus adhaerentes: Giant adherens cell junction systems connect and attract human mesenchymal stem cells.Cell Tissue Res. 2007; 328: 499-514Crossref PubMed Scopus (73) Google Scholar]. Individual donor variability represents yet another challenge. Although MSCs can be isolated from various tissues, such as umbilical cord blood and adipose tissue [12Kern S. Eichler H. Stoeve J. Kluter H. Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.Stem Cells. 2006; 24: 1294-1301Crossref PubMed Scopus (2434) Google Scholar], human bone marrow remains the most common source. After isolation, the initial cell population must be expanded in vitro before further use in a clinical setting. Most clinical studies have used 1.0–2.0 × 106 MSCs/kg body weight [13Sato K. Ozaki K. Mori M. Muroi K. Ozawa K. Mesenchymal stromal cells for graft-versus-host disease: Basic aspects and clinical outcomes.J Clin Exp Hematop. 2010; 50: 79-89Crossref PubMed Scopus (75) Google Scholar]; therefore, the target range after expansion is approximately 1.4 × 108 cells. To reduce any risk of contamination and complications related to aging processes, the expansion period of MSCs should be kept as short as possible [14Bieback K. Hecker A. Schlechter T. et al.Replicative aging and differentiation potential of human adipose tissue-derived mesenchymal stromal cells expanded in pooled human or fetal bovine serum.Cytotherapy. 2012; 14: 570-583Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar]. As the expansion medium has a significant impact on the characteristics and proliferation kinetics of MSCs, the choice of medium is a critical decision for each MSC-based cell product. Fetal calf serum (FCS) has been used extensively as a standard medium supplement for MSC expansion in research settings. However, FCS has several disadvantages that make it inadequate for clinical use: high lot-to-lot variability [15Honn K.V. Singley J.A. Chavin W. Fetal bovine serum: A multivariate standard.Proc Soc Exp Biol Med. 1975; 149: 344-347Crossref PubMed Scopus (138) Google Scholar, 16Zheng X. Baker H. Hancock W.S. Fawaz F. McCaman M. Pungor E. Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs.Biotechnol Prog. 2006; 22: 1294-1300Crossref PubMed Scopus (203) Google Scholar], risk of contamination with infectious agents [17Halme D.G. Kessler D.A. Regulation of stem-cell-based therapies.N Engl J Med. 2006; 355: 1730-1735Crossref PubMed Scopus (294) Google Scholar], and potential for anaphylactic reactions [18Sundin M. Ringden O. Sundberg B. Nava S. Gotherstrom C. Le Blanc K. No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients.Haematologica. 2007; 92: 1208-1215Crossref PubMed Scopus (236) Google Scholar]. Thus, FCS is rated critically by the European Medicines Agency and should be avoided for clinical applications [19European Medicines Agency (EMA) EMAGuideline on the use of bovine serum in the manufacture of human biological medicinal products. Author, London2013Google Scholar]. In a recent consensus statement, standards and requirements for Good Manufacturing Practice (GMP)-compliant MSC production were delineated [20Wuchter P. Bieback K. Schrezenmeier H. et al.Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.Cytotherapy. 2015; 17: 128-139Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar]. Xeno-free supplements, particularly human platelet lysate (HPL), have been developed for clinical applications [21Fekete N. Rojewski M.T. Fürst D. et al.GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC.PLoS One. 2012; 7: e43255Crossref PubMed Scopus (132) Google Scholar, 22Kazemnejad S. Allameh A. Gharehbaghian A. Soleimani M. Amirizadeh N. Jazayeri M. Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone marrow-derived mesenchymal stem cells to functional hepatocytes-like cells.Vox Sang. 2008; 95: 149-158Crossref PubMed Scopus (37) Google Scholar, 23Bieback K. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.Transfus Med Hemother. 2013; 40: 326-335Crossref PubMed Scopus (131) Google Scholar, 24Bieback K. Hecker A. Kocaomer A. et al.Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow.Stem Cells. 2009; 27: 2331-2341Crossref PubMed Scopus (374) Google Scholar, 25Kinzebach S. Dietz L. Kluter H. Thierse H.J. Bieback K. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells.BMC Cell Biol. 2013; 14: 48Crossref PubMed Scopus (36) Google Scholar]. However, the establishment of new protocols is not trivial, and the feasibility of GMP-compliant production of MSC preparations is often ambiguous. Therefore, the aim of this study was to contribute to a stepwise development of GMP-compliant protocols that can be adjusted individually to local needs depending on the respective therapeutic purpose. In an academic setting, we optimized the proliferation kinetics of MSCs grown in HPL-containing expansion medium and compared the efficiency of this medium with that of two commercially available media: (i) the GMP-compliant StemPro MSC SFM CTS medium and (ii) the non-xeno-free MSCGM medium, which contains 10% FCS and is not approved for clinical applications (i.e., research only), but has been widely used for scientific research for many years [26Gong Z. Calkins G. Cheng E.C. Krause D. Niklason L.E. Influence of culture medium on smooth muscle cell differentiation from human bone marrow-derived mesenchymal stem cells.Tissue Eng. 2009; 15: 319-330Crossref Scopus (70) Google Scholar, 27Mark P. Kleinsorge M. Gaebel R. et al.Human mesenchymal stem cells display reduced expression of CD105 after culture in serum-free medium.Stem Cells Int. 2013; 2013: 1-8Crossref Scopus (67) Google Scholar]. This study was approved by the ethics committee of the Medical Faculty of Heidelberg. All bone marrow samples were acquired from healthy voluntary donors after obtaining written informed consent according to guidelines approved by the ethics committee of the Medical Faculty of Heidelberg (File No. S-384/2004). Donors ranged between 24 and 52 years (median: 40 years) of age. MSCs were isolated from human bone marrow aspirates as described previously [28Wagner W. Wein F. Seckinger A. et al.Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.Exp Hematol. 2005; 33: 1402-1416Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar]. Briefly, mononuclear cells (MNCs) were isolated from bone marrow aspirate by density gradient centrifugation using Ficoll–Paque (GE Healthcare, Munich, Germany) and seeded in plastic culture flasks (Nunc EasYFlasks Nunclon, Δ 25 or 75 cm2, Thermo Fisher Scientific, Roskilde, Denmark) at a density of 100,000 MNCs/cm2 (HPL medium and MSCGM) or 250,000 MNCs/cm2 (StemPro MSC SFM CTS), as recommended by the manufacturer. This time point was defined as passage 0. Replicates were performed with MSCs from different donors in all experiments. To minimize donor dependence when comparing different groups within one experiment, MSCs in all compared groups originated from the same donors. Cells were cultured in a custom-made HPL medium, or the two commercially available media: StemPro MSC SFM CTS (GMP conform; Life Technologies, Thermo Fisher Scientific, Waltham, MA) or MSCGM (Mesenchymal Stem Cell Growth Medium, Catalog No. PT-3001, Lonza, Basel, Switzerland). The HPL medium contained ATMP-ready DMEM-LG (Dulbecco's modified Eagle's medium-low glucose, Advanced Therapy Medicinal Products) with 2 mmol/L L-glutamine (PAA, Pasching, Austria) as the basal medium. Additionally, alternative use of ATMP-ready α-MEM (α-minimum essential medium) and ATMP-ready DMEM–F12 Ham's medium (PAA, Pasching, Austria) as basal medium was examined. These basal media were supplemented with 10% pooled HPL if not otherwise indicated. HPL was provided by the Institute of Transfusion Medicine and Immunology, German Red Cross Donor Services Mannheim and prepared from outdated (5-day-old) pooled buffy coat-derived platelet concentrates in T-sol (pool of four donors). The HPL used herein is academic grade and not yet produced under GMP conditions. However, the German Red Cross Blood Donor Services Baden-Württemberg–Hessen now has a manufacturing license for HPL production, and this is fully characterized according to GMP standards. To derive platelet lysate, concentrates were shock-frozen and thawed pooling two concentrates (final pool of eight donors). HPL was centrifuged for 10 min at 2,000 g. Aliquots were stored at −30°C. To avoid thrombus formation, 1 U/mL heparin (Ratiopharm, Ulm, Germany) was added, and remaining debris was removed by additional centrifugation and filtration. In addition, the final medium contained penicillin/streptomycin (PAA). StemPro MSC SFM CTS (referred to as "StemPro" here) and MSCGM were prepared and stored according to the corresponding manufacturers' instructions. Briefly, StemPro Supplement (15 mL) was mixed with 80.5 mL StemPro MSC SFM Basal Medium, 1 mL GlutaMAX–I CTS (LifeTechnologies, Thermo Fisher Scientific), 1 mL antibiotic solution (penicillin + streptomycin, 100×, PAA), and 2.5 mL human serum AB (Catalog No. 14-491E, Lonza). As prescribed by the manufacturer, prior to MSC expansion with StemPro, plastic surfaces were coated with CELLstart CTS (LifeTechnologies, Thermo Fisher Scientific) according to the manufacturers' protocols. After reaching 70%–80% confluence, cells were detached from culture flasks using trypsin (TrypLE Select CTS, LifeTechnologies, Thermo Fisher Scientific) for 2–4 min and replated at a density of 5,000 cells/cm2 in all media if not indicated otherwise. This procedure is in line with the manufacturers' instructions for StemPro and MSCGM and has been used for MSC cultures in HPL medium [29Bernardo M.E. Emons J.A.M. Karperien M. et al.Human mesenchymal stem cells derived from bone marrow display a better chondrogenic differentiation compared with other sources.Connect Tissue Res. 2007; 48: 132-140Crossref PubMed Scopus (101) Google Scholar, 30Carrancio S. López-Holgado N. Sánchez-Guijo F.M. et al.Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification.Experimental Hematology. 2008; 36: 1014-1021Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar]. Media were changed every third day. Cells were counted with a hemocytometer (improved Neubauer cell chamber, Laboroptik, Lancing, UK) at each passage. To improve comparability between data from different experiments with different amounts of MNCs, all proliferation results for MSCs refer to a starting population of one million MNCs if not otherwise indicated. To determine the expansion time until reaching a sufficient number of MSCs for transplantation, for practical reasons, only fractions of the cell number were passaged under standardized conditions. These results were than extrapolated to the appropriate number of MNCs. To assess cell density and exclude any contamination, the cells were visually inspected daily by microscopy (Leitz, Diavert, Leica Microsystems, Wetzlar, Germany). Pictures for documentation of morphology were taken of randomly chosen areas at passage 4 (Olympus IX 70, Olympus, Hamburg, Germany) to reflect representative growth patterns. For the quantitative analysis of cell size, representative images of MSCs from all of the different media were acquired at a cell density of approximately 30% confluency. Image analysis was performed using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, http://imagej.nih.gov/ij/, 1997–2014). Briefly, the outline of each cell was marked and saved as a region of interest (ROI), and finally, mean and standard deviation of the area of all ROIs were calculated. For each of the conditions (different media, early passages 2 and 4), at least 50 cells from three different images of each donor were analyzed. The expression of surface markers was assessed by flow cytometry at passage 4 (FACScan flow cytometry system from BD Biosciences, Heidelberg, Germany; Software: CellQuest 3.3 software). Nonviable cells were excluded from measurements using propidium iodide (PI) staining. The following antibodies were used: CD14-PE (BD Pharmingen), CD19-APC-H7 (BD Biosciences), CD34-APC (Novitec), CD45-APC-H7 (BD), CD73-PE (BD Pharmingen), CD90-APC (BD Pharmingen), CD105-PE (Caltag), CD146PE (BD Pharmingen), and HLA-DR-FITC (BD). After culture and expansion in the respective media, cells were transferred into adipogenic, osteogenic, or chondrogenic differentiation medium at passage 4 for 21 days. Media were renewed every third day. The compositions of adipogenic and osteogenic media were described previously [9Wagner W. Feldmann R.E.J. Seckinger A. et al.The heterogeneity of human mesenchymal stem cell preparations: Evidence from simultaneous analysis of proteomes and transcriptomes.Exp Hematol. 2006; 34: 536-548Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 28Wagner W. Wein F. Seckinger A. et al.Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.Exp Hematol. 2005; 33: 1402-1416Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar, 31Horn P. Bork S. Wagner W. Standardized isolation of human mesenchymal stromal cells with red blood cell lysis.Methods Mol Biol. 2011; 698: 23-35Crossref PubMed Scopus (34) Google Scholar]. For chondrogenic differentiation, the commercial StemPro Chondrogenesis Differentiation Kit (LifeTechnologies, Thermo Fisher Scientific) was used according to the manufacturers' instructions. Briefly, 80,000 cells in 5 μL were placed in the middle of 24-well plates to induce formation of cell aggregates. Aggregates were cultured in chondrogenic differentiation medium and sectioned for staining (Cryostat 2800 Frigocut-E, Leica, Bensheim, Germany) after 21 days. Chondroid tissue was detected by Alcian blue (Sigma-Aldrich, St. Louis, MO) staining. Because of similar growth patterns, the sizes of cell aggregates were estimated by measuring the area using the ImageJ software package. Adipogenic differentiation was demonstrated by oil red O staining (Sigma-Aldrich), whereas Alizarin Red S (Sigma-Aldrich) was used to detect osteoid extracellular matrix. Isolation of human hematopoietic progenitor cells from umbilical cord blood was performed as described before [32Wein F. Pietsch L. Saffrich R. et al.N-Cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells.Stem Cell Res. 2010; 4: 129-139Crossref PubMed Scopus (55) Google Scholar, 33Ludwig A. Saffrich R. Eckstein V. et al.Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.Cytotherapy. 2014; 16: 111-121Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar], after obtaining written informed consent according to guidelines approved by the ethics committee of the Medical Faculty of Heidelberg (File No. 251/2002). Briefly, mononuclear cells were isolated by density gradient centrifugation on Ficoll-Hypaque (Biochrom KG, Berlin, Germany). CD34+ cells were purified by positive selection with a monoclonal anti-CD34 antibody using magnetic microbeads on an affinity column with a MACS CD34 isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Flow cytometric analysis of isolated CD34+ cells revealed a purity of >90%. CD34+ cells were stained with carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich) and analyzed by flow cytometry to monitor cell division kinetics after several days of (co-)culture, as described previously [33Ludwig A. Saffrich R. Eckstein V. et al.Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.Cytotherapy. 2014; 16: 111-121Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 34Walenda T. Bork S. Horn P. et al.Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells.J Cell Mol Med. 2010; 14: 337-350Crossref PubMed Scopus (119) Google Scholar]. Statistical analyses were performed using the two-tailed Student t test for independent samples. The results were considered significantly different at a p value < 0.05. The proliferation kinetics after addition of 5% or 10% HPL to three different basal media were compared systematically, using a cell density of 5,000 cells/cm2 (Fig. 1A). DMEM-LG with 10% HPL led to higher proliferation of MSCs compared with DMEM-LG/5% HPL, α-MEM/5% HPL, and α-MEM/10% HPL. In contrast, DMEM-LG–F12 Ham's was not sufficient for proliferation of MSCs. On the basis of these data, the DMEM-LG combined with 10% HPL was used in all subsequent comparative experiments (HPL medium). Different initial MNC seeding densities at passage 0 were tested, and only minor effects on the final cell yield were observed (Fig. 1B). To assess the effect of MSC plating density during passaging, cell densities of 5,000, 500, and 50 cells/cm2 were compared in a simultaneous long-term approach in HPL medium. Cells were passaged at approximately 80% confluence. A reduction in cell density resulted in largely enhanced growth kinetics (Fig. 1C). For better comparability, a seeding density of 5,000 MSC/cm2 was used in all further experiments in accordance with the protocols of commercially available media. MSCs cultured in HPL medium, StemPro, and MSCGM were plastic adherent and had the characteristic spindle-shaped morphology expected (Fig. 2A). Cells in HPL medium formed large cell protrusions, especially when cultured at low cell densities, whereas cells in MSCGM had a rather round morphology at higher passage numbers. Cultivation in StemPro led to slightly smaller cells with a very homogeneous morphology. In addition, a minor fraction of very large cells was observed, indicating the usual intrinsic heterogeneity of the cell population. Because of the overall smaller cell size in StemPro, the cells were able to reach a higher density on a given surface area. Quantitative analysis of the cell size measuring the area of the adherent cells revealed a significantly smaller cell size for MSCs grown in StemPro in early passages (1,922 ± 1,171 μm2) compared with the other conditions. MSCs cultured in HPL medium were significantly smaller (2,681 ± 1,892 μm2) compared with cells grown in FCS-containing MSCGM (3,249 ± 1,700 μm2) (Fig. 2B). Adipogenic and osteogenic differentiation capacity were confirmed for MSCs cultivated in all media (Fig. 3A, B). Any MSC preparation could be differentiated toward adipogenic lineages. Osteogenic differentiation was confirmed for all donors in at least one of the three tested media. Individual samples failed to exhibit osteogenic differentiation in MSCGM (one of four donors), StemPro, and HPL medium (two of four donors respectively), which was most likely a result of individual donor variability. Alcian blue staining for the detection of chondroid tissue was positive in MSCs from all tested media (Fig. 3C). Measurement of the area of those cell aggregates, though, revealed that the largest aggregates were formed by MSCs expanded in HPL medium. This might indicate a positive effect of HPL on differentiation of MSCs toward chondrogenic lineages (Fig. 3D). All tested MSC populations fulfilled the immunophenotypic ISCT criteria for the definition of MSCs [8Dominici M. Le Blanc K. Mueller I. et al.Minimal criteria for defining multipotent mesenchymal stromal cells: The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (11642) Google Scholar]. Additionally, expression of CD146, a marker that may be used to detect more primitive mesenchymal cells with presumed significance for the hematopoietic niche [35Sorrentino A. Ferracin M. Castelli G. et al.Isolation and characterization of CD146+ multipotent mesenchymal stromal cells.Exp Hematol. 2008; 36: 1035-1046Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar], was similar for all three media tested (Fig. 3E). Supportive effects of the three different media were tested simultaneously on long-term culture (50 days) of MSCs from seven different donors. Expansion of MSCs was possible in all tested media, and no decrease in proliferation (e.g., as a result of senescence) in any medium was observed within 50 days. MSCs expanded in StemPro had the highest proliferation kinetics. The number of cells after 50 days was slightly higher for MSCs cultured in HPL medium than for those cultured in MSCGM (Fig. 4). We have described the supportive potential of MSCs expanded in MSCGM medium in detail [33Ludwig A. Saffrich R. Eckstein V. et al.Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.Cytotherapy. 2014; 16: 111-121Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar]. In analogy, we analyzed MSCs expanded in StemPro and HPL medium. The support of HPC expansion was confirmed for both MSC populations without any statistically significant difference (Fig. 5). For therapeutic purposes, usually approximately 1.4 × 108 MSCs are used per patient. Because of the limited amount of bone marrow aspirate, the in vitro expansion of MSCs is essential to reach this number of cells. We calculated the time until a transplantation could be performed, starting with 10 mL bone marrow aspirate (BMA). Cultivation with StemPro led to a significantly shorter in vitro expansion period until the estimated target number of cells was reached (StemPro: 13 days, range: 11–18 days; HPL medium: 23 days, range: 18–30 days; MSCGM: 26.5 days, range:
Referência(s)