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TECHNOLOGY DEVELOPMENT |
aDepartment of Orthopaedic Surgery,
bDepartment of Surgery, Center for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, United Kingdom
Key Words. Mesenchymal stem cells • Hematopoietic stem cells • Bioreactor • Differentiation
Correspondence: Gang Li, M.D., Ph.D., Department of Orthopaedic Surgery, School of Biomedical Sciences, Queen’s University Belfast, Musgrave Park Hospital, Belfast, BT9 7JB, United Kingdom. Telephone: +44 (0)289090 2830; Fax: +44 (0)289090 2825; e-mail: g.li{at}qub.ac.uk
Received on November 25, 2005;
accepted for publication on May 16, 2006.
First published online in STEM CELLS EXPRESS May 25, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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(CCAAT/enhancer-binding protein-) (adipogenesis), were not detected. Upon induction, the bioreactor-expanded MSCs were able to differentiate into osteoblasts, chondrocytes, and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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The static amplification of MSCs is a time-consuming procedure and prone to contamination [1]. The increasing clinical applications appeal for an alternative to rapidly expand MSCs [9]. In previous studies, MSCs have been cultured alongside HSCs in spinner flasks without losing their multiple mesenchymal-lineage differentiation potentials [10, 11]. Such a mixed culture system is mutually beneficial for both MSC and HSC growth because the suspension system mimics the in vivo bone marrow microenvironment/niche containing various cytokines, chemokines, and growth factors [12]. The three-dimensional rotary bioreactor is considered to be the next generation of spinner flask with the unique environment of minimal shear stress and microgravity, which is particularly suitable for the mammalian cell survival. The large-scale expansion of HSCs has been previously achieved using the bioreactor system [13].
The aim of the present study was to examine the feasibility of expanding MSCs from isolated bone marrow mononuclear cells (MNCs) in a rotary bioreactor system. After 8 days of culture in a bioreactor with modified Myelocult medium (StemCell Technologies Inc., London, http://www.stemcell.com), the numbers of Stro-1+CD34+CD44+ MSCs, CD34+Stro-1–CD44+ HSCs, and total cells increased by 29-, 8-, and 9-fold, respectively. The bioreactor-expanded MSCs expressed primitive mesenchymal cell markers, maintained a high level of colony-forming efficiency-fibroblast per day (CFE-F/day), and were capable of differentiating into chondrocytes, osteoblasts, and adipocytes upon appropriate inductions. The results confirmed that the rapid expansion of (primitive) MSCs together with HSCs can be achieved using the rotary bioreactor system.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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MSCs in Static Culture
Freshly isolated MNCs were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 2 mM L-glutamine (Invitrogen Corporation, Paisley, U.K., http://www.invitrogen.com) and seeded into T75 flasks at a density of 1 x 105 cells per cm2. The cells were incubated at 37°C in a humidified atmosphere with 5% CO2. The first feeding was at day 7 and twice weekly thereafter until confluence.
MSCs in Bioreactor Culture
Freshly harvested MNCs were inoculated into two 50-ml RCCS bioreactors (Synthecon, Inc., Houston, TX, http://www.synthecon.com) at a concentration of 1 x 106 cells per milliliter. Two different medium preparations were used. Medium one is Myelocult medium, supplemented with 50 ng/ml stem cell factor (SCF), 10 ng/ml interleukin (IL)-3 and IL-6 (PeproTech EC Ltd., London, http://www.peprotechec.com), and freshly dissolved 10–6 M hydrocortisone (StemCell Technologies Inc.). This is hereafter referred to as modified Myelocult medium. Medium two (control) is DMEM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 2 mM L-glutamine and hereafter is referred to as DMEM. The rotation speed used for the bioreactor was 20 rpm. The cells were incubated at 37°C for 8 days in a humidified atmosphere with 5% CO2. The first feeding was at day 3 with 20% medium exchange without retention of cells. In addition, the growth factor supplements were reduced to 10 ng/ml of SCF and 2 ng/ml of IL-3 and IL-6 from the first medium exchange. The feeding was performed daily afterward. The limiting dilution feeding (LDF) protocol used here is based on the assumption that the changes of total cell and progenitor cell numbers measured can be extended to those removed during the feeding procedure [10]. The cells removed each time were used either for total cell counts (during the first 2 days of culture, cells were counted by randomly removing 50 µl from the filling port) or for flow cytometric analysis.
Tri-Color Flow Cytometry
All antibodies (Abs) were purchased from DakoCytomation Ltd. (Cambridgeshire, U.K., http://www.dako.co.uk) unless otherwise specified. Sample cells from day-1, -4, and -8 bioreactor culture were first treated with ammonium chloride solution to lyse any potential contaminated red blood cells prior to flow cytometric analysis. The remaining cells were washed in cold PBS buffer containing 5% human AB serum (Sigma). Separate aliquots of 1 x 105 cells in a final volume of 50 µl were used for each binding for 30 minutes at 4°C in the dark. The Abs used were 10 µl of monoclonal mouse anti-human Stro-1 (extracted from hybridoma provided by Dr. P.J. Simmons, Matthew Roberts Laboratory, Adelaide, South Australia, Australia), 2 µl of phycoerythrin (PE)-conjugated anti-human HCAM/CD44 (DF1485; Insight Biotechnology Limited, Wembley, U.K., http://www.insightbio.com), 2 µl of R-phycoerythrin-cyanin 5 (RPE-Cy5)-conjugated anti-human CD34 (Clone BIRMA-K3), 2 µl of secondary fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse Abs, and equal volume of isotype-matched FITC-, PE-, and RPE-Cy5-conjugated negative controls. Finally, all the cells were resuspended in 1 ml of 1% paraformaldehyde (Sigma) and kept in the dark at 4°C. The analysis was performed on a Partec Cytoflow Space flow cytometer (Partec GmbH, Münster, Germany, http://www.partec.com) equipped with the Partec FloMax software.
CFE-F Assay
CFE-F assay was used to examine the functional property of modified Myelocult bioreactor-expanded MSCs. Both freshly harvested MNCs (control) and bioreactor output cells were resuspended in DMEM and seeded into six-well plates (NUNC A/S, Roskilde, Denmark, http://www.nuncbrand.com) at a density of 1 x 105 cells per cm2. A third group was also included with MNCs suspended in the modified Myelocult medium to examine the effect of this medium on static culture. The cells were incubated at 37°C in a humidified atmosphere with 5% CO2, and the first feeding was 1 week later and twice weekly thereafter until colonies appeared. The cells were then fixed with 95% ethanol and stained with hematoxylin. The colonies were counted manually under a light box and presented as CFE-F = colony counts per 106 MNCs. Because the colony-forming time was slightly different among groups (e.g., colonies appeared sooner in bioreactor output cells than in the static control), the CFE-F data were further corrected for time differences and presented as CFE-F/day.
Western Blot
Total proteins of both bioreactor-expanded and static control MSCs were extracted using standard methods and quantified using a BCA (bicinchoninic acid) protein assay kit (Pierce Technology, Perbio Science UK Ltd., Cramlington, U.K., http://www.piercenet.com). The procedure was carried out as previously described [14]. Briefly, nitrocellulose membranes were incubated with the following mouse monoclonal anti-human Abs: endoglin (SH-2), vimentin (DakoCytomation Ltd.), collagen type II, CCAAT/enhancer-binding protein- (C/EBP-) (Insight Biotechnology Limited), osteocalcin (OC) (Abcam, Cambridge, U.K., http://www.abcam.com), and ß-actin (Sigma) overnight at 4°C, followed by immune detection with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (Igs) (DakoCytomation Ltd.). The antigen-Ab complexes were visualized with western blotting luminal reagent (Insight Biotechnology Limited).
Tri-Lineage Differentiation of the Bioreactor-Expanded MSCs
The reagents were all purchased from Sigma unless otherwise specified. Because the induction procedure is routinely performed on monolayer or pellet cells, the bioreactor output cells were allowed to attach prior to tri-lineage inductions. The passage-one MSCs were plated onto Petri dishes or LAB-TEK chamber slides (NUNC A/S) at a density of 2 x 104 cells per cm2. For chondrogenic induction, additional aliquots of 1 x 106 cells were pelleted down and cultured in 15-ml polypropylene tubes (Iwaki brand; Scitech Division, Asahi Glass Co. Ltd., Chiba, Japan, http://www.agc.co.jp/english) alongside the chamber slide cultures. The chondrogenic induction medium consisted of serum-free DMEM supplemented with 0.1 µM dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.merck.com), 0.2 mM ascorbic acid-2 phosphate, 1 mM sodium pyruvate, and 1:100 diluted ITS+ Premix (BD Biosciences, Oxford, U.K., http://www.bdbiosciences.com). Transforming growth factor-ß-1 (PeproTech) was freshly added at a final concentration of 10 ng/ml each time. The medium was changed every other day. After 28 days, the cells cultured in chamber slides were stained for collagen type II, and pellet cells were embedded in paraffin for alcian blue staining or processed for transmission electron microscopy (TEM). For osteogenesis, 0.1 µM dexamethasone, 0.2 mM ascorbic acid-2 phosphate, and 10 mM ß-glycerophosphate were added to the DMEM containing 10% FBS. The medium was changed twice weekly. After 18 days, the cells were subject to Von Kossa and alkaline phosphatase (ALP) staining and immunocytochemistry (ICC) examinations for OC, type I collagen, vimentin, and CD105 expressions. The adipogenic supplements consisted of 10–6 M dexamethasone, 0.50 mM methylisobutylxantine, and 50 uM indomethacin. The medium was changed twice a week, and after 12 days of culture, oil red O staining was performed to detect the accumulated lipid vacuoles in the cells.
ICC
A standard three-step indirect ICC method was used. In brief, after fixation in 95% ethanol, the cells were treated with 10% goat serum and incubated with primary monoclonal mouse anti-human collagen type II (1:200) and OC (1: 200), biotinylated secondary polyclonal goat anti-mouse Igs (1:100; DakoCytomation Ltd.), and ALP-streptavidin (1:100; Vector Laboratories, Ltd., Peterborough, U.K., http://www.vectorlabs.com). The positive staining was visualized with a Fast Red substrate pack (ZYMED Laboratories Inc., Cambridge, U.K., http://www.zymed.com), and the cells were counterstained with hematoxylin (Sigma). Negative controls were obtained by omitting the primary Abs.
TEM
To determine whether the cells underwent chondrogenic differentiation, the pellets were fixed for TEM, washed in 0.1 M sodium cacodylate buffer, postfixed in 0.1 M osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated through graded ethanol solutions, and embedded in epoxy resin (Araldite; Sil-Mid Limited, Coleshill, West Midlands, U.K., http://www.silmid.com). Semithin sections were stained with 1% toluidine blue (Sigma) to select appropriate fields; ultrathin sections were cut with diamond knives, placed on mesh hexagonal copper (3.5 mm) grids, contrasted with uranyl acetate and lead citrate, and examined with a JEM-100CX II electron microscope (JEOL Ltd., Tokyo, http://www.jeol.com.au).
Statistics
The means of different populations were compared by analysis of variance using software SPSS version 13.0 (SPSS Inc., Chicago, http://www.spss.com), and p < .05 was considered statistically significant. The data are presented as the mean ± SD of the pooled six independent experiments.
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RESULTS |
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(adipogenesis) were not detected (Fig. 4, lane A). The expression pattern of the above markers was similar in the MSCs cultured in static control (Fig. 4, lane B).
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DISCUSSION |
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It is well known that microgravity conditions promote cell growth as seen in outer space. Colvin et al. were the first to introduce the concept that microgravity conditions promoted stem cell expansion [15]. From then on, numerous publications have provided evidence that microgravity positively regulates cell growth. The bioreactor used here was invented by NASA to simulate tissue culture condition in outer space and is considered to be the next generation of spinner flasks. It offers the unique environment of microgravity and minimal shear stress, which enables cells to grow much as they would in vivo. The mammalian cells are particularly shear-sensitive and cannot survive at higher agitation rates. In this sense, the bioreactor is superior to the spinner flask because the latter employs continuous agitation and may disrupt the well-being and further expansion of the stem cells.
In the bioreactor system, three important parameters affecting cell survival are feeding regime, shear force effect, and culture medium with supplements. The LDF protocol used in the present study has certain advantages. First, as the culture progresses, rapid stem cell expansion may lead to the depletion of stimulatory cytokines and the production of suppressive factors in the culture system, compromising the further expansion of primitive stem cells [16, 17]. Medium exchange by LDF allows the dilution of inhibitory factors and retention of stimulatory ones at the same time. It has been proven that, both in bioreactor and static cultures, daily feeding with dilution of cells (without retention of the cells in the discarded medium) will improve the expansion of hematopoietic progenitors as well as total cell counts compared with feeding every other day with total cell retention [18–20]. Second, to achieve the optimal cell expansion in vitro, the interchange of metabolic waste and nutrients in the bioreactor culture system should be comparable with that occurring in vivo. A daily exchange of 20% of the total medium in the suspension culture system mimics the plasma perfusion rate in bone marrow, which is approximately 0.1 ml/ml marrow per minute [21]. Current results confirm that 20% dilution feeding is suitable for rapid expansion of primitive MSCs (Figs. 4 and 5).
The cytokine cocktail used in the present study is a combination of SCF, IL-3, and IL-6. The concentration of the cytokines used in the cocktail was determined in our preliminary experiments by screening the effects on MSC proliferation by various concentrations of SCF (1, 5, 10, 50, and 100 ng/ml) and IL-3 and IL-6 (1, 5, 10, 25, and 50 ng/ml). SCF, the ligand for the tyrosine kinase receptor c-kit, is an early-acting cytokine for hematopoiesis. It maintains and preserves long-term hematopoietic progenitors per se compared with the synchronized effect that many other cytokines need. Tong et al. have shown that the administration of SCF increased the absolute number of CD34+ cells and primitive myeloid progenitor cells after chemotherapy in patients with breast cancer [22]. IL-3 is a controversial cytokine; reports are conflicting in regard to its ability to inhibit or stimulate stem cell expansion. It has been demonstrated that IL-3 abrogated the expansion and self-renewal of primitive stem cells in a concentration-dependent manner [23]. In both human and murine models, IL-3 showed inhibitory effects on HSC reconstitution [1, 24]. On the other hand, IL-3 also promotes HSC proliferation at a high level of quality [25]. In the current study, IL-3 exhibited positive effects on the proliferation and preservation of both MSCs and HSCs, which may be partially explained by the finding of a significant increase of CD45–CD123+ (IL-3- receptor) cells with osteogenic potential after suspension culture [11]. Unlike in previous studies [10, 11], IL-6 was also added into the cocktail after a screening test showed that IL-6 alone stimulated MSC expansion. It has been suggested that IL-6 improves the transplantation outcome of human stem cells and stimulates hematopoiesis (especially thrombopoiesis) [22, 26]. Interestingly, it has been demonstrated that IL-6 has an antitumor effect [27]. The presence of IL-6 may help to eliminate the possible contamination of malignant cells in bone marrow harvests [28]. This may further strengthen the purging effect of the bioreactor during clinical practice [29].
Upon inoculation, we used a boost dose for the cytokine cocktail and switched to a lower concentration after the first medium exchange. Our preliminary data showed that the cell expansion was more rapid when the boost dose with higher concentrations of cytokine was used. The boost dose may help the primitive stem cells move from the quiescent state into the cell proliferation cycle, thus shortening the culture period. However, continuous addition of high doses of cytokines in HSC culture promotes rapid cell proliferation as well as cell differentiation [30]. Therefore, a combination of high (upon inoculation) and low (from first feeding) doses of cytokine supplements was used in the present study to ensure the expansion and preservation of primitive stem cell subpopulations.
Apart from the added exogenous cytokines, the endogenous factors produced by MSCs and HSCs in the suspension configuration also help to prevent the apoptosis and differentiation of the progenitor cells. As within the bone marrow cavities, MSCs and HSCs are always in close contact and support each other through the release of various growth factors, cytokines, and chemokines. In HSC culture, a stromal feeding layer is required for the growth of long-term culture-initiating HSCs by providing cytokines and growth factors such as leukemia inhibitory factor (LIF), Flt-3 ligand (FL), and granulocyte-macrophage colony-stimulating factor [31, 32]. LIF is constitutively expressed by bone marrow stroma cells and plays several roles in preventing differentiation of HSCs [20]; FL is capable of recruiting HSCs into the cell proliferation cycle and promoting cell survival through inhibition of apoptosis [33]. The understanding of the complex cytokine profiles in the rotary bioreactor suspension culture system will provide insights for ex vivo stem cell manipulation.
In the present study, the rotary speed of the bioreactor was 20 rpm, which was determined after different tests using 10, 15, 20, and 23 rpm. We found that 20 rpm was the optimal speed to prevent cell aggregation during the culture period in the bioreactor. We also found that cells started forming aggregates after 8 days in the rotary bioreactor. It has been postulated that microgravity conditions favor high-quality stem cell expansion for only a short period [15]. As culture progressed, extra efforts such as filtering the aggregates using 70-µm cell strainers and repeated pipetting had to be adopted to separate the cells; this was not practical after day 13, because the cells in the bioreactor became heavily aggregated. Because the total cell counts reached a peak at approximately day 8 and started declining afterward, the duration of 8 days was chosen as an average time for keeping the cells in the rotary bioreactor. In addition, the serological factors in the culture system have been reported to accelerate the differentiation of stem cells and may have a negative effect on retention of progenitor cells [34]. Even in serum-free conditions, the early-stage long-term repopulating HSCs were lost after 9 days of culture despite a further increase of the later-stage colony-forming cells and CD34+ cells [35]. Based on above observations, the slow-down of cell proliferation after approximately 8 days in the current bioreactor suggests the exhaustion or arrest of the early progenitor cells (Fig. 1). In clinical applications, the potential side effects related to serum supplementation have to be avoided. Baksh et al. optimized their culture configuration by using 10-fold-higher cytokine supplements in the absence of serum [10, 11]. The quality of MSC output after such a culture remained high. Efforts to define an alternative serum-free culture condition for MSC bioreactor expansion are under way.
In static culture, a small fraction of MSCs are actively engaged in the proliferation stage of the cell cycle (S+G2+M), whereas nearly 80% MSCs are in the quiescent state (G0) [36–38]. The average colony-forming time for the current bioreactor outputs was 12 days compared with 16 days for the non-treated control cells (Table 1), implying that the bioreactor treatment has increased the proliferation ability via promoting quiescent progenitors into the cell cycle and, consequently, shortened the time needed for colony formation upon plating.
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CONCLUSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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REFERENCES |
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) or DMEM (
) at a density of 1 x 106 cells per milliliter. Sample cells removed from the culture were counted manually throughout the culture period. By the end of 8 days, the total cell expansion upon input was 8.93 ± 0.41-fold and 0.11 ± 0.03-fold for the modified Myelocult bioreactor culture and DMEM bioreactor culture, respectively. The data are corrected for the dilution effect, and each time point represents the mean of six independent experiments, with error bars representing SD. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; Myelo, Myelocult medium.
