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ABSTRACT: While chondrogenesis of mesenchymal stem cells (MSCs) in vitro has been extensively studied, their participation in cartilage tissue repair remains unresolved. This study was designed to elucidate if MSCs affect the phenotype of articular chondrocytes (ACs). A combination of non-contact coculture modes were developed. Human or rabbit MSCs (hMSCs or rMSCs) and rabbit ACs (rACs) were encapsulated in alginate hydrogel beads (three-dimensional, 3D) or cultured in monolayer (two-dimensional, 2D) and subsequently cocultured in the Transwell® system. After coculture, cell morphology, growth, deposition of cartilaginous extracellular matrix (ECM) and gene expression of rACs were investigated. It was found that upon coculture without cell-cell contact, both 2D and 3D cultured MSCs dramatically induced the morphological transformation of 2D-cultured rACs from round to a spindle-like shape, and however inhibited the generation of cellular aggregates of 3D-cultured rACs. Most strikingly, coculture resulted in a significantly less deposition of cartilaginous ECM including glycosaminoglycans (GAG) and collagen type II by both 2D- and 3D-cultured rACs. Importantly, similar observations were achieved for rACs cultured in MSCs-conditioned medium, confirming the definite paracrine interactions between MSCs and rACs. Based on the analysis of gene expression, this phenotypic change of rACs was not identical as the dedifferentiation. To the best of our knowledge, this is a first study demonstrating that MSCs could downregulate chondrocytic differentiation of ACs and warrants considerations in cartilage tissue repair.
Stem cells and development 01/2013; · 4.15 Impact Factor
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ABSTRACT: Bottom-up approaches have emerged as a new philosophy in tissue engineering, enabling precise control over tissue morphogenesis at the cellular level. We previously prepared large bone-like tissues using cell-laden microbeads (microtissues) by following a modular approach to ensure cell viability. However, a long-term culture of such avascular macroscopic tissues (macrotissues) has not been evaluated. In the present study, microtissues were fabricated by cultivating human fibroblasts on Cytopore-2 microbeads in spinner flasks for 16 days. We then examined the long-term perfusion culture for macrotissues. Specifically, following assembly in a perfusion chamber for 15 days, cell death was found to be prominent at a depth of 500 µm from the surface of macrotissues towards the interior, suggesting that there was a new mass transfer limit leading to cell death instead of tissue maturation. Subsequently, we developed a strategy by incorporating microchannel structures in centimeter-sized tissue constructs to promote mass transport. By installing glass rods (1 mm diameter, 1 mm wall-to-wall spacing) in the perfusion chamber, stable microchannel architectures were introduced during the microtissue assembly process. Based on live/dead assay and scanning electron microscopy (SEM), these channelled macrotissues (length × diameter, 1.6 × 2.0 cm) demonstrated high cell viability and compact packing of microbeads. Comparative biochemical analysis further suggested a more homogeneous spatial distribution of cells and extracellular matrix (ECM) in the channelled macrotissues than in solid ones. Viable 3D large tissues can therefore be prepared by assembling cell-laden microbeads in conjunction with microchannel carving, meeting clinical needs in tissue repair. Copyright © 2012 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 07/2012; · 3.28 Impact Factor
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ABSTRACT: In an effort to improve TNFR-Fc production in GS-CHO cells, batch cultures were performed to investigate the effects of low
culture temperature on TNFR-Fc production. It was observed that low culture temperatures resulted in cell cycle arrest in
the G1 phase, led to suppressed cell growth, and prolonged the culture period. Although the highest TNFR-Fc concentration
was achieved with a culture temperature of 32°C at 247.4 mg/L, the maximum q
TNFR-Fc of 15.7 pg/cells/day was achieved at 30°C. Because the inhibition effect on cell growth at 30°C compromised its beneficial
effects specifically to TNFR-Fc productivity, TNFR-Fc concentration at this temperature was not significantly increased. Furthermore,
the increase in productivity of specific TNFR-Fc at low culture temperatures was also found to be due to an increase in the
transcriptional level of the TNFR-Fc gene, determined by RT-PCR analysis. In addition, low culture temperatures had no significant effect on the degree of sialylation
of TNFR-Fc. Taken together; a biphasic cultivation process developed in a fed-batch mode with a low temperature-production
phase enhanced TNFR-Fc production by GS-CHO cells and therefore offers major potential for bioprocess optimization.
Keywordscell growth arrest–GS-CHO cells–low temperature–TNFR-Fc–transcriptional level
Biotechnology and Bioprocess Engineering 04/2012; 16(1):136-143. · 1.28 Impact Factor
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ABSTRACT: A functional relationship between the growth and the progression of events associated with osteogenic differentiation of human
amniotic mesenchymal stem cells (hAMSCs) has been a fundamental question, which remains unclear. This study is aimed at investigating
the effects of low temperature and lactate individually, and in combination on the growth and osteogenic differentiation of
hAMSCs. It was shown that the growth of hAMSCs in growth medium was inhibited by both low-cultivation temperature and lactate.
By extending culture period at low temperature, cell growth declined gradually, while the ALP expression and calcium deposition
increased progressively. However, the growth of hAMSCs induced in osteogenic medium at 37°C was markedly enhanced by additional
lactate. The ALP expression and calcium deposition, on the contrary, were significantly depressed. Furthermore, the synergistic
actions of long-term low temperature and lactate resulted in more intense inhibition on both cell growth and osteogenic differentiation.
Therefore, these findings may imply the co-contribution of the culture environment on the selective manipulation on the growth
capacity and osteogenic differentiation potential of hAMSCs.
Biotechnology and Bioprocess Engineering 04/2012; 14(6):708-715. · 1.28 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) derived from human amnion have both self-renewal capability and multipotency and are an attractive
cell source for cell-based therapy. However, these cells have been shown to be heterogeneous, and as of yet no single-cell-derived
MSCs clone has been established from human amnion. This study was carried out to isolate MSCs clones by limiting dilution
method and compare their characteristics in vitro. Three clones (namely, 8B, 11D, and 11F) were established from a heterogeneous population of human amnion-derived cells (h-hAMCs).
The clones and h-hAMCs successfully proliferated while demonstrating different cumulative population doublings (CPD) during
an 80-day culture. In addition, the colony-forming efficiency (CFE) of h-hAMCs was significantly lower than those of 8B and
11F and higher than that of 11D. Clones 8B and 11F were tripotent, whereas 11D did not undergo chondrogenic differentiation.
All cells expressed surface markers including CD29, CD44, and CD105 and notably, the clones expressed higher levels of CD105
than h-hAMCs (95.96, 97.05, 98.14% and 72.81% for 8B, 11D, 11F and h-hAMCs, respectively). In addition, the expression of
stem cell gene Nanog-3 was associated with the differential differentiation potential of 11D from 8B, 11F, and h-hAMCs. These
results suggested that significant differences existed between individual hAMCs. Further studies for developing novel methods
to select sub-populations of hAMSCs are warranted for their clinical applications, in which CD105 and stem cell gene Nanog-3
are possible candidate markers.
Keywordsclone–differentiation–heterogeneous cells–human amniotic mesenchymal stem cells–phenotype–proliferation
Biotechnology and Bioprocess Engineering 04/2012; 15(6):1047-1058. · 1.28 Impact Factor
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Applied Microbiology and Biotechnology 02/2012; 94(2):563. · 3.42 Impact Factor
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ABSTRACT: The chemical approach to controlling stem cell fates is emerging as a powerful tool, holding great promise in tissue engineering and regenerative medicine. Various small molecules have been demonstrated capable of modulating stem cell differentiation. In this paper, we studied the effects of 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAR), an activator of AMP-activated protein kinase (AMPK), on mesenchymal stem cells (MSCs). AICAR at high concentrations (1.0-2.0 mM) significantly inhibited proliferation of both human amnion-derived MSCs (hAMSCs) and rabbit bone marrow-derived MSCs (BM-MSCs). Most importantly, AICAR efficiently promoted the osteogenic differentiation of hAMSCs and BM-MSCs in both growth medium and osteogenic medium. However, Metformin, another AMPK activator, showed no such effects. Meanwhile, AICAR significantly inhibited adipogenic differentiation of hAMSCs and BM-MSCs. Our data suggests that AICAR represents a potent molecule, which can be applied in bone tissue regeneration.
The International journal of artificial organs 12/2011; 34(12):1128-36. · 1.86 Impact Factor
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ABSTRACT: Tissue engineering of clinical-relevant large tissue constructs remains a big challenge due to the mass transfer limit. A modular approach via the assembling of modular tissues thus eliminating the mass transfer limit holds great promise for fabricating centimeter-sized constructs. In the present study, we investigated the feasibility of using microcarriers seeded with adult mesenchymal stem cells (MSCs) to fabricate a large bone tissue. It was demonstrated that human amniotic MSCs (hAMSCs) were efficiently seeded onto CultiSpher S microcarriers (made of porcine gelatin) in a spinner flask and quickly proliferated while retaining a great viability. Within a total culture period of 28 days, using a two-stage culture strategy, hAMSCs-laden microcarriers with a high cell density were prepared at the first stage and the cells were then directly induced to undergo osteogenic differentiation in the same culture flask. During this cultivation process, the aggregation of cell-laden microcarriers was apparent, which resulted in aggregates of 700-800 μm, a size permissive for maintaining high cell viability. The osteogenic differentiation of hAMSCs on microcarriers was confirmed with increased mineral deposition (Alizarin red S staining and quantification of calcium content), ALP activity as well as gene expression of osteogenic markers (collagen type I and osteocalcin). These modular bone-like tissues were used as building blocks to fabricate a macroscopic bone construct in a cylindrical perfusion culture chamber (2 cm in diameter). After a 7-day perfusion culture, these modular tissues readily assembled into a centimeter-sized construct (diameter × height: 2 cm × 1 cm). Both good cell viability and fairly homogenous distribution of cellular content and bone-characteristic ECM within the macrotissue were elaborated. This paper provided a proof-of-concept study for modularly engineering clinical-relevant large tissue replacements with cell-laden microcarriers.
Biomaterials 10/2011; 32(30):7532-42. · 7.40 Impact Factor
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ABSTRACT: The potential of hypoxanthine and thymidine (H&T) to promote growth of CHO cells and production of monoclonal antibody (mAb) was explored in this study. It was demonstrated that H&T stimulated the initial cell growth and enhanced volumetric production of anti-human CD20 mAb by 22%, mainly through the elevated integrated viable cell concentration (IVCC). The moderate alteration in cell cycle distribution might partially account for the increased cell growth. Subsequent long-term stability studies indicated that H&T did not accelerate decay kinetics in mAb productivity. Specifically, cells under both nucleic acids-replete (H&T supplementation) and nucleic acids-hungry (methotrexate treatment) culture conditions showed similar stable mAb production during the first 2 months, followed by a gradual decline in the specific production rate (q(mAb)) with a 40% drop at the fourth month. In addition, the decreased transcript level of intracellular heavy chain (HC) of anti-human CD20 mAb correlated well with the decreased q(mAb). Furthermore, genomic mutation rate regarding the loss-of-function occurrence of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was determined, showing that H&T repressed the HPRT spontaneous mutation rate while methotrexate (MTX) provoked the mutation rate. Collectively, our data illustrated that H&T as potential medium additives promoted both initial cell growth and volumetric production of mAb, while not affecting the long-term stability of antibody-producing CHO cells.
Applied Microbiology and Biotechnology 07/2011; 93(1):169-78. · 3.42 Impact Factor
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ABSTRACT: The specific productivity of tumor necrosis factor receptor-immunoglobulin G1 Fc fusion (TNFR-Fc) (q(TNFR-Fc)) in Chinese hamster ovary (CHO) cells at 30°C was approximately 5-fold higher than that at 37°C. To investigate reasons for increased q(TNFR-Fc) at low culture temperature, TNFR-Fc mRNA levels were determined by real-time PCR. It was found that like q(TNFR-Fc), the relative TNFR-Fc mRNA level was increased by lowering culture temperature, and more importantly, the kinetics of the increase in TNFR-Fc mRNA levels were in accordance with the changes in q(TNFR-Fc). The results demonstrated that the increased transcriptional level of TNFR-Fc was responsible for the increased q(TNFR-Fc) at low culture temperature. Enhanced levels of mRNA could derive from increased gene copy number, improved mRNA stability, or enhanced transcriptional rate. There was not a big change of gene copy number by lowering culture temperature. The transcriptional rate of TNFR-Fc was slightly decreased at 30°C, compared to 37°C. However, mRNA stability of TNFR-Fc was significantly improved by lowering culture temperature. The half-life of TNFR-Fc mRNA was 5.55 h at 30°C, whereas that was 3.69h at 37°C. Taken together, the reasons for the increased q(TNFR-Fc) in CHO cells at low culture temperature were mainly the enhanced TNFR-Fc mRNA levels, which resulted from the improved mRNA stability, rather than the changes in the gene copy number or the transcriptional rate.
Journal of Bioscience and Bioengineering 12/2010; 111(3):365-9. · 1.79 Impact Factor
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ABSTRACT: A recently developed protocol, "microtissue assembly" holds great promise to address the issue of limited mass transfer within engineered large tissue replacements (macrotissues), wherein small "building blocks" (microtissues) are prepared and then assembled into macrotissues. Previous studies suggested that aggregation behavior of microcarrier-based microtissues were very important for macrotissue engineering. However, a systematic study on the aggregation behavior of microtissues is still missing. In this study, to examine the aggregation behavior of microtissues, effects of key operation parameters in dynamic culture including cell seeding density, microcarrier concentration, L-ascorbic acid 2-phosphate (V(c)) and agitating speed were investigated. The aggregation process could be divided into three phases (i.e., lag, growth and stable). Aggregation efficiency (S) was found to be modulated by cell seeding density, microcarrier concentration, addition of V(c) and agitating speed. A mathematical model correlating the operation parameters with S at different phases of aggregation was developed and experimentally proved to be able to predict S with varied operation parameters. In the end, a cylindrical macrotissue (diameter × height: 2.0 cm × 0.8 cm) with fairly good integrity and cellularity and uniform cell distribution was successfully engineered through perfusion assembling microtissues with controlled S under selected culture conditions. Our study showed that aggregation of microtissues could be precisely modulated, which would definitely facilitate engineering macrotissues with high quality.
Journal of biotechnology 09/2010; 150(3):438-46. · 2.88 Impact Factor
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ABSTRACT: Lowering the culture temperature is often useful to improve the production of many recombinant proteins in Chinese hamster ovary (CHO) cells. Batch cultivation of GS-CHO cells expressing TNFR-Fc antibody was therefore carried out at 30, 33.5 and 37 degrees C. TNFR-Fc productivity, q(TNFR-Fc), increased as culture temperature decreased; and the maximum q(TNFR-Fc) was 20 mg/(10(9) cells.day) at 30 degrees C which was three times that at 37 degrees C. Increasing the viable cell density (VCD) to above 2.2 x 10(6) cells/ml, however, decreased the q(TNFR-Fc) at 30 degrees C, which was due to a reduction in transcription of the TNFR-Fc gene. Taken together, lowering temperature can improve q(TNFR-Fc) but the negative effect of increasing VCD compromises this effect. Further process development addressing the issue of cell density-dependent TNFR-Fc productivity is therefore needed.
Biotechnology Letters 09/2010; 32(9):1239-44. · 1.68 Impact Factor
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ABSTRACT: An animal-component-free and chemically defined fed-batch process for GS-engineered cell lines producing recombinant antibodies has been developed. The fed-batch process relied on supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of by-products (lactate and osmolality). The proportionalities of nutritional consumption were determined by direct analysis. The robust, metabolically responsive feeding strategy was based on the offline measurement of glucose. The fed-batch process was shown to perform equivalently in GS-CHO and GS-NS0 cultures. Compared with batch cultures, the fed-batch technology generated the greater increase in cell yields (5-fold) and final antibody concentrations (4-8 fold). The majority of the increase in final antibody concentration was a function of the increased cell density and the prolonged culture time. This generic and high-yielding fed-batch process would shorten development time, and ensure process stability, thereby facilitating the manufacture of therapeutic antibodies by GS-engineered cell lines.
Journal of Microbiology and Biotechnology 12/2009; 19(12):1695-702. · 1.38 Impact Factor
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ABSTRACT: Lactic acid, originated from degradation of biomaterials, cell cultures, and so on, would be a toxic compound in acute states. The present study was undertaken to ascertain whether the proliferation, metabolism, and differentiation of rabbit mesenchymal stem cells (rMSCs) were affected by additional lactic acid. Furthermore, this study aimed to determine whether this influence was due to decreasing pH, increasing osmotic pressure, or chemical action of lactate ion. It was shown that the proliferation and metabolism of MSCs were inhibited by decreasing pH or increasing lactate. However, when osmolarity was adjusted to the same level as that of sodium lactate using sodium chloride, cell proliferation was little affected by osmotic pressure. We also concluded that colony-forming potential and osteogenic differentiation capacity were significantly depressed by decreasing pH or increasing lactate. As was shown, this inhibition of lactate was not only due to osmotic pressure, but also mainly due to chemical action of lactate ion. However, we observed that acidifying extracellular medium and lactate ion promoted the retention of adipogenic differentiation potential of MSCs during in vitro expansion, which suggested that growth arrest and the decrease of osteogenic differentiation potential did not affect the adipogenic conversion of MSCs.
Cell Biology and Toxicology 02/2009; 25(6):573-86. · 2.51 Impact Factor
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ABSTRACT: Tissue engineering is a promising technique to repair or reconstruct the damaged cartilage in clinical use. However, chondrocyte growth is limited by the mass transport in scaffolds as diffusion is likely to be the primary mechanism. In this study, a mathematical model was developed based on oxygen diffusion and reaction to simulate chondrocyte growth. In order to accord with the fact, effective diffusion coefficients and space limitation were considered in this model and good agreement was found between experimental data and mathematical simulations. Furthermore, relationships established in the model system can be used to optimize the situation in real bioreactors and the design of three-dimensional scaffolds.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2007; 23(1):171-5.
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ABSTRACT: To investigate the effects of ex-vivo expansion on proliferative ability, pluripotentiality and other biologic characteristics of human bone marrow mesenchymal stem cells(MSCs).
MSCs were isolated from human costal bone and passaged under the same culture conditions. At each passage, the characteristics of proliferation kinetics, osteogenic, chondrogenic, adipogenic differentiation potential were analyzed, and cell morphology, surface markers and cell cycle were investigated as well.
The proliferative ability and osteogenic, adipogenic differentiation potential decreased during culture expansion, while chondrogenic differentiation potential had no significant changes. MSCs maintained their multiple differentiation potential during their life-span. For each passage, the positive ratio of CD29, CD44, CD105 were all above 90% and the negative ratio of CD14, CD34, CD45 were below 4%.
Culture expansion causes MSCs to gradually lose their stem cell properties. During ex-vivo expansion of hMSCs, the osteogenic and adipogenic differentiation potential are more likely to lose than chondrogenic differentiation potential. Multiple differentiation potential is conserved longer than self-renewal capacity. MSCs before 7th passage can be a valuable subject for basic research and clinical application.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 02/2006; 22(1):7-10.
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ABSTRACT: Cell seeding of three-dimensional scaffolds is the first step of the cultivation of engineered tissues. The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. In the present work, human fibroblasts were seeded to collagen-chitosan sponges by static seeding, stirred seeding and perfusion seeding. The effects of seeding conditions on the resulting seeding density, the seeding efficiency and the initial cell distribution were studied. The seeding efficiency was relatively high (88.9%) at low inoculation cell density, and decreased rapidly wjth the increasing of inoculation cell density in static seeding. Stirred seeding yielded the lowest seeding efficiency. Nonuniform initial cell distribution was observed in both static and stirred seeding. The perfusion seeding, which has a characteristic of high seeding efficiency (>77%) , high initial cell density and uniform initial cell distribution in 3-D scaffolds, is the optimum method for cell seeding to 3-D scaffold.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 07/2005; 21(4):649-53.
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ABSTRACT: The effect of the calcium ion (Ca2+) on the growth and differentiation of the mouse epidermal keratinocytes cultured in serum-free medium was investigated. It was found that the optimal level of calcium ion in the medium was about 0.2 mmol/L. Under such a culture condition the colony forming efficiency, attachment percentage, percentage of the cells with cornified envelops, and percentage of the senesced cells were measured to be about 10.8%, 30.8%, 5.1%, and 26.8%, respectively. However, the Ca2+ concentrations in the medium above 0.6 mmol/L resulted in significant differentiation and senescence of the keratinocytes, which was found to be harmful for keratinocyte growth and expansion in vitro.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 10/2002; 18(5):626-9.
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ABSTRACT: The effect of antioxidants on the in vitro life span of mouse keratinocytes was investigated in this work. It was found that the life span of the keratinocytes cultured in the medium supplemented with antioxidants was extended significantly. The most beneficial antioxidant used in this work was the mercaptoethanol, followed by the catalase and SOD. However, the growth rates of keratinocytes in vitro under all the experimental conditions still declined with the culture time. It was also found that the antioxidants added in the medium were also helpful to enhance the keratinocyte colony formation. In addition, the aging kinetics of the mouse epidermal keratinocytes in vitro were analyzed, and finally the aging rate constants corresponding to antioxidants used were calculated.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 10/2002; 18(5):630-3.
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ABSTRACT: The effect of trypsin on the separation an subculture of the keratinocytes was investigated in this work. It was found that when 0.25% trypsin was employed for 5 minutes to separate keratinocytes, the number of active keratinocytes and the cells capable of forming colony were higher than those of other experimental conditions. The maximum attached ratio of primary keratinocytes was obtained when skin tissues were treated at 0.05% concentration of trypsin. With the increase of the trypsin concentrations, the attached ratio, attachment rate constant, and colony forming efficiency were all increased. Thus, 0.25% concentration of trypsin was recommended for separating and subculturing the keratinocytes.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2002; 18(1):59-62.
