Displaying publications 41 - 60 of 68 in total

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  1. Analysis of Terpenoid Indole Alkaloids, Carotenoids, Phytosterols, and NMR-Based Metabolomics for Catharanthus roseus Cell Suspension Cultures
    Saiman MZ, Mustafa NR, Verpoorte R
    Methods Mol Biol, 2018;1815:437-455.
    PMID: 29981141 DOI: 10.1007/978-1-4939-8594-4_31
    The plant Catharanthus roseus is a rich source of terpenoid indole alkaloids (TIA). Some of the TIA are important as antihypertensive (ajmalicine) and anticancer (vinblastine and vincristine) drugs. However, production of the latter is very low in the plant. Therefore, in vitro plant cell cultures have been considered as a potential supply of these chemicals or their precursors. Some monomeric alkaloids can be produced by plant cell cultures, but not on a level feasible for commercialization, despite extensive studies on this plant that deepened the understanding of the TIA biosynthesis and its regulation. In order to analyze the metabolites in C. roseus cell cultures, this chapter presents the method of TIA, carotenoids, and phytosterols analyses. Furthermore, an NMR-based metabolomics approach to study C. roseus cell culture is described.
    Matched MeSH terms: Cell Culture Techniques/methods*
  2. Chondrocyte-alginate constructs with or without TGF-β1 produces superior extracellular matrix expression than monolayer cultures
    Ab-Rahim S, Selvaratnam L, Raghavendran HR, Kamarul T
    Mol Cell Biochem, 2013 Apr;376(1-2):11-20.
    PMID: 23238871 DOI: 10.1007/s11010-012-1543-0
    Tissue engineering approaches often require expansion of cell numbers in vitro to accelerate tissue regenerative processes. Although several studies have used this technique for therapeutic purposes, a major concern involving the use of isolated chondrocyte culture is the reduction of extracellular matrix (ECM) protein expressed due to the transfer of cells from the normal physiological milieu to the artificial 2D environment provided by the cell culture flasks. To overcome this issue, the use of alginate hydrogel beads as a substrate in chondrocyte cultures has been suggested. However, the resultant characteristics of cells embedded in this bead is elusive. To elucidate this, a study using chondrocytes isolated from rabbit knee articular cartilage expanded in vitro as monolayer and chondrocyte-alginate constructs was conducted. Immunohistochemical evaluation and ECM distribution was examined with or without transforming growth factor (TGF-β1) supplement to determine the ability of cells to express major chondrogenic proteins in these environments. Histological examination followed by transmission electron microscopy and scanning electron microscopy was performed to determine the morphology and the ultrastructural characteristics of these cells. Results demonstrated a significant increase in glycosaminoglycan/mg protein levels in chondrocyte cultures grown in alginate construct than in monolayer cultures. In addition, an abundance of ECM protein distribution surrounding chondrocytes cultured in alginate hydrogel was observed. In conclusion, the current study demonstrates that the use of alginate hydrogel beads in chondrocyte cultures with or without TGF-β1 supplement provided superior ECM expression than monolayer cultures.
    Matched MeSH terms: Cell Culture Techniques/methods*
  3. The differential effects of commercial specialized media on cell growth and transforming growth factor beta 1-induced epithelial-mesenchymal transition in bronchial epithelial cells
    Hasan NAHM, Harith HH, Israf DA, Tham CL
    Mol Biol Rep, 2020 May;47(5):3511-3519.
    PMID: 32279207 DOI: 10.1007/s11033-020-05439-x
    Epithelial-mesenchymal transition (EMT) is one of the mechanisms that contribute to bronchial remodelling which underlie chronic inflammatory airway diseases such as chronic obstructive pulmonary disorder (COPD) and asthma. Bronchial EMT can be triggered by many factors including transforming growth factor β1 (TGFβ1). The majority of studies on TGFβ1-mediated bronchial EMT used BEGM as the culture medium. LHC-9 medium is another alternative available which is more economical but a less common option. Using normal human bronchial epithelial cells (BEAS-2B) cultured in BEGM as a reference, this study aims to validate the induction of EMT by TGFβ1 in cells cultured in LHC-9. Briefly, the cells were maintained in either LHC-9 or BEGM, and induced with TGFβ1 (5, 10 and 20 ng/ml) for 48 h. EMT induction was confirmed by morphological analysis and EMT markers expression by immunoblotting. In both media, cells induced with TGFβ1 displayed spindle-like morphology with a significantly higher radius ratio compared to non-induced cells which displayed a cobblestone morphology. Correspondingly, the expression of the epithelial marker E-cadherin was significantly lower, whereas the mesenchymal marker vimentin expression was significantly higher in induced cells, compared to non-induced cells. By contrast, a slower cell growth rate was observed in LHC-9 compared to that of BEGM. This study demonstrates that neither LHC-9 nor BEGM significantly influence TGFβ1-induced bronchial EMT. However, LHC-9 is less optimal for bronchial epithelial cell growth compared to BEGM. Thus, LHC-9 may be a more cost-effective substitute for BEGM, provided that time is not a factor.
    Matched MeSH terms: Cell Culture Techniques/methods
  4. Discovery of α-Glucosidase Inhibitors from Marine Microorganisms: Optimization of Culture Conditions and Medium Composition
    Trang NTH, Tang DYY, Chew KW, Linh NT, Hoang LT, Cuong NT, et al.
    Mol Biotechnol, 2021 Nov;63(11):1004-1015.
    PMID: 34185249 DOI: 10.1007/s12033-021-00362-3
    Various studies showed that the suppression of α-glucosidase activity can impede the glucose absorption in our body, and therefore, it can be used to treat type 2 diabetes. Hence, the compounds with anti-α-glucosidase have gained considerable attention because of their potential application in diabetes treatment. In previous literature studies, these anti-α-glucosidase compounds were extracted from plants and fungus. Less studies are being conducted to identify the anti-α-glucosidase compounds in the microbial community. In this study, 23 marine bacterial strains were screened for their potential to suppress the α-glucosidase activity. The highest inhibitory activity was exhibited by isolated L06 which was identified as Oceanimonas smirnovii EBL6. The cultivation conditions, such as temperature and pH, were optimized to increase the production of α-glucosidase inhibitors by Oceanimonas smirnovii EBL6 strain. The result findings showed that the highest yield of α-glucosidase inhibitors can be obtained at the culture time of 120 h, fermentation temperature of 30 °C, and pH 4.6. Under these conditions, the inhibitory activity of α-glucosidase can reach 81%. The IC50 of n-butanol extract was 13.89 μg/ml, while standard acarbose was 31.16 μg/ml. Overall, these findings suggest that Oceanimonas smirnovii produces α-glucosidase inhibitors and could been applied in the biochemical and medicinal fields in the future.
    Matched MeSH terms: Cell Culture Techniques/methods
  5. Immobilised Sarawak Malaysia yeast cells for production of bioethanol
    Zain MM, Kofli NT, Rozaimah S, Abdullah S
    Pak J Biol Sci, 2011 May 01;14(9):526-32.
    PMID: 22032081
    Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.
    Matched MeSH terms: Cell Culture Techniques/methods
  6. Fulltext Protective effects of stem cells from human exfoliated deciduous teeth derived conditioned medium on osteoarthritic chondrocytes
    Muhammad SA, Nordin N, Hussin P, Mehat MZ, Abu Kasim NH, Fakurazi S
    PLoS One, 2020;15(9):e0238449.
    PMID: 32886713 DOI: 10.1371/journal.pone.0238449
    Treatment of osteoarthritis (OA) is still a major clinical challenge due to the limited inherent healing capacity of cartilage. Recent studies utilising stem cells suggest that the therapeutic benefits of these cells are mediated through the paracrine mechanism of bioactive molecules. The present study evaluates the regenerative effect of stem cells from human exfoliated deciduous teeth (SHED) conditioned medium (CM) on OA chondrocytes. The CM was collected after the SHED were cultured in serum-free medium (SFM) for 48 or 72 h and the cells were characterised by the expression of MSC and pluripotency markers. Chondrocytes were stimulated with interleukin-1β and treated with the CM. Subsequently, the expression of aggrecan, collagen type 2 (COL 2), matrix metalloproteinase-13 (MMP-13), nuclear factor-kB (NF-kB) and the level of inflammatory and anti-inflammatory markers were evaluated. SHED expressed mesenchymal stromal cell surface proteins but were negative for haematopoietic markers. SHED also showed protein expression of NANOG, OCT4 and SOX2 with differential subcellular localisation. Treatment of OA chondrocytes with CM enhanced anti-inflammation compared to control cells treated with SFM. Furthermore, the expression of MMP-13 and NF-kB was significantly downregulated in stimulated chondrocytes incubated in CM. The study also revealed that CM increased the expression of aggrecan and COL 2 in OA chondrocytes compared to SFM control. Both CM regenerate extracellular matrix proteins and mitigate increased MMP-13 expression through inhibition of NF-kB in OA chondrocytes due to the presence of bioactive molecules. The study underscores the potential of CM for OA treatment.
    Matched MeSH terms: Cell Culture Techniques/methods
  7. Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3)
    Chew FN, Tan WS, Boo HC, Tey BT
    Prep Biochem Biotechnol, 2012;42(6):535-50.
    PMID: 23030465 DOI: 10.1080/10826068.2012.660903
    An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.
    Matched MeSH terms: Cell Culture Techniques/methods
  8. The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system
    Abu ML, Mohammad R, Oslan SN, Salleh AB
    Prep Biochem Biotechnol, 2021;51(4):350-360.
    PMID: 32940138 DOI: 10.1080/10826068.2020.1818256
    A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.
    Matched MeSH terms: Cell Culture Techniques/methods
  9. Comparative study on human and bovine AT-SC isolation methods
    Reshak AH, Shahimin MM, Buang F
    Prog Biophys Mol Biol, 2013 Nov;113(2):295-8.
    PMID: 24080186 DOI: 10.1016/j.pbiomolbio.2013.09.001
    Mammalian adipose tissue derived stem cells (AT-SC) have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 h and 2 h under 37 °C in 5% CO2 incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 h under 37 °C in CO2 incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 h under 37 °C in CO2 incubator.
    Matched MeSH terms: Cell Culture Techniques/methods
  10. Fulltext Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity
    Higuchi A, Kao SH, Ling QD, Chen YM, Li HF, Alarfaj AA, et al.
    Sci Rep, 2015 Dec 14;5:18136.
    PMID: 26656754 DOI: 10.1038/srep18136
    The tentative clinical application of human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human induced pluripotent stem cells, is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore, we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.
    Matched MeSH terms: Cell Culture Techniques/methods*
  11. Fulltext Factors Affecting Cell Biomass and Flavonoid Production of Ficus deltoidea var. kunstleri in Cell Suspension Culture System
    Haida Z, Syahida A, Ariff SM, Maziah M, Hakiman M
    Sci Rep, 2019 07 02;9(1):9533.
    PMID: 31267036 DOI: 10.1038/s41598-019-46042-w
    A study was conducted to establish in vitro culture conditions for maximum production of biomass and flavonoid content for Ficus deltoidea var. kunstleri, locally named as Mas Cotek, known to have a wide variety of potential beneficial attributes for human health. Size of initial inoculum, cell aggregate and initial pH value have been suggested to influent content of biomass and flavonoid for cell suspension culture in several plant species. In the present study, leaf explants were cultured by cell suspension culture procedures in MSB5 basal medium supplemented with predetermined supplements of 30 g/L sucrose, 2.75 g/L gelrite, 2 mg/L picloram and 1 mg/L kinetin with continuous agitation of 120 rpm in a standard laboratory environment. Establishment of cell suspension culture was accomplished by culturing resulting callus in different initial fresh weight of cells (0.10, 0.25, 0.50, 1.0, and 2.0 g/25 mL of media) using similar basal medium. The results showed that the highest production of biomass (0.65 g/25 mL of media) was recorded from an initial inoculum size of 2.0 g/25 mL media, whereas the highest flavonoid (3.3 mg RE/g DW) was found in 0.5 g/25 mL of media. Cell suspension fractions classified according to their sizes (500-750 µm, 250-500 µm, and <250 µm). Large cell aggregate size (500-750 µm) cultured at pH 5.75 produced the highest cell biomass (0.28 g/25 mL media) and flavonoid content (3.3 mg RE/g DW). The study had established the optimum conditions for the production of total antioxidant and flavonoid content using DPPH and FRAP assays in cell suspension culture of F. deltoidea var. kunstleri.
    Matched MeSH terms: Cell Culture Techniques/methods*
  12. Fulltext Continuous Butanol Fermentation of Dilute Acid-Pretreated De-oiled Rice Bran by Clostridium acetobutylicum YM1
    Al-Shorgani NKN, Al-Tabib AI, Kadier A, Zanil MF, Lee KM, Kalil MS
    Sci Rep, 2019 03 15;9(1):4622.
    PMID: 30874578 DOI: 10.1038/s41598-019-40840-y
    Continuous fermentation of dilute acid-pretreated de-oiled rice bran (DRB) to butanol by the Clostridium acetobutylicum YM1 strain was investigated. Pretreatment of DRB with dilute sulfuric acid (1%) resulted in the production of 42.12 g/L total sugars, including 25.57 g/L glucose, 15.1 g/L xylose and 1.46 g/L cellobiose. Pretreated-DRB (SADRB) was used as a fermentation medium at various dilution rates, and a dilution rate of 0.02 h-1 was optimal for solvent production, in which 11.18 g/L of total solvent was produced (acetone 4.37 g/L, butanol 5.89 g/L and ethanol 0.92 g/L). Detoxification of SADRB with activated charcoal resulted in the high removal of fermentation inhibitory compounds. Fermentation of detoxified-SADRB in continuous fermentation with a dilution rate of 0.02 h-1 achieved higher concentrations of solvent (12.42 g/L) and butanol (6.87 g/L), respectively, with a solvent productivity of 0.248 g/L.h. This study showed that the solvent concentration and productivity in continuous fermentation from SADRB was higher than that obtained from batch culture fermentation. This study also provides an economic assessment for butanol production in continuous fermentation process from DRB to validate the commercial viability of this process.
    Matched MeSH terms: Batch Cell Culture Techniques/methods*
  13. Fulltext Molecular organization of recombinant human-Arabidopsis chromosomes in hybrid cell lines
    Liu Y, Liaw YM, Teo CH, Cápal P, Wada N, Fukui K, et al.
    Sci Rep, 2021 Mar 30;11(1):7160.
    PMID: 33785802 DOI: 10.1038/s41598-021-86130-4
    Although plants and animals are evolutionarily distant, the structure and function of their chromosomes are largely conserved. This allowed the establishment of a human-Arabidopsis hybrid cell line in which a neo-chromosome was formed by insertion of segments of Arabidopsis chromosomes into human chromosome 15. We used this unique system to investigate how the introgressed part of a plant genome was maintained in human genetic background. The analysis of the neo-chromosome in 60- and 300-day-old cell cultures by next-generation sequencing and molecular cytogenetics suggested its origin by fusion of DNA fragments of different sizes from Arabidopsis chromosomes 2, 3, 4, and 5, which were randomly intermingled rather than joined end-to-end. The neo-chromosome harbored Arabidopsis centromeric repeats and terminal human telomeres. Arabidopsis centromere wasn't found to be functional. Most of the introgressed Arabidopsis DNA was eliminated during the culture, and the Arabidopsis genome in 300-day-old culture showed significant variation in copy number as compared with the copy number variation in the 60-day-old culture. Amplified Arabidopsis centromere DNA and satellite repeats were localized at particular loci and some fragments were inserted into various positions of human chromosome. Neo-chromosome reorganization and behavior in somatic cell hybrids between the plant and animal kingdoms are discussed.
    Matched MeSH terms: Cell Culture Techniques/methods
  14. Fulltext Microencapsulated Aliivibrio fischeri in alginate microspheres for monitoring heavy metal toxicity in environmental waters
    Futra D, Heng LY, Surif S, Ahmad A, Ling TL
    Sensors (Basel), 2014 Dec 05;14(12):23248-68.
    PMID: 25490588 DOI: 10.3390/s141223248
    In this article a luminescence fiber optic biosensor for the microdetection of heavy metal toxicity in waters based on the marine bacterium Aliivibrio fischeri (A. fischeri) encapsulated in alginate microspheres is described. Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(II) were selected as sample toxic heavy metal ions for evaluation of the performance of this toxicity microbiosensor. The loss of bioluminescence response from immobilized A. fischeri bacterial cells corresponds to changes in the toxicity levels. The inhibition of the luminescent biosensor response collected at excitation and emission wavelengths of 287 ± 2 nm and 487 ± 2 nm, respectively, was found to be reproducible and repeatable within the relative standard deviation (RSD) range of 2.4-5.7% (n = 8). The toxicity biosensor based on alginate micropsheres exhibited a lower limit of detection (LOD) for Cu(II) (6.40 μg/L), Cd(II) (1.56 μg/L), Pb(II) (47 μg/L), Ag(I) (18 μg/L) than Zn(II) (320 μg/L), Cr(VI) (1,000 μg/L), Co(II) (1700 μg/L), Ni(II) (2800 μg/L), and Fe(III) (3100 μg/L). Such LOD values are lower when compared with other previous reported whole cell toxicity biosensors using agar gel, agarose gel and cellulose membrane biomatrices used for the immobilization of bacterial cells. The A. fischeri bacteria microencapsulated in alginate biopolymer could maintain their metabolic activity for a prolonged period of up to six weeks without any noticeable changes in the bioluminescence response. The bioluminescent biosensor could also be used for the determination of antagonistic toxicity levels for toxicant mixtures. A comparison of the results obtained by atomic absorption spectroscopy (AAS) and using the proposed luminescent A. fischeri-based biosensor suggests that the optical toxicity biosensor can be used for quantitative microdetermination of heavy metal toxicity in environmental water samples.
    Matched MeSH terms: Cell Culture Techniques/methods
  15. Fulltext Effect of cell density on formation of three-dimensional cartilaginous constructs using fibrin & human osteoarthritic chondrocytes
    Shamsul BS, Chowdhury SR, Hamdan MY, Ruszymah BHI
    Indian J Med Res, 2019 05;149(5):641-649.
    PMID: 31417032 DOI: 10.4103/ijmr.IJMR_45_17
    Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage.

    Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development.

    Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II.

    Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.

    Matched MeSH terms: Cell Culture Techniques/methods
  16. Fulltext Comparison of invasion by human microvascular endothelial cell lines in response to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in a threedimensional (3D) cell culture system
    Ng CT, Yip WK, Mohtarrudin N, Seow HF
    Malays J Pathol, 2015 Dec;37(3):219-25.
    PMID: 26712666 MyJurnal
    Immortalized human endothelial cells are widely used as in vitro models for debilitating conditions such as cancer, cardiovascular and ocular diseases. Human microvascular endothelial cell (HMEC-1) is immortalized via stable transfection with a gene encoding SV40 large antigen whilst telomerase-immortalized human microvascular endothelial (TIME) cells is immortalized by engineering the human telomerase catalytic protein (hTERT) into primary microvascular endothelial cells. Here, we established a three-dimensional (3D) spheroid invasion assay with HMEC-1 and TIME and compared the difference in their ability to invade through the collagen matrix in response to exogenous growth factors, namely vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
    Matched MeSH terms: Cell Culture Techniques/methods*
  17. Fulltext Experimental production of clinical-grade dendritic cell vaccine for acute myeloid leukemia
    Tan YF, Sim GC, Habsah A, Leong CF, Cheong SK
    Malays J Pathol, 2008 Dec;30(2):73-9.
    PMID: 19291915 MyJurnal
    Dendritic cells (DC) are professional antigen presenting cells of the immune system. Through the use of DC vaccines (DC after exposure to tumour antigens), cryopreserved in single-use aliquots, an attractive and novel immunotherapeutic strategy is available as an option for treatment. In this paper we describe an in vitro attempt to scale-up production of clinical-grade DC vaccines from leukemic cells. Blast cells of two relapsed AML patients were harvested for DC generation in serum-free culture medium containing clinical-grade cytokines GM-CSF, IL-4 and TNF-alpha. Cells from patient 1 were cultured in a bag and those from patient 2 were cultured in a flask. The numbers of seeding cells were 2.24 x 10(8) and 0.8 x 10(8), respectively. DC yields were 10 x 10(6) and 29.8 x 10(6) cells, giving a conversion rate of 4.7% and 37%, respectively. These DC vaccines were then cryopreserved in approximately one million cells per vial with 20% fresh frozen group AB plasma and 10% DMSO. At 12 months and 21 months post cryopreservation, these DC vaccines were thawed, and their sterility, viability, phenotype and functionality were studied. DC vaccines remained sterile up to 21 months of storage. Viability of the cryopreserved DC in the culture bag and flask was found to be 50% and 70% at 12 months post cryopreservation respectively; and 48% and 67% at 21 months post cryopreservation respectively. These DC vaccines exhibited mature DC surface phenotypic markers of CD83, CD86 and HLA-DR, and negative for haemopoietic markers. Mixed lymphocyte reaction (MLR) study showed functional DC vaccines. These experiments demonstrated that it is possible to produce clinical-grade DC vaccines in vitro from blast cells of leukemic patients, which could be cryopreserved up to 21 months for use if repeated vaccinations are required in the course of therapy.
    Matched MeSH terms: Cell Culture Techniques/methods*
  18. Fulltext A modified mycological medium for isolation and culture of Malassezia furfur
    Chua KB, Chua IL, Chua IE, Chong KH, Chua KH
    Malays J Pathol, 2005 Dec;27(2):99-105.
    PMID: 17191392
    A mycological medium was developed for primary isolation and culture of lipophilic yeasts. It was initially based on published information of nutrients and trace components that would promote the growth of these yeasts. It was subsequently modified and adjusted to specifically promote the growth of lipophilic yeasts and simultaneously avoid the luxurious growth of other fungi and bacteria. With this medium, the conventional bacteriological procedures such as microbial streaking for pure culture and anti-microbial sensitivity testing could be carried out for these lipophilic yeasts.
    Matched MeSH terms: Cell Culture Techniques/methods*
  19. Fulltext Generation of dendritic cells from acute myeloid leukaemia cells and monocytes: our local experience
    Lim MN, Leong CF, Cheong SK, Seow HF
    Malays J Pathol, 2003 Dec;25(2):107-12.
    PMID: 16196366
    Dendritic cells (DC) are efficient and potent antigen-presenting cells. Pilot clinical trials indicated that DC loaded with tumour antigen could induce tumour-specific immune responses in various cancers including B-cell lymphoma, melanoma and prostate cancer. Owing to extensively low number of DC in the blood circulation, a variety of sources have been used to generate DC including monocytes, CD34+ stem cells and even with leukaemic blast cells. We demonstrate here a simple method to generate DC from acute myeloid leukaemia (AML) cells and monocytes from healthy donor or remission samples. AML cells or monocytes were cultured in RPMI 1640 media supplemented with foetal bovine serum or autologous serum where possible and different combinations of cytokines GM-CSF, IL-4 and TNF-alpha. The generated DC were evaluated for their morphology by phase contrast microscopy and May Grunwald Giemsa staining. Viability of cells was determined by trypan blue dye exclusion. Percentage of yields and immunophenotypes were carried out by flow cytometry. We found that cultured AML cells and monocytes developed morphological and immuno-phenotypic characteristics of DC. Monocytes are better than AML blast in generating DC and serve as a ready source for dendritic cell vaccine development.
    Matched MeSH terms: Cell Culture Techniques/methods*
  20. Isolation techniques of murine bone marrow progenitor cells and their adipogenic, neurogenic and osteogenic differentiation capacity
    Ng AM, Kojima K, Kodoma S, Ruszymah BH, Aminuddin BS, Vacanti AC
    Med J Malaysia, 2008 Jul;63 Suppl A:121-2.
    PMID: 19025015
    Bone marrow derived progenitor cells have been widely studied for its multipotent property and have proofed to be an important resource in regenerative medicine. However, the propagation of murine bone marrow appeared to be a great challenge as compared to other mammalian species. In this study, various isolation techniques and the plasticity of the isolated cells were evaluated. Our result shows that magnetic sorting technique yielded the most viable cells and displayed wider differentiation capacity.
    Matched MeSH terms: Cell Culture Techniques/methods
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