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.
The therapeutic potential of human mesenchymal stromal stem cells (hMSCs) for cell-based therapeutic is greatly influenced by the in vitro culture condition including the culture conditions. Nevertheless, there are many technical challenges needed to be overcome prior to the clinical use including the quantity, quality, and heterogeneity of the cells. Therefore, it is necessary to develop a stem cell culture procedure or protocol for cell expansion in order to generate reproducible and high-quality cells in accordance with good manufacturing practice for clinical and therapeutic purposes. Here we assessed the MSCs characteristic of human Wharton's jelly mesenchymal stromal cells in in vitro culture according to the criteria established by the International Society for Cellular Therapy. Besides, the viability of the WJMSCs was determined in order to increase the confidence that the cells are employed to meet the therapeutic efficacy.
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.
Several research groups have utilized dental pulp stem cells for numerous studies as treatment modality for Parkinson's disease (PD). However, the roles of dental pulp stem cells in governing the Parkinson's disease inflammatory microenvironment remain to be evaluated. In this article, we elaborate the method where we can investigate the effects of dental pulp stem cells on neurons and microglia in an in vitro inflammatory microenvironment.
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.
Stem cells isolated from dental pulp possess the capacity for self-renewal and the potential for multi-lineage differentiation. However, dental pulp stem cells have different characteristics in terms of their culture conditions. The success of stem cells culture is governed by its micro-environmental niche. Therefore, we studied the effects of culture niche on long-term expansion of dental pulp stem cells in terms of cell morphology, growth kinetics, senescence pattern, cell surface marker expression differentiation capacity, and seeding plating density of dental pulp stem cells in four different, widely used media composition Among the various basal media tested, α-minimum essential media and knock out-minimum essential media supplemented with 10% fetal bovine serum were found to be the most optimal media composition in preserving the phenotypic characteristics and differentiation potential for prolonged periods as compared with DMEM-F12 and DMEM-LG. Plating density has been shown to affect overall yield. As a conclusion, the adoption of an appropriate culture system significantly improved cell yield, thus enabling the attainment of sufficient yields for therapeutic applications economizing in terms of cost of production and minimizing seeding cell density for maximum yield.
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.
Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.
In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability.
Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.
Various explants (stem, leaf, and root) of Citrus assamensis were cultured on MS media supplemented with various combinations and concentrations (0.5-2.0 mg L(-1)) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mg L(-1) NAA and 2.0 mg L(-1) BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL(-1) sucrose and pH of 5.8 was most optimum for in vitro regeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy of in vivo and in vitro/ex vitro Citrus assamensis were also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, but in vitro plantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.
The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.
The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.
In this study, endoglucanase was produced from oil palm empty fruit bunch (OPEFB) by a locally isolated aerobic bacterium, Bacillus pumilus EB3. The effects of the fermentation parameters such as initial pH, temperature, and nitrogen source on the endoglucanase production were studied using carboxymethyl cellulose (CMC) as the carbon source. Endoglucanase from B. pumilus EB3 was maximally secreted at 37 degrees C, initial pH 7.0 with 10 g/l of CMC as carbon source, and 2 g/l of yeast extract as organic nitrogen source. The activity recorded during the fermentation was 0.076 U/ml. The productivity of the enzyme increased twofold when 2 g/l of yeast extract was used as the organic nitrogen supplement as compared to the non-supplemented medium. An interesting finding from this study is that pretreated OPEFB medium showed comparable results to CMC medium in terms of enzyme production with an activity of 0.063 U/ml. As OPEFB is an abundant solid waste at palm oil mills, it has the potential of acting as a substrate in cellulase production.
Bio-hydrogen production from wastewater using sludge as inoculum is a sustainable approach for energy production. This study investigated the influence of initial pH and temperature on bio-hydrogen production from dairy wastewater using pretreated landfill leachate sludge (LLS) as an inoculum. The maximum yield of 113.2 ± 2.9 mmol H2/g chemical oxygen demand (COD) (12.8 ± 0.3 mmol H2/g carbohydrates) was obtained at initial pH 6 and 37 °C. The main products of volatile fatty acids were acetate and butyrate with the ratio of acetate:butyrate was 0.4. At optimum condition, Gibb's free energy was estimated at -40 kJ/mol, whereas the activation enthalpy and entropy were 65 kJ/mol and 0.128 kJ/mol/l, respectively. These thermodynamic quantities suggest that bio-hydrogen production from dairy wastewater using pretreated LLS as inoculum was effective and efficient. In addition, genomic and bioinformatics analyses were performed in this study.
The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.
The present study focused on lipopeptide biosurfactant production by Streptomyces sp. PBD-410L in batch and fed-batch fermentation in a 3-L stirred-tank reactor (STR) using palm oil as a sole carbon source. In batch cultivation, the impact of bioprocessing parameters, namely aeration rate and agitation speed, was studied to improve biomass growth and lipopeptide biosurfactant production. The maximum oil spreading technique (OST) result (45 mm) which corresponds to 3.74 g/L of biosurfactant produced, was attained when the culture was agitated at 200 rpm and aeration rate of 0.5 vvm. The best aeration rate and agitation speed obtained from the batch cultivation was adopted in the fed-batch cultivation using DO-stat feeding strategy to further improve the lipopeptide biosurfactant production. The lipopeptide biosurfactant production was enhanced from 3.74 to 5.32 g/L via fed-batch fermentation mode at an initial feed rate of 0.6 mL/h compared to that in batch cultivation. This is the first report on the employment of fed-batch cultivation on the production of biosurfactant by genus Streptomyces.
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.
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.
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.