62 research outputs found
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Enzyme-free release of adhered cells from standard culture dishes using intermittent ultrasonic traveling waves.
Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process
Establishment and characterisation of cell lines from the caerulean damsel, Pomacentrus caeruleus
Cell lines and primary cell cultures from fish tissues have been used for
research in disease diagnosis and cytotoxicity evaluation of environmental
pollutants. The principal aim of this work was the development of continuous
cell lines from the caerulean damsel, Pomacentus caeruleus. To achieve this,
tissues were first sourced from donor fishes, devoid of contaminants using
standardised disinfection protocols. Primary cultures were then initiated and
suitable media, additives, dissociation reagents as well as optimum incubation
temperature were determined. Successful primary cultures were subcultured
and passaged to derive continuous cell lines which were cryopreserved for
long term storage. The continuous cell lines established were characterised by
immunotyping using cell type markers and authenticated to confirm the
species of origin using previously established barcoding techniques.
Preliminary applications such as gene transfection studies and cytotoxicity
assays using bacterial extracellular products were done in addition to ensuring
that the cultures and cryostocks were contamination-free
Thermophilic mixed culture degradation of Miscanthus x giganteus as a guide to strategies for consolidated bioprocessing
The successful development of consolidated bioprocessing requires microorganisms capable of degrading lignocellulosic biomass and fermenting the resulting sugars. Commercial cellulases and hemicellulases are currently being used to access these sugars, adding to the cost of producing useful products from lignocellulose. This study reports the enrichment of thermophilic, miscanthus degrading bacterial cultures from a municipal composting facility. The detected and isolated bacteria were characterized by 16S rRNA gene sequence analysis and were mostly Chitinophagaceae family, Meiothermus spp. and Geobacillus spp. Other isolated species included Cohnella spp., Brevibacillus sp., Chelatococcus spp., Thermobacillus spp., Thermoanaerobacterium spp., Thermobispora bispora, Bacillus spp., Staphylococcus sp. and Micrococcus sp.
After enrichment, the mixed population was able to degrade greater than 50% of an ammonium hydroxide pre-treated Miscanthus x giganteus sample (1 g) over a six week incubation period at 55oC, with a reduction in the amounts of all components, including acid soluble and acid insoluble lignin. The glycoside hydrolases and other enzymes identified in the culture supernatants included endo-1,4-β-glucanase A, glucoamylase, xylan 1,4-β-xylosidase, xylose isomerase, xylulokinase, superoxide dismutase, transaldolase, Mn-catalase, Δ-1-pyrroline-5-carboxylate dehydrogenase and endo-β-N-acetylglucoseaminidase H. The HPLC analysis showed that fermentation products formate and lactate were present in the culture supernatant.
Expression of an endoglycoside hydrolase (Csac_0137 from Caldicellulosiruptor saccharolyticus) gene in Geobacillus thermoglucosidasius strains, NCIMB 11955 and DL33, improved their β-glucosidase specific activity on cellobiose, and improved glycoside hydrolase activities of recombinant DL33 strain when grown on pre-treated M. x giganteus. Co-culturing of either transformed or wild-type NCIMB 11955 and DL33 with some of the isolated strains improved their glycoside hydrolase activity and growth on pretreated M. x giganteus.Open Acces
Epithelial cell adhesion to a planar substratum: Quantitative studies using a miniaturised parallel-plate shearing apparatus
The nature and quantitative aspects of epithelial cell adhesion are reviewed, and their relevance discussed in relation to the homeostasis of the dento-epithelial junction. The design, construction and testing of a miniaturised parallel-plate shearing apparatus for the measurement of cell adhesion based on the radial flow chamber principle of Fowler & McKay (1980) is described. Cultures of an established epithelial cell line on glass coverslips were exposed to flow conditions for varying times in the shearing chamber at 37°, 8° and 4°C, and subsequently photographed under standardized conditions. The critical shear radius (CSR) was determined by densitometry from a negative film and the minimum distraction force (MDF) at the CSR calculated using the measured flow rate and predetermined viscosity values of the medium. The calculated mean MDF values at 37°C ranged from 1.04 to 1.36 pascals, and was independent of the culture inoculation density (2.5 x 10
5 to 10
6 cells ml-1) and thetime (range: 5 to 20 min) of exposure to shearing conditions. Cell-to-cell adhesion in multilayer cultures was assessed by a cell-separation index (O) which represented the proportion of cells detached in a specific annulus per unit shearing force. The minimal force necessary to separate the upper layer(s) of cells was calculated to be significantly less than cell-to-substratum adhesion (MDF) being in the range of 0.43 to 0.64 Pa. Measurements of cell-to-substratum adhesion at 4°C demonstrated a three to four fold increase of the MDF (6.19 Pa) compared to that at 37°C (1.35 Pa). Part of this adhesion was resistant to mild trypsinisation and was stabilised by low temperature, and by treatment with concanavalin A or colchicine. A classification of cellular adhesion as trypsin-sensitive (TSA) and trypsin-resistant adhesion (TRA) on the basis of the different temperature and protease susceptibility is proposed with the corresponding physiological functions of "frictional" and "tractional" adhesion respectively. The implications of the dual adhesion hypothesis are discussed with respect to the integrity of the dento-gingival junction
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The Impact of substrate texture on cell behavior
Traditionally, cell studies have focused mainly on the effects of biochemical signals on cellular behaviour; however, there is a growing interest in investigating the effects of physical cues on the behaviour of the cells. Development of fabrication techniques has allowed researchers to design topographical cues at the micro and nanometer scale. In this study, we fabricated fibronectin stripes, which mimic some of the frequent patterns that cells are exposed to in their natural microenvironment, and studied the effects of these patterns on 3T3 fibroblasts and human mesenchymal stem cells which are of significant importance in the field of regenerative medicine and tissue engineering. Our results show that cells align and elongate in the direction of the patterns and the morphological features of cell, nucleus and architecture of force-bearing stress fibers of the actin cytoskeleton are all highly dependent on the stripe width. 3D confocal measurements showed that the cell thickness and nuclear shape is regulated through the different arrangement of perinuclear actin stress fibers depending on the stripe sizes. We also observed that when the size of stripes (5 µm) is significantly smaller than the size of cell nuclei (subnuclear stripes), cells exhibit 3 different morphological responses to the patterns. Minimal confinement, branching on the stripes and elongation on a single stripe. The majority of cells and nuclei align themselves with the subnuclear stripes; however, a substantial number of hMSCs nuclei are elongated perpendicular to the direction of the pattern, a finding which we explained using a geometrical model based on cell size. The behaviour of 3T3 cell lines and the hMSCs were compared for the different types of pattern.
We designed setups that allow us to perform Raman microspectroscopy both in 2D and 3D topographical patterns in order to be able to study their morpho-chemical effects on cells. We also genetically modified 3T3 fibroblasts to express fluorescent-tagged proteins that visualize the actin cytoskeleton and the nucleus which allow us to perform live cell imaging to study the dynamics of cell behaviour on topographical patterns.Peterhouse College research studentship,
Cavendish laboratory research scholarship,
Cambridge Trust- IDB 3 years full PhD scholarshi
Large scale in vitro expansion of mesenchymal stem cells.
Mesenchymal stem cells (MSCs) can undergo self-renewal and differentiation into a
variety of mature cell types. Thus, using MSCs for tissue engineering and other
medical applications holds many promising advantages over normal somatic cells.
However, exactly these characteristics make MSCs more difficult to grow and
control in vitro. The aim of this research project was to investigate different culture
systems for their utility to expand bone marrow derived MSCs in large quantities. A
large scale expansion of MSCs is especially of interest since only a small number of
bone marrow derived MSCs are present in donor derived samples, which do not meet
the demand for medical applications.
In this thesis three different culture systems, static monolayer cultures, stirred
suspension cultures, and pour-off cultures, were compared with each other for their
ability to support MSCs proliferation while allowing them to keep their full
differentiation capacity. Cell samples derived from these cultures were used for cell
count, to start a CFU-f assay and to start osteogenic and adipogenic differentiation
assays. The highest MSC numbers obtained from the static monolayer cultures were
about 460% of the initial cells. The differentiation capacity of these cells was
restricted, so they only formed osteoblasts. Furthermore, MSC samples obtained
from this culture system were used for proteomic analysis on an electrospray
ionisation quadrupol (ESI-qQ-STAR) mass spectrometer with the isobaric tag for
relative and absolute quantitation (iTRAQ) method. This analysis revealed a
difference in the proteome of MSCs from different passage levels which is involved
in a changing proliferation and differentiation behaviour.
In stirred suspension cultures, the increase in MSC number varied for the different
culture media. The best result was achieved in MyeloCult® medium with Pluronic F-
68, IL-3 and SCF, however, reaching only 140% of the initial cell density, this result
was significantly worse than in the control monolayer cultures. The pour-off cultures
supported an increase in MSC number, which resulted in 860% of the initial cell
number. In addition, MSCs expanded in this culture system were able to differentiate
into ostoblasts and adipocytes. Thus, pour-off cultures are the most promising culture
system for large scale expansion of MSCs with high differentiation potential
Isolation and characterisation of medicinal compounds from Phyllanthus Niruri L
In many countries, Phyllanthus niruri L is one of the most popular alternatives of natural herbal remedy to overcomemany symptoms due to its wide range of therapeutic uses.Even though a considerable number of research projectshave been conducted in order to reveal the pharmacological activities of Phyllanthus niruri L, and that a number of reports have been produced mentioning its pharmacological effect, the rich constituents of this plant are yet to be comprehensively studied, particularly with regards to the nature of the biological activities that the compounds have.Accordingly, the main focus of this study is the exploration of Phyllanthus niruri L as a new candidate of natural compound. This was done through a series of bioassay- guided plant extraction and isolation protocols.Phyllanthus niruri L crude extracts were tested againstplasmodium, cancer cell lines, and platelet aggregation. Guided by the bioassay results, the isolation procedureswere performed using advance chromatography techniques, in order to purify the most - active substances thatrepresent the final candidate natural product.This study also employed flow cytometry and proteomics studies, as an attempt to identify the fundamental principlesof the mechanism of action of the isolated compounds.The results demonstrated that Phyllanthus niruri Lextracts showed a potency as antiplasmodial, anti-cancer, as well as anti-platelet agent. In inhibiting plasmodiumfalciparum growth, the potency of the extracts from the most to the least potent activity was methanol > water > ethanol > chloroform > hexane (IC 50values were 1.6 μg/ml, 9.6 μg/ml, 25 μg/ml, and 141 μg/ml, respectively).With regards to its anti-cancer effect, Phyllanthus niruri Lextracts showed a significant cytotoxic effect on human Caucasian lung large cell carcinoma (COR-L23), human acute T lymphoblastic leukaemia (MOLT-4), andhuman caucasian chronic myelogenous leukaemia (K562).Among all extracts, methanol extract demonstrated the strongest cytotoxicity effect with a low IC 50 values for all cell lines tested (IC 50 values for COR-L23, MOLT- 4, and K562 was 48.92 ± 0.52 μg/ml,42.21 ± 4.98 μg/ml, and 139.28 ± 19.02 μg/ml, respectively). Methanol, water, ethanol, and hexane extracts did not show any toxicity towards normal fibroblast cell line (3T3). However,chloroform extract demonstrated a toxic effect to the normal cells line (IC 50 value 164.3 ± 8.4 μg/ml).x The antiplatelet activity of Phyllanthus niruri Lwas further explored in this study due to a remarkable inhibitory effect of methanolic extract observed on ADP- induced platelet aggregation. With the aid of bioassay- guided isolation protocol, the study has isolated fourcompounds from the methanolic extract, which demonstrated a potency in preventing in-vitro platelet aggregation induced by ADP (compound 1, 2 , 3, and 6). The IC 50of each compound was 179.9 ± 2.67 μg/ml (compound1), 31.91 ± 1.86 μg/ml (compound 2),77.68±6.44 μg/ml (compound 3), and 43.35 ± 6.44 μg/ml (compound 6).Compound 2 was identified as corilagin or [3,5-dihydroxy-2-(3,4,5-trihydroxybenzoyl)oxy -6-[(3,4,5-trihydroxybenzoyl)oxymethyl]oxan-4-yl]3,4,5-trihydroxybenzoate. The finding of this study demonstrated that corilagin altered the G-protein signalling pathway in a selective manner by impeding Gq-protein cascade. Corilagin might act through G13-mediated signalling;however it showed no significant effect on Gi-mediated signalling pathway. Consequently, corilagin inhibited platelet shape changes, granule secretion, and platelet aggregate formation, which was suggested to take place by the inhibition of the elevation of intracellular Ca2+ level due to the inactivation of PLCβ. Although corilaginshowed no observable inhibitory effect on the initial activation of the major platelet glycoprotein, GPIIb/IIIa, the findings suggested that the interaction between the activated integrin and the related ligands is affected, which results in the inhibition of further amplification of platelet aggregation. Overall,this study confirmed the antiplatelet activity of corilagin and explainedits coherent mode of actions, which support the future development of corilagin, isolated from Phyllanthus niruriL as a natural-sourced antiplatelet compound
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