Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.
The objectives of this study were: (1) to investigate the role of mixed culture of biomass in the regeneration of mono-amine modified silica (MAMS) and granular activated carbon (GAC) loaded with Acid Orange 7 (AO7), (2) to quantify and compare the bioregeneration efficiencies of AO7-loaded MAMS and GAC using the sequential adsorption and biodegradation approach and (3) to evaluate the reusability of bioregenerated MAMS. The results show that considerably higher bioregeneration efficiency of AO7-loaded MAMS as compared to that of AO7-loaded GAC was achieved due to higher reversibility of adsorption of MAMS for AO7 and favorable pH factor resulting in more AO7 desorption. The progressive loss of adsorption capacity of MAMS for AO7 with multiple cycles of use suggests possible chemical and microbial fouling of the adsorption sites.
This study was undertaken in order to identify the best culture strategy to expand and osteogenic differentiation of human bone marrow stem cells (hBMSCs) for subsequent bone tissue engineering. In this regard, the experiment was designed to evaluate whether it is feasible to bypass the expansion phase during hBMSCs differentiation towards osteogenic lineages by early induction, if not identification of suitable culture media for enhancement of hBMSCs expansion and osteogenic differentiation. It was found that introduction of osteogenic factors in alpha-minimum essential medium (αMEM) during expansion phase resulted in significant reduction of hBMSCs growth rate and osteogenic gene expressions. In an approach to identify suitable culture media, the growth and differentiation potential of hBMSCs were evaluated in αMEM, F12:DMEM (1:1; FD), and FD with growth factors. It was found that αMEM favors the expansion and osteogenic differentiation of hBMSCs compared to that in FD. However, supplementation of growth factors in FD, only during expansion phase, enhances the hBMSCs growth rate and significantly up-regulates the expression of CBFA-1 (the early markers of osteogenic differentiation) during expansion, and, other osteogenic genes at the end of induction compared to the cells in αMEM and FD. These results suggested that the expansion and differentiation phase of the hBMSCs should be separately and carefully timed. For bone tissue engineering, supplementation of growth factors in FD only during the expansion phase was sufficient to promote hBMSCs expansion and differentiation, and preferably the most efficient culture condition.
The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo-, chondro-, and adipo-lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy.
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.
A bioflocculant-producing bacterial strain with highly mucoid and ropy colony morphological characteristics identified as Bacillus spp. UPMB13 was found to be a potential bioflocculant-producing bacterium. The effect of cation dependency, pH tolerance and dosage requirement on flocculating ability of the strain was determined by flocculation assay with kaolin as the suspended particle. The flocculating activity was measured as optical density and by flocs formation. A synergistic effect was observed with the addition of monovalent and divalent cations, namely, Na⁺, Ca²⁺, and Mg²⁺, while Fe²⁺ and Al³⁺ produced inhibiting effects on flocculating activity. Divalent cations were conclusively demonstrated as the best cation source to enhance flocculation. The bioflocculant works in a wide pH range, from 4.0 to 8.0 with significantly different performances (P < 0.05), respectively. It best performs at pH 5.0 and pH 6.0 with flocculating performance of above 90%. A much lower or higher pH would inhibit flocculation. Low dosage requirements were needed for both the cation and bioflocculant, with only an input of 50 mL/L for 0.1% (w/v) CaCl₂ and 5 mL/L for culture broth, respectively. These results are comparable to other bioflocculants produced by various microorganisms with higher dosage requirements.
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.
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.
Nine aerobic cellulolytic bacterial cultures were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ) and the American Type Culture Collection (ATCC). The objectives of this study were to characterize the cellulolytic bacteria and to determine the optimum moisture ratio required for solid state fermentation (SSF) of palm kernel cake (PKC). The bacteria cultures were grown on reconstituted nutrient broth, incubated at 30°C and agitated at 200 rpm. Carboxymethyl cellulase, xylanase, and mannanase activities were determined using different substrates and after SSF of PKC. The SSF was conducted for 4 and 7 days with inoculum size of 10% (v/w) on different PKC concentration-to-moisture ratios: 1 : 0.2, 1 : 0.3, 1 : 0.4, and 1 : 0.5. Results showed that Bacillus amyloliquefaciens 1067 DSMZ, Bacillus megaterium 9885 ATCC, Paenibacillus curdlanolyticus 10248 DSMZ, and Paenibacillus polymyxa 842 ATCC produced higher enzyme activities as compared to other bacterial cultures grown on different substrates. The cultures mentioned above also produced higher enzyme activities when they were incubated under SSF using PKC as a substrate in different PKC-to-moisture ratios after 4 days of incubation, indicating that these cellulolytic bacteria can be used to degrade and improve the nutrient quality of PKC.
To determine the optimum light intensity per cell required for rapid growth regardless of cell density, continuous cultures of the microalga Chlorella zofingiensis were grown with a sufficient supply of nutrients and CO2 and were subjected to different light intensities in the range of 75-1000 μE m(-2) s(-1). The cell density of culture increased over time for all light conditions except for the early stage of the high light condition of 1000 μE m(-2) s(-1). The light intensity per cell required for the high specific growth rate of 0.5 day(-1) was determined to be 28-45 μE g-ds(-1) s(-1). The specific growth rate was significantly correlated to light intensity (y=0.721×x/(66.98+x), r(2)=0.85, p<0.05). A high specific growth rate was maintained over a range of light intensities (250-1000 μE m(-2) s(-1)). This range of light intensities suggested that effective production of C. zofingiensis can be maintained outdoors under strong light by using the optimum specific light intensity.
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.
A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.
Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.
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.
Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1(+) cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs.
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.
Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.
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).
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.
Cancer is a major health problem worldwide. The failure of current treatments to completely eradicate cancer cells often leads to cancer recurrence and dissemination. Studies have suggested that tumor growth and spread are driven by a minority of cancer cells that exhibit characteristics similar to those of normal stem cells, thus these cells are called cancer stem cells (CSCs). CSCs are believed to play an important role in initiating and promoting cancer. CSCs are resistant to currently available cancer therapies, and understanding the mechanisms that control the growth of CSCs might have great implications for cancer therapy. Cancer cells are consist of heterogeneous population of cells, thus methods of identification, isolation, and characterisation of CSCs are fundamental to obtain a pure CSC populations. Therefore, this chapter describes in detail a method for isolating and characterizing a pure population of CSCs from heterogeneous population of cancer cells and CSCs based on specific cell surface markers.