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Showing papers in "Cytotechnology in 2008"


Journal ArticleDOI
TL;DR: A snapshot of current knowledge regarding the potential of cells from the amniotic membrane of human term placenta to address current shortcomings in the field of regenerative medicine is provided.
Abstract: An urgent current need in regenerative medicine is that of identifying a plentiful, safe and ethically acceptable stem cell source for the development of therapeutic strategies to restore functionality in damaged or diseased organs and tissues. In this context, human term placenta represents a prime candidate, as it is available in nearly unlimited supply, is ethically problem-free and easily procured. Placental cells display differentiation capacity toward all three germ layers, while also displaying immunomodulatory effects, therefore supporting the possibility that they could be applied in an allogeneic transplantation setting. Although promising data have been reported to date, further study is required to fully characterize the differentiation potential of placenta-derived cells and to identify their possible clinical applications. Here, we provide a snapshot of current knowledge regarding the potential of cells from the amniotic membrane of human term placenta to address current shortcomings in the field of regenerative medicine.

114 citations


Journal ArticleDOI
TL;DR: The results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.
Abstract: Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days’ culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days’ culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-β signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.

106 citations


Journal ArticleDOI
TL;DR: Two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO2) and pH, measured with fluorescent sensors are studied thus allowing the study of the metabolic state of cells in culture without removing or damaging cells during cultivation.
Abstract: Here, we have studied two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO2) and pH, measured with fluorescent sensors thus allowing the study of the metabolic state of cells in culture without removing or damaging cells during cultivation. Two cell lines, namely, NS0 and CHO were batch-grown in 24-well plates at different serum concentrations with the sensors implemented in the bottom of each well. The data showed a good relationship between the dO2 and pH data obtained from fluorescent probes and the growth and death characteristics of cells. The method has provided a high throughput on-line multi-parametric analysis of mammalian cell cellular activity.

104 citations


Journal ArticleDOI
TL;DR: It is demonstrated that homogenous S-MSC populations can be reproducibly isolated from individual donors of different age and conditions for optimal in vitro propagation of human skin-derived mesenchymal stem cells are found.
Abstract: This study investigated conditions for optimal in vitro propagation of human skin-derived mesenchymal stem cells (S-MSC). Forty primary skin-derived precursor cell (SKP) cultures were established from both male and female donors (age 29–65 years) and eight of them were randomly selected for in-depth characterization. Effects of basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), leukemia inhibiting factor (LIF) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on S-MSC proliferation were investigated. Primary SKP cultures were >95% homogenous for CD90, CD73, and CD105 marker expression enabling to classify these cells as S-MSC. FGF-2 dose-dependent stimulation was observed in low serum medium only, whereas EGF neither stimulated S-MSC proliferation nor potentates the effect of FGF-2. Pronounced donor to donor differences among S-MSC cultures were observed in 3-day proliferation assay. This study demonstrates that homogenous S-MSC populations can be reproducibly isolated from individual donors of different age. Optimal cell culture conditions for in vitro propagation of S-MSC are B27 supplemented or low serum media with FGF-2 (4 ng/ml). EGF and LIF as well as db-cAMP are dispensable for S-MSC proliferation.

102 citations


Journal ArticleDOI
TL;DR: The SVF represents a rich source of mesenchymal cells potentially able to differentiate into adipocytes, chondrocytes, osteoblasts, myocytes, cardiomyocytes, hepatocytes, and neuronal, epithelial and endothelial cells, which are ideal candidates for use in regenerative medicine, tissue engineering, including gene therapy and cell replacement cancer therapies.
Abstract: Cell encapsulation, a strategy whereby a pool of live cells is entrapped within a semipermeable membrane, represents an evolving branch of biotechnology and regenerative medicine. For example, over the last 20 years, male and female gametes and embryos have been encapsulated with or without somatic cells for different purposes, such as in vitro gametogenesis, embryo culture, cell preservation and semen controlled release. Beside that, cell encapsulation technology in alginate, which is a natural biodegradable polymer that mimics the extracellular matrix and supports both cell functions and metabolism, has been developed with the aim of obtaining three-dimensional (3D) cultures. In this context, adipose-derived stromal vascular fraction (SVF) has attracted more and more attention because of its enormous potential in tissue regeneration. In fact, the SVF represents a rich source of mesenchymal cells (ADSCs), potentially able to differentiate into adipocytes, chondrocytes, osteoblasts, myocytes, cardiomyocytes, hepatocytes, and neuronal, epithelial and endothelial cells. These cells are ideal candidates for use in regenerative medicine, tissue engineering, including gene therapy and cell replacement cancer therapies. As long as technological resources are available for large-scale cell encapsulation intended for advanced therapies (gene therapy, somatic cell therapy and tissue engineering), the state-of-the-art in this field is reviewed in terms of scientific literature.

99 citations


Journal ArticleDOI
TL;DR: A method to measure in vivo the mitochondrial mass and activity, in sea urchin Paracentrotus lividus eggs and embryos revealed a variation of mitochondrial distribution and an increase of activity after fertilization.
Abstract: Mitochondria, responsible for the energy-generating process essential for the cell metabolism, differ for the number, localization and activity in animal cells and tissues in relation to the energetic needs. Using fluorescent probes specific for mitochondria, Mitotracker Green (MTG) and Orange (MTO), and Confocal Laser-Scanning Microscope (CLSM), we elaborated a method to measure in vivo the mitochondrial mass and activity, in sea urchin Paracentrotus lividus eggs and embryos. The analysis of captured images, revealed a variation of mitochondrial distribution and an increase of activity after fertilization.

97 citations


Journal ArticleDOI
TL;DR: Surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules and it is shown that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment.
Abstract: Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory.

97 citations


Journal ArticleDOI
TL;DR: Water extract of dried fruit of Zyzyphus Jujube was tested for its possible anticancer effect and induction of apoptosis on human tumor cell lines, HEp-2, HeLa and Jurkat cell lines and showed cytotoxic activity of ZyzophusJujube on tumor cells.
Abstract: In the present study, water extract of dried fruit of Zyzyphus Jujube was tested for its possible anticancer effect and induction of apoptosis on human tumor cell lines, HEp-2, HeLa and Jurkat cell lines. The inhibitory effect of water extract of this fruit on cell proliferation was assessed by MTT colorimetric assay. The induction of apoptosis of this extract was analyzed by DNA fragmentation analysis. Zyzyphus Jujube extract showed inhibitory effects on mentioned cell lines. Jurkat leukemic line was found the most sensitive cells with IC50 of 0.1 μg mL−1. Our study also showed a typical DNA laddering in this cell line. The present study showed cytotoxic activity of Zyzyphus Jujube on tumor cells. Although Zyzyphus Jujube has useful compounds for medical applications.

72 citations


Journal ArticleDOI
TL;DR: DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells, therefore A. hirtifolium might be a candidate for tumor suppression.
Abstract: Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.

58 citations


Journal ArticleDOI
TL;DR: The results suggest that the suppression of hepatoma cell proliferation by [6]-gingerol may be due to cell cycle arrest and apoptosis induction, and it is suggested that the anti-oxidative property of [6], Gingerol, may be involved in its anti-invasive activity of hepatomas cells.
Abstract: Effect of [6]-gingerol, a major pungent component in ginger, on the proliferation of a rat ascites hepatoma AH109A cells was investigated by measuring [3H]thymidine incorporation into acid-insoluble fraction of the cultured cells and that on the invasion by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells. [6]-Gingerol inhibited both the proliferation and invasion of hepatoma cells in a dose-dependent manner at concentrations of 6.25–200 μM (proliferation) and 50–200 μM (invasion). [6]-Gingerol accumulated cells in S phase and elongated doubling time of hepatoma cells, and increased the rate of apoptosis. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. [6]-Gingerol suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with [6]-gingerol, HX and XO or with [6]-gingerol and hydrogen peroxide. Furthermore, [6]-gingerol reduced the intracellular peroxide levels in AH109A cells. These results suggest that the suppression of hepatoma cell proliferation by [6]-gingerol may be due to cell cycle arrest and apoptosis induction. They also suggest that the anti-oxidative property of [6]-gingerol may be involved in its anti-invasive activity of hepatoma cells.

56 citations


Journal ArticleDOI
TL;DR: Human epidermal keratinocytes were shown to maintain 93.0%–99.6% viability at 72 h under medium perfusion ranging from 0.025–0.4 μl min−1, a level not possible in 96 well plates.
Abstract: Human epidermal keratinocytes (HEK) are skin cells of primary importance in maintaining the body’s defensive barrier and are used in vitro to assess the irritation potential and toxicity of chemical compounds. Microfluidic systems hold promise for high throughput irritant and toxicity assays, but HEK growth kinetics have yet to be characterized within microscale culture chambers. This research demonstrates HEK patterning on microscale patches of Type I collagen within microfluidic channels and maintenance of these cells under constant medium perfusion for 72 h. HEK were shown to maintain 93.0%–99.6% viability at 72 h under medium perfusion ranging from 0.025–0.4 μl min−1. HEK maintained this viability while ∼100% confluent—a level not possible in 96 well plates. Microscale HEK cultures offer the ability to precisely examine the morphology, behavior and viability of individual cells which may open the door to new discoveries in toxicological screening methods and wound healing techniques.

Journal ArticleDOI
TL;DR: It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%.
Abstract: Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

Journal ArticleDOI
TL;DR: Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions and it was possible to conclude that hepatocyte growth factor, epidermal growth factors, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.
Abstract: Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Journal ArticleDOI
TL;DR: A generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfections reagent for the production of recombinant IgG is developed, showing to be reproducible at 10, 50, and 100-L scale expressing between 5 and 8 mg L−1 IgG.
Abstract: We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7–8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L−1 IgG.

Journal ArticleDOI
TL;DR: Quantitative reverse-transcription PCR analysis revealed that the expression of CXCR4, a chemokine receptor essential for migration of PGCs from the bloodstream to the gonads, was reduced after the circulating PGC stage (day 2.5).
Abstract: Primordial germ cells (PGCs) are precursors of germline cells. Although avian PGCs have been used to produce transgenic birds, their characteristics largely remain unknown. In this study, we isolated PGCs from chicken embryos at various developmental stages and analyzed the gene expression. Using the expression of stage-specific embryonic antigen-1 (SSEA-1) as a marker of chicken PGCs, we purified PGCs from embryos by fluorescence-activated cell sorting after incubation for 2.5–8.5 days. The number of SSEA-1+ cells was almost unchanged during days 2.5–8.5 of incubation in females but continuously increased in male. Expression of several genes, including Blimp1, SOX2, and CXCR4, was observed in SSEA-1+ cells but not in SSEA-1− cells in both female and male embryos. Quantitative reverse-transcription PCR analysis revealed that the expression of CXCR4, a chemokine receptor essential for migration of PGCs from the bloodstream to the gonads, was reduced after the circulating PGC stage (day 2.5).

Journal ArticleDOI
TL;DR: Efficient protein delivery with a non-peptide based reagent is demonstrated, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts and a step-by-step method to optimize conditions for successful intracellular protein delivery is presented.
Abstract: Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.

Journal ArticleDOI
TL;DR: These and other biotechnological approaches for the expansion of hematopoietic progenitor progenitors and their utility to study several aspects ofHematopOietic stem cell biology are discussed here.
Abstract: Umbilical cord blood transplantation is clinically limited by its low progenitor cell content. Ex vivo expansion has become an alternative to increase the cell dose available for transplants. Expansion has been evaluated in several ways such as static cultures combining growth factors or mimicking the natural microenvironment using co-culture systems. However, static cultures have a small volume capacity and therefore large-scale expansion has been addressed using bioreactors. These and other biotechnological approaches for the expansion of hematopoietic progenitors and their utility to study several aspects of hematopoietic stem cell biology are discussed here.

Journal ArticleDOI
TL;DR: Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes and induced a significant increase of chromosomal aberrations at all the concentrations and treatment periods tested.
Abstract: Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 μg ml−1 concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 μg ml−1 concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant.

Journal ArticleDOI
TL;DR: The results show that use of DMEM:F12 and several-fold lower than conventional concentrations of KOSR can efficiently support maintenance of mESC and that K OSR should be dose as well as lot optimized.
Abstract: The expansion of stem cell numbers while retaining their developmental properties is a bioprocess challenge. We compared the growth rates and embryoid body (EB) formation yields of R1 and EFC murine embryonic stem cells (mESC) cultured in two basal media (DMEM or DMEM:F12) with additions of 1.7–15% fetal bovine serum (FBS) or serum replacer (KOSR). Whereas the basal medium or KOSR dose did not have a significant effect on growth rate for either cell line, increasing doses of KOSR had a significant negative effect on the EB yield of EFC cells. Use of DMEM:F12 and increasing doses of FBS independently and significantly increased the growth rate for both cell lines. DMEM:F12 also significantly increased EB yields for both cell lines. The results show that use of DMEM:F12 and several-fold lower than conventional concentrations of KOSR can efficiently support maintenance of mESC and that KOSR should be dose as well as lot optimized.

Journal ArticleDOI
TL;DR: Evaluating engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the unfolded protein response (UPR) is evaluated.
Abstract: Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR.

Journal ArticleDOI
TL;DR: BA showed a cytotoxic effect by decreasing the replication index (RI) and mitotic index (MI), and decreased the MI in a dose-dependent manner for the 24 h treatment period.
Abstract: The aim of this study was to determine the possible genotoxic effects of boric acid (BA) (E284), which is used as an antimicrobial agent in food, by using sister chromatid exchange (SCEs) and chromosome aberration (CAs) tests in human peripheral lymphocytes. The human lymphocytes were treated with 400, 600, 800, and 1000 μg/mL concentrations of BA dissolved in dimethyl sulfoxide (DMSO), for 24 h and 48 h treatment periods. BA did not increase the SCEs for all the concentrations and treatment periods when compared to control and solvent control (DMSO). BA induced structural and total CAs at all the tested concentrations for 24 and 48 h treatment periods. The induction of the total CAs was dose dependent for the 24 h treatment period. However, BA did not cause numerical CAs. BA showed a cytotoxic effect by decreasing the replication index (RI) and mitotic index (MI). BA decreased the MI in a dose-dependent manner for the 24 h treatment period.

Journal ArticleDOI
TL;DR: The Drosophila Schneider S2 (S2) Expression System enables expression of recombinant proteins constitutively, as well as inductively, with significant acceleration of the selection time, and a new plasmid is generated that contains both the expression cassette and puromycin selection marker in a single plasmids.
Abstract: The Drosophila Schneider S2 (S2) Expression System enables expression of recombinant proteins constitutively, as well as inductively. This system can establish both transient and stable transformants with various selection markers. The generation of stable cell lines for increased expression or large scale expression of the desired protein is currently accomplished by cotransfection of both the expression and selection vectors. The selection vectors, pCoHYGRO and pCoBLAST, are commercially available using hygromycin-B and blasticidin S, respectively. Recently, we generated a plasmid, pCoPURO, for selection of transfected S2 cells using puromycin, which allows significant acceleration of the selection time. Although co-transfection of the selection marker with the plasmid for heterologous protein expression is functional in stable expression at short culture periods, the expression levels of stable transformants are continuously decreased during long culture times. To overcome this limitation, we generated pMT-PURO, a new plasmid that contains both the expression cassette and puromycin selection marker in a single plasmid. This system allows rapid selection and maintenance of the transformed S2 lines for extended culture periods.

Journal ArticleDOI
TL;DR: The use of two food grade proteases are described to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum.
Abstract: The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.

Journal ArticleDOI
TL;DR: This research optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment.
Abstract: The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO2), or L-15 (cultured without 5% CO2) medium then cultured at 17, 27, or 37 °C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 × 108 per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO2) or L-15 (cultured without 5% CO2). The optimum culture temperature was 27 °C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.

Journal ArticleDOI
TL;DR: This study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin and aggregation of actin including disrupted fibrous actin in the cells was observed.
Abstract: A methanol extract of Alsomitra macrocarpa leaves and branches induced a marked alteration of cell morphology in a human stellate cell line (LX-2). Similar morphologic alterations were observed in several other cell lines. Active compound was purified from the extract and determined to be cucurbitacin E (Cuc E). It has been known that Cuc E causes marked disruption of the actin cytoskeleton, supporting our observation, but how Cuc E altered the actin cytoskeleton has not been elucidated. By using the standard fluorescence assay using copolymerization and depolymerization of native and pyrene labelled actin, this study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin. When NIH-3T3 cells exogenously expressing YFP-labeled actin were treated with Cuc E, firstly the aggregation of globular actin and secondly the aggregation of actin including disrupted fibrous actin in the cells was observed.

Journal ArticleDOI
TL;DR: By loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.
Abstract: Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.

Journal ArticleDOI
TL;DR: This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice and finds no reduction in cell viability after short term (<6 months) storage at −80 °C.
Abstract: Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies.

Journal ArticleDOI
TL;DR: The results remark the necessity to perform the karyotype analysis during culture in order to develop new culture conditions to maintain the correct chromosome complement in long-term culture of mESC lines.
Abstract: Although mouse embryonic stem cell lines (mESCs) have been established since 1981, systematic studies about chromosomal changes during culture are lacking. In this study, we report the results of a cytogenetic analysis performed on three mESC lines (named UPV02, UPV06 and UPV08) cultured for a period of 3 months. At time intervals, the variation of the chromosome number together with the expression of markers of the undifferentiated status, i.e., OCT-4, SSEA-1, FOM-1 and alkaline phosphatase activity, were determined. The three mESC lines showed a progressive loss of euploid metaphases during the 3 months period of culture. Chromosome abnormalities were accumulated at the latest passages analysed. Metacentric chromosomes were the most frequent chromosome abnormality observed throughout the period of culture. Interestingly, in coincidence with, or few passages after, the drop of euploidy, the alkaline phosphatase activity was partially or totally lost, whereas the OCT-4, SSEA-1 and FOM-1 stem markers were always positive throughout the period of culture. Our results remark the necessity to perform the karyotype analysis during culture in order to develop new culture conditions to maintain the correct chromosome complement in long-term culture of mESC lines.

Journal ArticleDOI
TL;DR: Bovine fibroblasts grown on aligned carbon nanotubes for a period of 2 weeks were found to have raised bodies and pronounced cell extensions for anchoring themselves to the substrate similar to that of the cells found in native tissues.
Abstract: Multiwall carbon nanotube templates formed on the surfaces of planar interdigitated microelectrode arrays by means of AC electric field-guided assembly are being explored as potential substrates for tissue engineering. The objective of the present study is to examine whether surface patterns of aligned multiwall carbon nanotubes can have an effect on cell growth, morphology, and alignment. Bovine fibroblasts grown on aligned carbon nanotubes for a period of 2 weeks were found to have raised bodies and pronounced cell extensions for anchoring themselves to the substrate similar to that of the cells found in native tissues. On the other hand, cells grown on various control surfaces had a flat, circular morphology. The cell cultures were visualized by means of SEM imaging and the resulting morphologies were statistically analyzed and compared.

Journal ArticleDOI
TL;DR: It was observed that significant aggregation of MSC into cellular clumps occurred when incubated in PBS at 0 °C, with a higher degree of aggregation occurring under calcium-free conditions, whereas no significant changes in cell viability were observed with PBS containing calcium.
Abstract: Bone marrow-derived mesenchymal stem cells (MSC) are being extensively studied as potential therapeutic agents for various diseases and have demonstrated tremendous promise to date. To reduce immunological and inflammatory reaction upon delivery of MSC in situ, the cells are often suspended in protein-free and nutrient-poor buffered saline solution at high titers and kept on ice (0 °C) until completion of the transplantation procedure. This study investigated the effects of suspending MSC (5 × 106 cells/mL) in phosphate buffered saline (PBS) with and without calcium, over a time course of 90 and 180 min, at temperatures of 0 and 37 °C. The results at 0 °C showed a small but significant decrease in cell viability within calcium-free PBS after 180 min, whereas no significant changes in cell viability were observed with PBS containing calcium. Additionally, it was observed that significant aggregation of MSC into cellular clumps occurred when incubated in PBS at 0 °C, with a higher degree of aggregation occurring under calcium-free conditions. By contrast at 37 °C, there was a more pronounced decrease in cell viability after 90 and 180 min, but lesser aggregation of MSC both in the presence and absence of calcium. The aggregation of MSC into cellular clumps could pose an embolic hazard if delivered into the arterial vasculature in cardiac applications, can clog-up injection or infusion catheters utilized for cell delivery during surgery, and can also possibly reduce the overall efficacy of transplantation therapy.