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Culture Environment (culture + environment)
Selected AbstractsThree-Dimensional Culture Environments Enhance Osteoblast DifferentiationJOURNAL OF PROSTHODONTICS, Issue 7 2008Jessica Boehrs BS Abstract Purpose: In previous work from our laboratory, we demonstrated that the three-dimensional (3D) cell cultures developed in simulated microgravity environments enhanced osseous-like aggregate formation and accelerated preosteoblast cell differentiation. Thus, as described here, we hypothesize that aggregate formation and mineralization would occur with fewer than 10 × 106 cells as previously described. Materials and Methods: Human preosteoblastic cells were cultured at different concentrations in a rotary wall vessel to simulate microgravity for 7 days. Aggregate size was assessed, and mineralization and collagen expression detected using Von Kossa and Masson Trichrome staining. Scanning electron microscopy was used for structural and elemental analysis. Immunohistochemistry was used to detect expression of the osteogenic markers BSPII and osteopontin (OP). Results: Size and calcium expression were dependent upon cultured cell number (p < 0.01). Calcium and collagen expression were detected throughout the aggregate, but organization was independent of cell number. Aggregates had similar microscopic structural patterns demonstrating organized development. Presence of BSPII and OP showed that the aggregates share common differentiation proteins with in vivo bone formation. Conclusions: These results may lead to novel bone engineering techniques associated with dental treatment. [source] Proteome analysis of the culture environment supporting undifferentiated mouse embryonic stem and germ cell growthELECTROPHORESIS, Issue 10 2007Nicolas Buhr Abstract The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth. [source] The use of probiotics in shrimp aquacultureFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2006Ali Farzanfar Abstract Shrimp aquaculture, as well as other industries, constantly requires new techniques in order to increase production yield. Modern technologies and other sciences such as biotechnology and microbiology are important tools that could lead to a higher quality and greater quantity of products. Feeding and new practices in farming usually play an important role in aquaculture, and the addition of various additives to a balanced feed formula to achieve better growth is a common practice of many fish and shrimp feed manufacturers and farmers. Probiotics, as ,bio-friendly agents' such as lactic acid bacteria and Bacillus spp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. In addition, probiotics are nonpathogenic and nontoxic microorganisms without undesirable side-effects when administered to aquatic organisms. These strains of bacteria have many other positive effects, which are described in this article. [source] In vitro studies on the influence of precultural conditioning method on osteoblast reactions of a new type of injectable calcium cement materialJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2006Feng Chai Abstract A new injectable dicalcium phosphate dehydrate (DCPD)-based cement material "PD" VitalOs Cement® was studied to elucidate the process of equilibrium occurring in the early stage of implantation. The present study investigated the pH variations of the cement sample-immersing culture medium at determined intervals, time-dependent calcium/phosphate release, cell proliferation, and vitality in the cells,cement coculture milieu, after different preculture conditionings of the samples. Measurement of pH variation showed that without renewing the medium, pH value of sample lixiviate medium first dropped and, after 70 h, gradually balanced. When medium was renewed each day, pH value of lixiviate medium first descended and, after 24 h, gradually returned to pH 7.2. The cell viability revealed an excellent cytocompatibility of the cement. Both cell proliferation and vitality test showed that the preculture conditioning treatment is important at least for good performance of osteoblasts growing on the surface of calcium phosphate hydraulic cement (CPHC) samples in vitro. The results of calcium and phosphate assays clearly showed that this cement material can continuously dissolve to release calcium and phosphate in the liquid cell culture environment. The decrease of proliferation in some experimental groups with short conditioning is due to an excess of acid, which still can have some influence on cell growth after 24 h, since the biological milieu is not continuously renewed as in in vivo conditions. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006 [source] Laminar variation in neuronal viability and trophic dependence in neocortical slicesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2001Mary M. Niblock Abstract Organotypic slices are used frequently in studies of central nervous system development and function because they provide excellent experimental access with significant preservation of cellular context and relationships. Within a slice, however, a variety of factors may cause individual classes of neurons to respond differently to the culture environment. Differences in deafferentation, cellular maturation, trophic dependence and ongoing naturally occurring cell death may produce changes in the neuronal population that are transparent to the experimenter but that could affect experimental results significantly. In this study, we examined the distribution and prevalence of cell death among neurons in each cortical layer in organotypic slices. In addition, we assessed the ability of several neurotrophic factors to ameliorate neuronal death in each cortical layer. Within the first 24 hr in culture, there was striking laminar variation in the extent of neuronal death in culture, which could not be accounted for by the pattern of programmed cell death in vivo. In addition, neurons in the six layers of the neocortex differed in the degree to which they could be rescued by neurotrophic factors. These data suggest that differential neuronal death and rescue are important considerations in studies utilizing organotypic slices and may represent particularly confounding variables in studies of effects of trophic factors in such preparations. J. Neurosci. Res. 65:455,462, 2001. © 2001 Wiley-Liss, Inc. [source] Intervertebral disc cell response to dynamic compression is age and frequency dependent,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2009Casey L. Korecki Abstract The maintenance of the intervertebral disc extracellular matrix is regulated by mechanical loading, nutrition, and the accumulation of matrix proteins and cytokines that are affected by both aging and degeneration. Evidence suggests that cellular aging may lead to alterations in the quantity and quality of extracellular matrix produced. The aims of this study were to examine the role of loading and maturation (a subset of aging), and the interaction between these two factors in intervertebral disc cell gene expression and biosynthesis in a controlled 3D culture environment. Cells were isolated from young (4,6 months) and mature (18,24 months) bovine caudal annulus fibrosus and nucleus pulposus tissue. Isolated cells were seeded into alginate and dynamically compressed for 7 days at either 0.1, 1, or 3 Hz or maintained as a free-swelling control. After 7 days, DNA and sulfated glycosaminoglycan contents were analyzed along with real time, quantitative reverse transcription-polymerase chain reaction analysis for collagen types I and II, aggrecan, and matrix metalloproteinase-3 gene expression. Results suggest that maturation plays an important role in intervertebral disc homeostasis and influences the cell response to mechanical loading. While isolated intervertebral disc cells responded to mechanical compression in 3D culture, the effect of loading frequency was minimal. Altered cellular phenotype and biosynthesis rates appear to be an attribute of the cell maturation process, potentially independent of changes in cellular microenvironment associated with lost nutrition and disc degeneration. Mature cells may have a decreased capacity to create or retain extracellular matrix components in response to mechanical loading compared to young cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 800,806, 2009 [source] Investigating the importance of flow when utilizing hyaluronan scaffolds for tissue engineeringJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 2 2010Gail C. Donegan Abstract Esterified hyaluronan scaffolds offer significant advantages for tissue engineering. They are recognized by cellular receptors, interact with many other extracellular matrix proteins and their metabolism is mediated by intrinsic cellular pathways. In this study differences in the viability and structural integrity of vascular tissue models cultured on hyaluronan scaffolds under laminar flow conditions highlighted potential differences in the biodegradation kinetics, processes and end-products, depending on the culture environment. Critical factors are likely to include seeding densities and the duration and magnitude of applied biomechanical stress. Proteomic evaluation of the timing and amount of remodelling protein expression, the resulting biomechanical changes arising from this response and metabolic cell viability assay, together with examination of tissue morphology, were conducted in vascular tissue models cultured on esterified hyaluronan felt and PTFE mesh scaffolds. The vascular tissue models were derived using complete cell sheets derived from harvested and expanded umbilical cord vein cells. This seeding method utilizes high-density cell populations from the outset, while the cells are already supported by their own abundant extracellular matrix. Type I and type IV collagen expression in parallel with MMP-1 and MMP-2 expression were monitored in the tissue models over a 10 day culture period under laminar flow regimes using protein immobilization technologies. Uniaxial tensile testing and scanning electron microscopy were used to compare the resulting effects of hydrodynamic stimulation upon structural integrity, while viability assays were conducted to evaluate the effects of shear on metabolic function. The proteomic results showed that the hyaluronan felt-supported tissues expressed higher levels of all remodelling proteins than those cultured on PTFE mesh. Overall, a 21% greater expression of type I collagen, 24% higher levels of type IV collagen, 24% higher levels of MMP-1 and 34% more MMP-2 were observed during hydrodynamic stress. This was coupled with a loss of structural integrity in these models after the introduction of laminar flow, as compared to the increases in all mechanical properties observed in the PTFE mesh-supported tissues. However, under flow conditions, the hyaluronan-supported tissues showed some recovery of the viability originally lost during static culture conditions, in contrast to PTFE mesh-based models, where initial gains were followed by a decline in metabolic viability after applied shear stress. Proteomic, cell viability and mechanical testing data emphasized the need for extended in vitro evaluations to enable better understanding of multi-stage remodelling and reparative processes in tissues cultured on biodegradable scaffolds. This study also highlighted the possibility that in high-density tissue culture with a biodegradable component, dynamic conditions may be more conducive to optimal tissue development than the static environment because they facilitate the efficient removal of high concentrations of degradation end-products accumulating in the pericellular space. Copyright © 2009 John Wiley & Sons, Ltd. [source] Standardization of In Vitro Macrophotography for Assessment of Cutaneous ResponsesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009Sergio G. Coelho The increased popularity of commercially available three-dimensional human skin equivalents in recent years has allowed for assessment of melanogenesis modulated by compounds topically applied to the skin or directly incorporated from the medium. These skin equivalents provide a suitable model for elucidating the mechanisms of action of various factors that modulate skin pigmentation or other properties of the skin. As such, researchers need to objectively quantify cutaneous responses at the macroscopic level. A simple method to standardize macrophotography images is reported that can quantify cutaneous responses in human skin equivalents of Asian, Black or African American, and Caucasian or White racial/ethnic origin. Macrophotographs are analyzed using the Commission Internationale de l'Eclairage L*a*b* color space system in combination with a personal computer and image editing software. Pigmentation changes monitored over a 9 day period showed a high correlation with melanin content evaluated in Fontana,Masson-stained sections. These results indicate the feasibility of using a macrophotography setup in a sterile tissue culture environment to objectively assess in vitro cutaneous responses in human skin equivalents. This serves as an adjunct tool to biochemical and morphological methods to effectively quantify changes in pigmentation over time. [source] Improving in vitro Maturation of Oocytes in the Human Taking Lessons from Experiences in Animal SpeciesREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2001J Smitz One to three per cent of infertile women develop severe ovarian hyperstimulation syndrome after superovulation for assisted reproduction treatment (ART). This severe complication can be avoided when oocytes are obtained at an immature stage (germinal vesicle stage) out of small or medium-sized follicles. This hypothesis has been tested in several infertile women, but clinical pregnancies are disappointlingly low. This new approach in ART is still at an experimental phase and this treatment has still to be improved before routine clinical application. Experimental work in animals and humans suggest a beneficial effect in providing a short preliminary pretreatment with follicle-stimulating hormone to select for a developing cohort of follicles. The aspiration of oocyte cumulus complexes is carried out with a short needle applying reduced aspiration pressure. A crucial point is to provide the appropriate culture environment for the immature oocytes. An optimal cumulus-enclosed human oocyte culture system needs to be defined. The composition of the culture medium could be suggested by in vitro work carried out in animal models. As developmental competence is established during the latest phases of oocyte growth and is dependent on the storage of RNA, a prolonged in vitro maturation period (before inducing nuclear maturation) could provide the necessary transcriptional and translational changes. The conditions to achieve this improved cytoplasmic maturation by prolonging the in vitro culture remain to be defined. More objective noninvasive parameters for oocyte maturity are also needed to pursue research in this field. [source] Cloning Adult Farm Animals: A Review of the Possibilities and Problems Associated with Somatic Cell Nuclear TransferAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2003J. L. Edwards In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of ,pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans. [source] Effects of stocking density on the nutrient budget and growth of the western king prawn (Penaeus latisulcatus Kishinouye) in a recirculating aquaculture systemAQUACULTURE RESEARCH, Issue 10 2010Le Van Khoi Abstract Intensification in the commercial culture of prawns can have a significant impact on the water quality and hence on the survival, growth and the surrounding environment. The present study aims to evaluate the effects of stocking density on the water quality and performance of the western king prawns (Penaeus latisulcatus) and the nutrient budget of the culture environment. Four stocking densities of 4, 8, 16 and 32 prawns m,2 were tested in 12 recirculating systems. Prawn weight and specific growth rate increased with decreasing stocking density, while the survival rate showed the reverse trend. The mean total ammonia nitrogen, nitrate nitrogen, total phosphorus and soluble reactive phosphorus were significantly higher (P<0.05) at the higher stocking density. The nutrient budget revealed that the prawns could assimilate only 9.34,20.13% nitrogen and 4.97,11.25% phosphorus of the total nutrient inputs. The drained water at harvest was the major sink of phosphorus and nitrogen at stocking densities of 4, 8 and 16, which accounted for 45.59,64.82% and 44.28,65.62% of the total inputs, respectively, while a significant proportion of nitrogen sunk into the sediment at 32 prawns m,2. The study suggested that the stocking densities of western king prawns can be up to 16 prawns m,2 in the recirculating water environment. [source] Protein requirements of Nile tilapia (Oreochromis niloticus) fry cultured at different salinitiesAQUACULTURE RESEARCH, Issue 8 2010Edvino Larumbe-Morán Abstract Effect of isolipidic (62.7 ± 5.0 g kg,1) diets with protein levels of 204.6 (T20), 302.3 (T30), 424.6 (T40) or 511.0 g kg,1 (T50) on growth and survival in Nile tilapia (Oreochromis niloticus Linnaeus 1758) fry cultured for 70 days at one of four salinities (0, 15, 20 and 25 g L,1) was evaluated. A bifactorial (4 × 4) design was used with 16 treatments run in triplicate and 20 fry (0.25 ± 0.04 g) per replicate under semi-controlled conditions. Four independent, recirculating systems (one per salinity level) were used, each one with 12 circular tanks (70 L capacity), filters and constant aeration. The different salinities had no significant effect on growth. Weight gain improved significantly as dietary protein content increased, although organisms fed the T50 diet had a lower growth rate. Survival was highest (98.33%) in the T50/15 (protein/salinity levels) treatment and lowest (71.0%) in the T20/20 treatment, with no pattern caused by the variables. The T40/25, T40/20 and T50/0 treatments produced the most efficient growth and feed utilization values while the T20 treatments at all the salinities resulted with the lowest performance. With the exception of the T50 treatments, a non-significant tendency to increased weight gain was observed as water salinity increased, suggesting that the salinity of the culture environment does not influence dietary protein requirements in Nile tilapia O. niloticus fry. [source] Growth and digestive enzymes of Macrobrachium rosenbergii juveniles: effect of different stocktypes and dietary protein levels under a similar culture environmentAQUACULTURE RESEARCH, Issue 12 2009Vidya Sagar Abstract A feeding trial was conducted to study the effect of dietary protein (DP) levels on the growth and digestive enzyme activities of different wild stocks of Macrobrachium rosenbergii juveniles. Wild juveniles of M. rosenbergii were collected from the west coast of India, Gujarat (G), Maharashtra (M) and from the east coast of India, Andhra Pradesh (A), and raised in culture ponds of 200 m2 at 1 juvenile m,2. All the animals were tagged individually with Elastomer tags of a particular colour assigned to their respective stock and acclimatized for 7 days before being released into the pond at a ratio of 70:65:65 (A:M:G). Each of the two feeds, the first with 27% DP, termed the suboptimum level (S), and the second 32% DP, termed the optimum level (O), was fed in duplicate ponds at 6% of the body mass for the first 30 days and 4% for the last 30 days. The average weight of stocked prawn, respectively, in O DP and S DP fed ponds was 0.90 ± 0.04 and 1.06 ± 0.08 g for the G stock, 0.80 ± 0.07 and 1.01 ± 0.1 g for the M stock and 3.06 ± 0.13 and 3.10 ± 0.23 g for the A stock. Both the protein level and the stock type had a significant (P<0.05) effect on the weight gain% of the prawn. There was an approximate 95% change in weight gain with a DP change. Similarly, G and M stocks exhibited significantly higher (P<0.05) growth rates of approximately 90% than the A stock, although no difference was noted between the G and the M stocks. However, for protein × stock (interaction) levels, there was no significant difference (P>0.05) among the groups. Although insignificant, the survival rates among the different stocks varied from 56% to 77%. Optimum protein level showed a significant increase (P<0.05) in the specific growth rate (SGR). Feed conversion ratio, feed efficiency ratio, protein efficiency ratio and net protein utilization were not affected either due to DP, stock type or their interaction. The O × A group exhibited the maximum variation in body weight. Digestive enzyme activities were similar in all the groups, but enzymes for phospho-monoesterase were significantly higher (P<0.05) at O DP. Both the G and the M stock showed a significantly higher (P<0.05) alkaline phosphatase activity while acid phosphatase activity was significantly higher (P<0.05) in the M stock only. Overall, the G and M stocks showed higher growth responses compared with the A stock. [source] Effect of settled sludge on dissolved ammonia concentration in tanks used to grow abalone (Haliotis midae L.) fed a formulated dietAQUACULTURE RESEARCH, Issue 2 2009Rowan D Yearsley Abstract The relative contribution that solid waste or ,sludge', which accumulates at the bottom of abalone (Haliotis midae L.) tanks, makes to dissolved ammonia has not been established. Sludge was allowed to accumulate in 10 fully stocked abalone tanks, fed a formulated feed (Abfeed®; Marifeed, South Africa), for 24 days. Sludge was subsequently siphoned from five of these tanks. Total ammonia nitrogen (TAN) production and toxic, free ammonia nitrogen (FAN) were recorded in the tanks from which sludge was removed and compared with those from which sludge was not removed over the subsequent 50 h. Tanks with neither abalone nor sludge present were used as a control. The mean production of TAN (±standard deviation) was an average of 44% higher in tanks from which sludge was not removed compared with those from which it was, indicating that the sludge was a significant contributor to dissolved ammonia in the tanks. The toxic FAN concentrations were correspondingly higher in tanks with sludge present (2.3±0.3 ,L,1) compared with cleaned tanks (1.9±0.1 ,L,1). Our results indicate that regular removal of sludge from abalone tanks should significantly reduce the dissolved ammonia levels, thereby improving water quality in the culture environment. [source] Combined effects of water exchange regimes and calcium carbonate additions on growth and survival of hatchery-reared juvenile spotted babylon (Babylonia areolata Link 1807) in recirculating grow-out systemAQUACULTURE RESEARCH, Issue 7 2006S Kritsanapuntu Abstract To determine a suitable culture environment to maximize growth and survival, the hatchery-reared juvenile spotted babylon, Babylonia areolata, were held in plastic rearing tanks at four calcium carbonate additions of 0, 100 and 300 g tonne,1, and four water exchange regimes of 0-, 15-, 30- and 60-day intervals in a recirculating grow-out system for 120 days. The results clearly showed that growth was greatest between water exchange regimes of 15- and 30-day intervals and all calcium carbonate additions, with water exchange regimes of 0- and 60-day intervals resulting in poor growth. Final survival was highest between water exchange regimes of 15- and 30-day intervals, and all calcium carbonate additions, with water exchange regimes of 0-day intervals and all calcium carbonate additions resulting in high mortalities. This study showed that water exchange regimes had a stronger influence on the growth of juvenile B. areolata than calcium carbonate additions. It is recommended that B. areolata juveniles be maintained within the water exchange regimes range of 15,30-day intervals and at calcium carbonate additions between 0 and 500 g tonne,1, providing optimum conditions for production of this species in a recirculating grow-out system. [source] Development of two cell culture systems from Asian seabass Lates calcarifer (Bloch)AQUACULTURE RESEARCH, Issue 1 2006Wazir S Lakra Abstract Two new cell culture systems namely epitheloid cells of Lates (LCE) and fibroblastic cells of Lates (LCF) have been developed from fry and fingerling of the economically important brackishwater fish Lates calcarifer. Primary cultures were initiated by explant technique using caudal fin of fingerling and whole body tissue of the fry. The nutritional requirements and the growth pattern in response to different culture environment were similar for the two cell cultures. The culture medium used was Leibovitz-15 supplemented with 20% fetal bovine serum (FBS) and 1% fish serum. The LCE comprised of epithelioid cells and LCF cells were fibroblastic. With a split ratio of 1:2, the confluency of cells was attained in 8,10 days at an incubation temperature of 28°C. The cells were found to grow well in a wide range of temperature (24,32°C) and stable at 20 and 36°C. The growth rate of LCF and LCE cells increased proportionately with the concentration of FBS from 5% to 20%. A decrease of serum level to 10% after eight subcultures produced no apparent change in cell morphology and growth rate. The viability of cells was found to be 70% when revived after a month of storage in liquid nitrogen (,196°C). [source] Design of Fibrin Matrix Composition to Enhance Endothelial Cell Growth and Extracellular Matrix Deposition for In Vitro Tissue EngineeringARTIFICIAL ORGANS, Issue 1 2009Divya Pankajakshan Abstract Tissue-engineered blood vessel substitutes should closely resemble native vessels in terms of structure, composition, mechanical properties, and function. Successful cardiovascular tissue engineering requires optimization of in vitro culture environment that would produce functional constructs. The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system to render elasticity to blood vessel wall, whereas collagen is responsible for providing mechanical strength. The objective of this study was to understand the significance of various ECM components on endothelial cell (EC) growth and tissue generation. We demonstrate that, even though fibrin is a good matrix for EC growth, fibronectin is the crucial component of the fibrin matrix that enhances EC adhesion, spreading, and proliferation. Vascular EC growth factor is known to influence in vitro growth of EC, but, so far, ECM deposition in in vitro culture has not been reported. In this study, it is shown that incorporation of a mixture of hypothalamus-derived angiogenic growth factors with fibrin matrix enhances synthesis and deposition of insoluble elastin and collagen in the matrix, within 10 days of in vitro culture. The results suggest that a carefully engineered fibrin composite matrix may support EC growth, survival, and remodeling of ECM in vitro and impart optimum properties to the construct for resisting the shear stress at the time of implantation. [source] Definition and validation of operating equations for poly(vinyl alcohol)-poly(lactide-co-glycolide) microfiltration membrane-scaffold bioreactorsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010R.J. Shipley Abstract The aim of this work is to provide operating data for biodegradable hollow fiber membrane bioreactors. The physicochemical cell culture environment can be controlled with the permeate flowrate, so this aim necessitates the provision of operating equations that enable end-users to set the pressures and feed flowrates to obtain their desired culture environment. In this paper, theoretical expressions for the pure water retentate and permeate flowrates, derived using lubrication theory, are compared against experimental data for a single fiber poly(vinyl alcohol),poly(lactide-co-glycolide) crossflow module to give values for the membrane permeability and slip. Analysis of the width of the boundary layer region where slip effects are important, together with the sensitivity of the retentate and permeate equations to the slip parameter, show that slip is insignificant for these membranes, which have a mean pore diameter of 1.1,µm. The experimental data is used to determine a membrane permeability, of k,=,1.86,×,10,16,m2, and to validate the model. It was concluded that the operating equation that relates the permeate to feed ratio, c, lumen inlet flowrate, Ql,in, lumen outlet pressure, P1, and ECS outlet pressure, P0, is (1) where A and B are constants that depend on the membrane permeability and geometry (and are given explicitly). Finally, two worked examples are presented to demonstrate how a tissue engineer can use Equation (1) to specify operating conditions for their bioreactor. Biotechnol. Bioeng. 2010;107: 382,392. © 2010 Wiley Periodicals, Inc. [source] Quantification of chemical,polymer surface interactions in microfluidic cell culture devicesBIOTECHNOLOGY PROGRESS, Issue 2 2009Hui Xu Abstract Microfluidic cell culture devices have been used for drug development, chemical analysis, and environmental pollutant detection. Because of the decreased fluid volume and increased surface area to volume ratio, interactions between device surfaces and the fluid is a key element that affects the performance and detection accuracy of microfluidic devices, particularly if fluid is recirculated by a peristaltic pump. However, this issue has not been studied in detail in a microfluidic cell culture environment. In this study, chemical loss and contaminant leakage from various polymer surfaces in a microfluidic setup were characterized. The effects of hydrophilic coating with Poly (vinyl alcohol), Pluronic® F-68, and multi-layer ionic coating were measured. We observed significant surface adsorption of estradiol, doxorubicin, and verapamil with PharMed® BPT tubing, whereas PTFE/BPT and stainless steel/BPT hybrid tubing caused less chemical loss in proportion to the fraction of BPT tubing in the hybrid system. Contaminants leaching out of the BPT tubing were found to be estrogen receptor agonists as determined by estrogen-induced green fluorescence expression in an estrogen responsive Ishikawa cell line and also caused interference with an estradiol enzyme-linked immunosorbent assay (ELISA) assay. Stainless steel/BPT hybrid tubing caused the least interference with ELISA. In summary, polymer surface and chemical interactions inside microfluidic systems should not be neglected and require careful investigations when results from a microfluidic system are compared with results from a macroscale cell culture setup. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Effects of Oxygen Transport on 3-D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures: Experiments and Mathematical ModelBIOTECHNOLOGY PROGRESS, Issue 4 2005Feng Zhao Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 ,mol/106 cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher YL/G in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering [source] Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa CellsBIOTECHNOLOGY PROGRESS, Issue 2 2005Nicole A. Bleckwenn Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus-HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6-well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage-dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell-covered microcarriers suspended in culture medium in spinner flasks, suggested a re-examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 °C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 ,g EGFP/106 infected cells. [source] An evaluation of potential diets for the culture of postpueruli spiny lobsters Panulirus argus (Palinuridae)AQUACULTURE NUTRITION, Issue 2 2009S.L. COX Abstract Provision of a suitable feed is paramount for the ongoing success of spiny lobster culture. This study compared and evaluated the performance of seven diets for first instar juvenile spiny lobster Panulirus argus [5,6 mm carapace length (CL)] based on growth rates, survival, and feed conversion ratio. Results demonstrated that a seafood-based juvenile formulation produced the fastest growth rate (3.49% weight gain day,1 and 0.90% CL increase day,1 over a 28-day period). These growth rates were also reflected by a low feed conversion rate (3.04) for this formulated feed. Similar results were also obtained for juveniles fed a frozen seafood diet, however, frozen brine shrimp, Artemia salina (both enriched and un-enriched), frozen enriched mysis shrimp, a dry pellet, and a meal-based juvenile formulation did not produce consistent growth rates. Survival rates ranged between 38% and 85% and demonstrate that juvenile lobsters have better likelihoods of survival in captive culture environments compared to the wild. Although further nutritional refinement is recommended, the results from this research have significant implication for the possible expansion of juvenile spiny lobster growout to a larger scale. [source] Survival and growth of hatchery-produced postlarvae and spat of the Caribbean scallops Argopecten nucleus and Nodipecten nodosusAQUACULTURE RESEARCH, Issue 3 2009Luz Adriana Velasco Abstract A study was made of the growth and survival of postlarvae and spat of Argopecten nucleus and Nodipecten nodosus produced in the laboratory and exposed to different conditions of settlement and culture. An evaluation was made of the different types of collection substrates (onion-bags, artificial-grass, fibreglass-plates), two types of settlement inducers (epinephrine, low temperature), and two culture environments (sea, laboratory). The results showed that larger numbers of postlarvae were obtained in artificial-grass collectors than on fibreglass-plates or in the onion-bags. The growth of the A. nucleus postlarvae was not affected by the collector type, while that of N. nodosus was higher on fibreglass-plates, and lesser in the onion-bags. Both thermal and chemical stimulation of the competent larvae resulted in recovery of a greater percentage of postlarvae. The growth of postlarvae of A. nucleus was better in groups exposed to the two methods of settlement induction, while those of N. nodosus remained unaffected by this treatment. The survival of the postlarvae and spat was greater in the laboratory than at sea except in the case of A. nucleus postlarvae attached to collectors, whose survival was similar in both environments. The growth of postlarvae and spat was greater in the sea than in the laboratory. [source] Dynamics of intracohort cannibalism in cultured fishAQUACULTURE RESEARCH, Issue 7 2002E Baras Abstract Cannibalism is a frequent phenomenon in fish, especially in culture environments where fish are unable to escape predation via habitat segregation or migration. Not all cultured fish species start to exhibit cannibalism at the same age or size, nor is cannibalism equally intense in different species or life stages. Predator to prey size ratios vary substantially between species and life stages, chiefly because cannibalism is governed by gape size limitations and allometric growth of mouthparts. The development of sense organs, hard body parts, swimming and escape capacities in both the predator and the prey also influence prey size selectivity. The dynamics of cannibalism are influenced by these, as well as by environmental, factors that have effects on feed intake, growth depensation and facilitate or complicate the displaying of cannibalistic behaviour. Knowledge about cannibalistic behaviour and the logistics of cannibalism along with environmental enhancement are prerequisites for the mitigation of cannibalism in aquaculture. Also, within the context of strain selection, it is of importance to determine whether cannibals are natural-born killers or just lottery winners. These factors are discussed, chiefly as they apply to intracohort cannibalism. In addition, guidelines are suggested for cannibalistic risk assessment, and methods for mitigation of cannibalism are discussed. [source] Three-dimensional culture systems for the expansion of pluripotent embryonic stem cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Michael P. Storm Abstract Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two-dimensional (2D) static culturing techniques are inadequate for large-scale production. The culture of mammalian cells in three-dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher-S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher-S out-performed the Solohill microcarriers. When cultured for successive passages on Cultispher-S microcarriers, mESCs maintained their pluripotency, demonstrated by self-renewal, expression of pluripotency markers and the ability to undergo multi-lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher-S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra-1,60, NANOG, and OCT-4. Our study highlights the importance of optimization of initial seeding parameters and provides proof-of-concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683,695. © 2010 Wiley Periodicals, Inc. [source] A Systematic Approach for Scale-Down Model Development and Characterization of Commercial Cell Culture ProcessesBIOTECHNOLOGY PROGRESS, Issue 3 2006Feng Li The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study. [source] | ||||||||||||||||