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  • Chemistry  (8)
  • Male
  • metabolic engineering
  • 1995-1999  (8)
  • 1
    Electronic Resource
    Electronic Resource
    Different measures of human hematopoietic cell culture performance are optimized under vastly different conditions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 505-513 
    Details
    ISSN: 0006-3592
    Keywords: bone marrow ; hematopoiesis ; perfusion ; culture optimization ; stroma ; stem cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Hematopoiesis, the formation of mature blood cells from stem (LTC-IC) and progenitor (CFU-GM) cells in the bone marrow, is a complex tissue-forming process that leads to many important physiological functionalities. Consequently, a functioning ex vivo hematopoietic system has a variety of basic scientific and clinical uses. The design and operation of such a system presents the tissue engineer with challenges and choices. In this study, three culture variables were used to control ex vivo human hematopoiesis. Systematic variation of inoculum density (ID), medium exchange interval (MEI), and the use of preformed stroma (PFS) showed that (1) all three variables significantly influenced culture performance, (2) the three variables interacted strongly, and (3) the variables could be manipulated to achieve the optimization of different performance criteria. Donor-to-donor variability in culture performance was great at low ID but was minimized at higher ID. PFS had a large positive effect on cell and CFU-GM output at low ID, but had minimal effect at higher ID. In fact, PFS caused a decrease in LTC-IC output at high ID. The effects of PFS indicated that stromal cell elements became more limiting than proliferative cell elements as ID was reduced.In cultures without PFS, maximum cell output was obtained with high ID using a short MEI, whereas the greatest cell expansion ratio was obtained at low ID with an intermediate MEI. Maximum CFU-GM output was obtained from cultures with high ID using a short to intermediate MEI, whereas the greatest CFU-GM expansion ratio was obtained at intermediate ID with an intermediate MEI. The addition of PFS altered the locations of these maxima. In general, PFS moved the maxima to lower ID, and culture output became more sensitive to MEI. Therefore, the optimization of one performance criterion always resulted in a decline of the others. This study demonstrates that ex vivo tissue function is sensitive to many culture variables in an interactive fashion and that systematic multivariable studies are required to characterize tissue function. Once the effects of individual variables and their interactions are known, this knowledge can be used to optimize tissue performance with respect to desired criteria. © 1996 John Wiley & Sons, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    Cell cycle dependence of retroviral transduction: An issue of overlapping time scales (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 272-281 
    Details
    ISSN: 0006-3592
    Keywords: gene transfer ; retrovirus ; cell cycle ; intracellular stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant retroviruses are currently used as gene delivery vehicles for the purpose of gene therapy. It is generally believed that the efficiency of retroviral transduction depends on the cell cycle status of the target cells. However, it has been reported that this is not the case for the transduction of human and murine fibroblasts, in contrast to other cell types such as lymphocytes. The predictions of a mathematical model that we constructed, offer an explanation of this contradiction, based on the dynamics of the underlying processes of target cell growth and the intracellular decay of retroviral vectors. The model suggests that the utility of synchronization experiments, that are usually employed to study cell cycle specificity, is severely limited when the time scales of the above kinetic events are comparable to each other. The predictions of the model also suggest the use of retroviral vectors as cell cycle markers, as an alternative way to detect cell cycle dependence of retroviral transduction. This method obviates the need for cell synchronization and therefore, it does not perturb the cell cycle or interfere with the life cycle of retroviral vectors. Moreover, it does not depend on the intracellular stability of retroviral vectors. Our results show that in contrast to previously reported results, transduction of murine fibroblasts is cell cycle dependent, and they are consistent with the current notion that mitosis is the phase that confers transduction susceptibility. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:272-281, 1998.
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  • 3
    Electronic Resource
    Electronic Resource
    Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 479-492 
    Details
    ISSN: 0006-3592
    Keywords: stem cell ; bioreactor ; stromal layer ; Graetz number ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10-1, 100, and 101) and P(10-2, 10-1, 100, 101, and 102). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, …, 0.1 and Gz = 0.1, …, 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions. © 1996 John Wiley & Sons, Inc.
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  • 4
    Electronic Resource
    Electronic Resource
    Membrane adsorption characteristics determine the kinetics of flow-through transductions (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 260-270 
    Details
    ISSN: 0006-3592
    Keywords: retrovirus ; gene therapy ; gene transfer ; virus adsorption ; membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Retrovirus-mediated gene transfer is currently limited by random Brownian motion of the retrovirus. This limitation can be overcome by flowing the retrovirus solution through a porous membrane that supports the target cells, leading to a significant increase in the transduction efficiency. This procedure is termed “flow-through transduction.” In this study, we characterized the effects of the fluid flowrate and the influence that membrane characteristics have on the flow-through transduction procedure. The transduction efficiencies increased with flowrate until a plateau was reached at average flow velocities exceeding 0.3 cm/h for flow times of 3 to 4 h, using a collagen-coated depth (COL) membrane. A correlation between the optimal time for maximal gene transfer using flow-through transductions and the optimal time for maximal virus activity on the membrane was found, suggesting that the membrane adsorption capacity for virus determined the amount of gene transfer that could occur.Membrane adsorption characteristics were further investigated using two different membrane types: a tracketched polyester screen (PE) membrane and the COL membrane. Flow-through transductions using the PE and COL membranes showed that a high level of gene transfer could be attained using the COL membrane while the PE membrane gave much lower transduction efficiencies. The addition of the polycation polybrene (PB) changed these results markedly, making transductions achieved on the PE membrane similar in number to those obtained on the COL membrane. Since PB is believed to influence the virus adsorption to PE membrane, these results further support the conclusion that the increase in gene transfer achieved by the flow-through transduction procedure is due to virus adsorption to the membrane. The flow-through transduction procedure thus leads to co-localization of the viral vector and the target cell that in turn leads to a high transduction efficiency. © 1996 John Wiley & Sons, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    Unilineage model of hematopoiesis predicts self-renewal of stem and progenitor cells based on ex vivo growth data (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 24-33 
    Details
    ISSN: 0006-3592
    Keywords: unilineage model ; tissue function ex vivo ; hematopoiesis ; stem cell expansion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Stem cell models are used to describe the function of several tissues. We present unilineage kinetic description of stem cell models and their application to the analysis of ex vivo hematopoietic cell expansion data. This model has the capability to simulate the total cell number and the number of cells at each stage of differentiation over time as a function of the stem cell self-renewal probability, the growth rate of each subpopulation, and the mature cell death rate. The model predicts experimental observations in perfusion-based hematopoietic bioreactor systems. To obtain net cell expansion ex vivo, the model simulations show that the stem cell self-renewal probability must exceed one-half, thus resulting in net expansion of the stem cell population. Experimental data on long-term culture-initiating cells (LTC-IC) confirm this prediction and the probability of self-renewal is estimated to be 0.62 to 0.73. This self-renewal probability, along with the death rate, define a relationship in which the apparent overall growth rate is less than the compartmental growth rate. Finally, the model predicts that cells beyond the stem cell stage of differentiation must self-renew to achieve the level of expansion within the time frame observed in experimental systems. © 1996 John Wiley & Sons, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    Parametric sensitivity of stoichiometric flux balance models applied to wild-type Escherichia coli metabolism (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 69-79 
    Details
    ISSN: 0006-3592
    Keywords: E. coli ; linear optimization ; metabolic fluxes ; stoichiometry ; sensitivity analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Stoichiometrically based flux balance models provide a method to quantify the metabolic pathway fluxes within a living cell. Predictions of flux balance models are expected to have applications in pathway engineering as well as in bioprocess design and control. These models utilize optimality principles applied to metabolic pathway stoichiometry along with the metabolic requirements for growth to determine the flux distribution in a metabolic network. A flux balance model has been developed for Escherichia coli W3110 using five experimentally determined strain-specific parameters. In this report, we determine the sensitivity of the predictions of the flux balance model to these five strain-specific parameters. Model predictions are shown to be sensitive to the two parameters describing metabolic capacity, while they are relatively insensitive to the three parameters that describe the metabolic requirements for growth. Thus, when stoichiometrically based models are formulated for additional strains one needs to measure the metabolic capacity (maximum rates of nutrient and oxygen utilization) accurately. Determination of metabolic capacity from batch experiments is relatively easy to perform. On the other hand, the harder to determine maintenance parameters need not be as accurately determined. © 1995 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260450110
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  • 7
    Electronic Resource
    Electronic Resource
    Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 605-611 
    Details
    ISSN: 0006-3592
    Keywords: Chlorella vulgaris ; algae ; elemental balancing ; medium design ; high-density cultures ; photoautotrophic growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The basic requirements for high-density photoautotrophic microalgal cultures in enclosed photobioreactors are a powerful light source and proper distribution of light, efficient gas exchange, and suitable medium composition. This article introduces the concept of balancing the elemental composition of growth medium with biomass composition to obtain high-density cultures. N-8 medium, commonly used for culturing Chlorella vulgaris was evaluated for its capacity to support high-density cultures on the basis of elemental stoichiometric composition of C. vulgaris. This analysis showed that the N-8 medium is deficient in iron, magnesium, sulfur, and nitrogen at high cell densities. N-8 medium was redesigned to contain stoichiometrically balanced quantities of the four deficient elements to support a biomass concentration of 2% (v/v). The redesigned medium, called M-8 medium, resulted in up to three- to fivefold increase in total chlorophyll content per volume of culture as compared to N-8 medium. Further experiments showed that addition of each of the four elements separately to N-8 medium did not improve culture performance and that balanced supplementation of all four deficient elements was required to yield the improved performance. Long-term (24 d) C. vulgaris culture in M-8 medium showed continuous increase in chlorophyll content and biomass throughout the period of cultivation. In contrast, the increase in chlorophyll content and biomass ceased after 7 and 12 d, respectively in N-8 medium, demonstrating the higher capacity of M-8 medium to produce biomass. Thus, the performance of high cell density photobioreactors can be significantly enhanced by proper medium design. The elemental composition of the biomass generated is an appropriate basis for medium design. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:605-611, 1998.
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  • 8
    Electronic Resource
    Electronic Resource
    How will bioinformatics influence metabolic engineering? (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 162-169 
    Details
    ISSN: 0006-3592
    Keywords: bioinformatics ; metabolic engineering ; genetic engineering ; mathematical analysis ; stoichiometry ; enzyme kinetics ; modal analysis ; genetic circuits ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:162-169, 1998.
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