Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry

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), 10(0), and 10(1)) and P(10(-2), 10(-1), 10(0), 10(1), and 10(2)). 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. (c) 1996 John Wiley & Sons, Inc.     

Different measures of human hematopoietic cell culture performance are optimized under vastly different conditions

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. (c) 1996 John Wiley & Sons, Inc.     

Spatial variation in polyphenolic content of Ascophyllum nodosum (Fucales,Phaeophyta)

Spatial variation in polyphenolic content in annual shoots of the brown alga Ascophyllum nodosum was quantified using a hierarchical sampling design. Three sampling levels, covering distances of 10⁰–10⁶ m, were used. Comparisons were made between two areas, Tjärnö on the Swedish west-coast and the Isle of Man in the Irish Sea, with very different types of environmental conditions. No significant differences in mean polyphenolic levels were found between the two study areas (6.6% of dry mass at Tjärnö and 9.2% at the Isle of Man), whereas significant and substantial differences were found among sites within areas (range 5.7%–11.4%) and among quadrats within sites (range 3.7%–13.1 %). The extensive variation at the smaller spatial scales points out the importance of using thorough sampling procedures at all levels in large-scale studies on algal polyphenolics, e.g. biogeographical comparisons, which have been neglected in several previous studies. Moreover, the results imply that experiments on causal factors of polyphenolic variation should be designed to explain the spatial scales on which the factors are impportant. This study also investigated the relationship between polyphenolic concentration and both plant size and mean area of annual shoots. The mean area was used as an estimate of the mean growth rate of the annual shoots within an individual. No significant relationships were found between shoot growth rate, or plant size, and polyphenolic levels in annual shoots at any of the three spatial scales that were investigated.     

Production of N2O in soil during decomposition of dead yeast cells with different spatial distributions

Production and sources of N₂O were determined in soil columns amended with autoclaved yeast cells either mixed into or added as 0.5 cm³ lumps to the soil in combination with no or 200 g NO₃ ^--N g^-1. At four occasions over a two-week study period, subsets of cores were measured for N₂O production during 4-hour incubations under atmospheres of ambient air, 10 Pa of C₂H₂, and N₂, respectively. Denitrification enzyme activity (DEA) was assessed in subsamples of cores that had been incubated continuously under air.Autoclaved yeast provided a C-source readily available for denitrifying bacteria in the soil. Nitrous oxide production was negligible in unamended columns whereas accumulated N₂O losses in the presence of yeast material were substantial, varying between 15 to 49 ng N₂O-N g^-1 h^-1. Mixing yeast into the soil caused the highest production of N₂O followed by the yeast lump and no yeast treatments. Incubation in the presence of 10 Pa C₂H₂ indicated that denitrification was the sole source of N₂O, in accordance with an increase in DEA. Nitrous oxide production and DEA peaked after 4–7 days of incubation, and both were unaffected by additional NO₃ ^-. Two-to four-fold responses to anaerobiosis and accumulation of NO₃ ^- and NH₄ ⁺ in proximity of the lumps indicated that N₂O production here was limited by relatively low C-availability. In contrast, 10- to 12-fold responses to anaerobiosis and no accumulation of inorganic N suggested a higher C-availability where yeast was mixed into the soil.     

DNA methylation is not involved in the structural alterations of Ornithine Decarboxylase or Total Chromatin of c-Ha-rasVal 12 Oncogene-Transformed NIH-3T3 Fibroblasts

The ornithine decarboxylase (odc) gene is an early response gene, whose increased expression and relaxed chromatin structure is closely coupled to neoplastic growth. In various tumour cells, the odc gene displays hypomethylation at the sequences CCGG. Hypomethylation of genes is believed to correlate with chromatin decondensation and gene expression. Since a given pattern of DNA methylation may not be preserved in neoplastic cells, we studied the methylation status of odc gene at the CCGG sequences in c-Ha-ras^{Val 12} oncogene-transformed NIH-3T3 fibroblasts during the growth cycle and relative to their normal counterparts. We found that the methylation state of the odc gene and its promoter and mid-coding and 3' regions remain unaltered during the cell cycle. We also found that in ras oncogene-transformed cells, which display a more decondensed nucleosomal organization of chromatin than the normal cells, the CCGG sequences in bulk DNA and at the odc gene were methylated to the same extent as in the nontransformed cells. These data suggest that DNA hypomethylation at the CCGG sequences is not a prerequisite for chromatin decondensation and cell transformation by the c-Ha-ras^{Val 12} oncogene.     

Enantioselective assay of β-receptor antagonists present in microdialysis and plasma samples of rats

The enantiomers of alprenolol, metoprolol, and propranolol have been separated on an enantioselective cellulase column and analysed using a fully automated HPLC system involving coupled column chromatography and fluorescence detection. The assays had sufficient selectivity and sensitivity to investigate the disposition of these β₂-receptor antagonists in blood and brain extracellular fluid of rats. A cellulase column was used as the first column to separate the enantiomers giving separation factors between 2.9 and 4.3. After the separation, the enantiomers were trapped on two small precolumns by the use of a switching valve and were then introduced on an achiral C₁₈ analytical column by eluting the small columns backward. The enantiomers in blood and brain tissue dialysates were analysed by direct injection of 8 μl samples. The limit of quantitation was 0.025–0.4 μg/ml of the different enantiomers. Plasma samples were analysed after a simple extraction procedure. The intraassay precision of the lowest quality control plasma samples (0.2–0.8 μg rac drug/ml) was 4–8% for the different enantiomers. © 1995 Wiley-Liss, Inc.     

Optimization of reproductive effort and foraging time in mammals: The influence of resource level and predation risk

Summary The trade-off between fitness benefits from foraging and associated costs in terms of predation risk is analysed by a simple model which takes into account the differential predation risk for reproducing and non-reproducing individuals. The currency that animals are assumed to maximize is their expected absolute fitness (probability of survival plus half of the expected litter size) after a potential reproductive period. Depending on resource levels and predation risk, this maximization can be achieved by (1) opting for individual survival and behaving as a strict time minimizer, (2) by reproducing at the maximal rate and behaving as a strict energy maximizer or (3) by submaximal reproductive effort and a behaviour intermediate between time minimization and energy maximization. Small changes in the availability of food or cover or in the density of predators can shift the optimum from one strategy to another. The shift is particularly abrupt, if predation pressure increases and the availability of resources remains high. This could explain the spatial and temporal variation in the reproductive effort and body weight observed in boreal small mammals with sustained, multiannual population fluctuations.     

The PKU mutation S349P causes complete loss of catalytic activity in the recombinant phenylalanine hydroxylase enzyme

The mutation S349P in exon 10 of the phenylalanine hydroxylase (PAH) gene was identified in one Norwegian and one Polish phenylketonuria (PKU) allele on a haplotype 1.7 background. This missense mutation in PAH codon 349 is a T to C transition in cDNA position 1267. This mutation has been reported both on haplotype 1 and 4, suggesting recurrent mutation. In two different expression systems, the pET and the pMAL systems of Escherichia coli, it was shown that the S349P mutation, introduced by site directed mutagenesis, results in complete loss of enzymatic activity. Thus, protein instability alone does not seem to be the direct cause of the lack of activity of this PKU mutation as previously reported.We have identified mutations in the PAH gene of 118 PKU patients in Norway. To obtain information about how the different mutations affect the catalytic properties of the PAH enzyme we have used two prokaryotic expression systems.We detected the mutation S349P (Forrest et al. 1991) in one Norwegian patient and one of Polish ancestry. This mutation has previously been reported on haplotype 4 in North-African Jews (Weinstein et al. 1993), and on haplotype 1 in French-Canadians (John et al. 1992) and in Danes (Guldberg et al. 1993a). Here we present gene expression data showing that the recombinant mutant enzyme has no measurable residual catalytic activity.     

Mixed photo-cross-linked polyvinyl alcohol and calcium-alginate gels for cell entrapment

Living cells may be immobilized by gel entrapment under very mild conditions. The ionotropic gelation of alginate with bivalent cations such as Ca²⁺, as well as photo-induced gelation of polyvinyl alcohol (PVA) bearing photosensitive stilbazolium (SbQ) groups, are procedures that are compatible with most bioactive materials. In the search for more stable and stronger alginate gel beads, experiments have been carried out to investigate mixed gels from alginate and PVA-SbQ. The swelling capacities, diffusion properties, and potential toxic effect of the binary gel beads have been evaluated. The gel beads of selected PVA-SbQ/alginate mixtures were applied successfully as carriers in a denitrification process with continuous feeding of unsterilized water medium. Under such conditions, the purely synthetic PVA-SbQ network is expected to have a longer lifespan than a natural biopolymer such as alginate.     

Copepod grazing on a phytoplankton community containing the toxic dinoflagellate Dinophysis acuminata

Copepod grazing on the toxic dinofiagellate Dinophysis acuminatafrom the west coast of France (La Rochelle) was studied witha concentrated (40–70 µm) phytoplankton assemblagedominated by Leptocylindrus danicus, D.acuminata, Ceratium fususand Ceratium furca. Copepod nauplii were also present. Threeto five copepods/copepodites (Acartia clausi, Isias clavipesand Centropages typicus) were incubated together with the phytoplankton.Dinophysis acuminata was grazed upon by all copepod species.However, to some extent, I.clavipes and C.typicus avoided itas food. Dinophysis acuminata cells represented for them only5–10% of total ingested carbon during the first 24 h,and almost all individuals survived and thrived well. In contrast,A.clausi did not avoid D.acuminata. which represented 30% ofingested carbon in 1 day. Acartia clausi then had a lower survivalthan the two other copepod species. However, the survival ofA.clausi was high in control incubations, where a plankton communitywithout D.acuminata was used as food. It is concluded that theokadaic acid of D acuminata is potentially toxic to some grazers,and/or might function as an allelopathic grazer repellent.