Is the Rehydrin TrDr3 from Tortula ruralis associated with tolerance to cold, salinity, and reduced pH? Physiological evaluation of the TrDr3-orthologue, HdeD from Escherichia coli in response to abiotic stress.

We have employed EST analysis in the resurrection moss Tortula ruralis to discover genes that control vegetative desiccation tolerance and describe the characterization of the EST-derived cDNA TrDr3 ( Tortula ruralis desiccation-stress related). The deduced polypeptide TRDR3 has a predicted molecular mass of 25.5 kDa, predicted pI of 6.7, and six transmembrane helical domains. Preliminary expression analyses demonstrate that the TrDr3 transcript ratio increases in response to slow desiccation relative to the hydrated control in both total and polysomal mRNA (mRNP fraction), which classifies TrDr3 as a rehydrin. Bioinformatic searches of the electronic databases reveal that Tortula TRDR3 shares significant similarities to the hdeD gene product ( HNS- dependent expression) from Escherichia coli. The function of the HdeD protein in E. coli is unknown, but it is postulated to be involved in a mechanism of acid stress defence. To establish the role of E. coli HdeD in abiotic stress tolerance, we determined the log survival percentage from shaking cultures of wild-type bacteria and the isogenic hdeD deletion strain (Delta hdeD) in the presence of low temperature (28 degrees C), elevated NaCl (5 % (w/v)), or decreased pH (4.5), or all treatments simultaneously. The Delta hdeD deletion strain was less sensitive, as compared to wild-type E. coli, in response to decreased pH ( p > 0.009), and the combination of all three stresses ( p > 0.0001).

Enhanced retroviral gene delivery in ultrasonic standing wave fields.

Enhancement of retroviral transduction efficiency has been achieved by several physical and chemical approaches. However, the application of those methods is hampered by not easily scalable configurations. In this study, instead of looking into the effect of sonoporation, the potential of ultrasonic standing wave fields (USWF) to facilitate retroviral transduction rate was explored. We reasoned that, driven by the primary acoustic radiation force, suspended cells moved to the pressure nodal planes first and formed cell bands. Nanometer-sized retroviruses, circulated between nodal planes by acoustic microstreaming, then used the preformed cell bands as the nucleating sites to attach on. As a result, the encounter opportunity between retroviruses and cells was increased and further facilitated the gene delivery efficiency. Our results showed that mega-Hertz USWF brought K562 erythroleukemia cells (10(6) cells/ml) and vesicular stomatitis virus G-protein (VSV-G) pseudotyped retroviruses (titer of 5 x 10(6) CFU/ml) into close contact at the pressure nodal planes, yielding a four-fold increment of enhanced green fluorescent protein transgene expression after 5-min USWF exposure in the presence of Polybrene. Furthermore, with a fixed titer of retrovirus, the transduction rate was augmented with the increase of cell concentration. In summary, USWF offer a feasible means to enhance retroviral transduction efficiency in large-scale settings.

Diminution of phagocytosed perfluorocarbon emulsions using perfluoroalkylated polyethylene glycol surfactant.

Perfluorocarbon emulsions have been considered as potential blood substitutes for years due to their high capacity of dissolving respiratory oxygen and carbon dioxide. However, they have been reported to associate with side effects (e.g., flu-like syndrome) after being injected into animal's bloodstream. The cause of these side effects is related to the phagocytosis of perfluorocarbon emulsions by cells (e.g., macrophages). Inspired by the approach of using polyethylene glycol (PEG) to camouflage liposomes, we synthesized a perfluoroalkylated PEG (R(F)-PEG) surfactant to provide steric hindrance for decreasing phagocytosis of perfluorocarbon emulsions. The R(F)-PEG surfactant along with Pluronic F-68 and egg yolk phospholipid mediated perfluorocarbon emulsions were incubated individually with J774A.1 macrophages to examine the degree of phagocytosis. 19F NMR studies were used to quantitatively determine the amount of perfluorocarbon emulsions phagocytosed by macrophages. Results showed that the degree of phagocytosis was diminished to a large extent for perfluorocarbon microparticles emulsified by the R(F)-PEG surfactant.

Impact of cell growth morphology on retroviral transduction: effect of contact inhibition.

Retrovirus-mediated gene transfer is one of the most commonly used methods to deliver, integrate, and express the gene of interest because the retrovirus can insert the desired gene into the chromosome of the target cells with high stability. However, to deliver the gene successfully, the retrovirus requires active division to integrate reversely transcribed DNA into the chromosome of target cells. In this study, we focused on the effect of cell-cell contact inhibition on the efficiency of retroviral transduction with two anchorage-dependent cell lines: NIH 3T3 and 293 cells. These two cell lines have very different cell morphologies and growth patterns on surfaces. Human embryonic kidney epithelial 293 cells tend to stick together after dividing, while NIH 3T3 cells migrate to occupy available surface and spread. Experimental data indicate that the abatement of the transduction rate of 293 cells was initiated in the early stage of the culture, whereas effect of contact inhibition of NIH 3T3 cells on the transduction rate became dominating at the end of the culture period. Experimental results were also quantitatively illustrated by plotting normalized multiplicity of infection (MOI) versus normalized cell density. According to the outcomes, cell inoculation density plays an important role in optimizing the retroviral transduction rate. The optimal time of retroviral transduction should be confined to the accelerating growth phase for 293 cells and at the exponential growth phase for NIH 3T3 cells. The implication drawn from this study is that contact inhibition effect on retroviral transduction should be taken into account for large-scale gene transfer systems such as the microcarrier bioreactor.

Enhancement of microalgal growth by using perfluorocarbon as oxygen carrier.

The contribution of green microalgae has been recognized in the production of useful products such as chemicals, fatty acids, proteins, carotenoids, pigments, polysaccharides, and pharmaceuticals. One of the challenges to the development of profitable bioproduct markets is the design, development, modeling, and evaluation of cost-effective production systems. The photobioreactors for microalgae can be either open or closed systems in large-scale production. In various situations, it has been reported that closed photobioreactors offer many advantages over open systems. These advantages include lower contamination, higher biomass densities, and better process control. Nevertheless, for the scale-up of enclosed tubular photobioreactors, the accumulation of oxygen as a photosynthetic byproduct has been a major limitation because it severely inhibits growth of microalgae. In this study, we use the distinctive feature of liquid perfluorocarbons (PFCs) as a carrier that efficiently removes the accumulated oxygen from algal medium phase in the spinner culture flasks. The results show the growth kinetics of microalgae cultivated by this approach is significantly improved.

Enhanced retroviral transduction of 293 cells cultured on liquid-liquid interfaces.

In clinical research, retrovirus-mediated gene therapy is one of the most commonly used methods to deliver and express the gene of interest due its ability to allow for stable gene integration into the chromosomes of target cells. To elevate the efficiency of viral transduction, several restrictions, such as low virus-cell encounters and the necessity for cell division, must be improved. In this study, we focused on the possibility of accelerating cell division and the ensuing increment of viral transduction on flexible substrata. Perfluorocarbon FC-40 was harnessed to form a liquid-liquid interface with culture medium. Enhanced green fluorescence protein (EGFP) was employed as the marker gene to quickly illustrate the percentage of viral infection. The results indicate that the gene transfer efficiency to 293 cells cultured on protein-precoated liquid-liquid interfaces was higher than in cells cultured on rigid polystyrene surfaces. This increased transduction rate on the liquid-liquid interface is consistent with the acceleration of division of 293 cells on a flexible interface, which exhibited less adhesiveness. The effect of cell-cell contact inhibition on the rate of gene transduction is also addressed in this study.

Fluoroalkylated polyethylene glycol as potential surfactant for perfluorocarbon emulsion.

So far, perfluorocarbon (PFC) emulsions have been manufactured based mainly on two surfactants, Pluronic F-68 and egg yolk phospholipids (EYP) for clinical use. However, they have been documented to induce inflammatory or allergic responses when PFC emulsions were injected into human bloodstream. The cause of these side effects is associated with the phagocytosis of emulsified PFC microparticles by cells such as macrophages. In order to lessen the side effects, it is logic to develop surfactants, which are more phagocytosis-resistant and biocompatible. In this study, a perfluoroalkylated polyethylene glycol (R(F)-PEG) surfactant was synthesized by reacting perfluorooctanoyl chloride (C7F15COCl) with PEG of molecular weigh 8000. Both R(F)-PEG 8000 and EYP were used to make PFC emulsions separately by an ultrasonic homogenizer. Individual PFC emulsions were then incubated with mouse macrophage J774A.1 cells to examine the degree of phagocytosis. From microscopic observation of cell morphology, our results showed that the process of phagocytosis was retarded to a large extend using the R(F)-PEG surfactant. We also harnessed 19F-NMR to quantitatively detect the amount of PFC emulsions phagocytosed by J774A.1 cells. 19F-NMR result was consistent with the qualitative microscopic observation aforementioned.

Unilineage model of hematopoiesis predicts self-renewal of stem and progenitor cells based on ex vivo growth data.

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

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.

Determination of specific oxygen uptake rates in human hematopoietic cultures and implications for bioreactor design.

Oxygen plays an important role in the cultivation of primary cells ex vivo. In this study, we used hermetically sealed tissue culture well inserts equipped with oxygen electrodes to measure the oxygen utilization of cultured human bone marrow mononuclear cells (BM MNCs). The oxygen uptake rate (OUR) of BM MNCs was determined during a 14-day culture in which both adherent and nonadherent cells were present. Early in the culture, the cells exhibited very low OURs. The specific OURs (uptake rate per cell) were at approximately 0.005 mumol/10(6) cells/hr shortly after the initiation of culture. The OUR then increased as the cultures developed. After about 8 to 10 days of cultivation the specific OURs had increased to 0.038 +/- 0.006 and 0.025 +/- 0.005 mumol/10(6) cells/hr for adherent and nonadherent cells, respectively, after which no further increase was observed. Based on these oxygen uptake rate data, a mathematical model of oxygen diffusion was formulated and use to investigate issues associated with hematopoietic bioreactor design, including initial cell density, medium depth, reactor configuration, and oxygen partial pressure. In situ OUR measurements confirmed predicted oxygen limitations based on the mathematical model and the experimentally determined OURs. High-density hematopoietic cultures present design challenges in terms of sufficient and uniform delivery of oxygen to an active hematopoietic culture. These challenges can be met by using parallel-plate bioreactors with thin liquid layers.