3-D computational modeling of media flow through scaffolds in a perfusion bioreactor

Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro. In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs. Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture. We have used the Lattice-Boltzmann method to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds. Microcomputed tomography imaging was used to define the scaffold microarchitecture for the simulations, which produce a 3-D fluid velocity field throughout the scaffold porosity. Shear stresses were estimated at various media flow rates by multiplying the symmetric part of the gradient of the velocity field by the dynamic viscosity of the cell culture media. The shear stress algorithm was validated by modeling flow between infinite parallel plates and comparing the calculated shear stress distribution to the analytical solution. Relating the simulation results to perfusion experiments, an average surface shear stress of 5x10(-5)Pa was found to correspond to increased cell proliferation, while higher shear stresses were associated with upregulation of bone marker genes. This modeling approach can be used to compare results obtained for different perfusion bioreactor systems or different scaffold microarchitectures and may allow specific shear stresses to be determined that optimize the amount, type, or distribution of in vitro tissue growth.     

Effect of carbon dioxide on the rheological behavior of submerged cultures of Chlorella minutissima in stirred tank reactors

The objective of this study was to quantify the effect of algal biomass concentration on the rheology of the algal culture broth. Batch cultivations of Chlorella minutissima were carried out with air and carbon dioxide in a stirred tank bioreactor with a working volume of 1.8 L. The apparent viscosity of the culture broth was significantly affected by the cell mass concentrations in the bioreactor. Culture broth containing 50 g/L cell mass from air fed was found to exhibit an apparent viscosity of 1.52 mPa.s. The apparent viscosity of the carbon‐dioxide‐fed cultivations was found to increase by 20% at a shear rate of 100 s^?1. The flow behavior of the system was adequately described by the Herschel–Bulkley model with a small yield stress.     

Rheology of enzyme liquefied corn stover slurries: The effect of solids concentration on yielding and flow behavior

The measurement of yield stress and shear thinning flow behavior of slurries formed from unpretreated corn stover at solids loadings of 100–300 g/L provides a key metric for the ability to move, pump, and mix this lignocellulosic slurry, particularly since corn stover slurries represent a major potential feedstock for biorefineries. This study compared static yield stress values and flow hysteresis of corn stover slurries of 100, 150, 200, 250, and 300 g/L, after these slurries were formed by adding pellets to a cellulase enzyme solution (Celluclast 1.5 L) in a fed-batch manner. A rotational rheometer was used to quantitate relative yield stress and its dependence on processing history at insoluble solids concentrations of 4%–21% (wt/vol). Key findings confirmed previous observations that yield stress increases with solids loadings and reaches ~3000 Pa at 25% (wt/vol) solids concentration compared to ~200 Pa after enzyme liquefaction. While optimization of slurry forming (i.e., liquefaction) conditions remains to be done, metrics for quantifying liquefaction extent are needed. The method for obtaining comparative metrics is demonstrated here and shows that the yield stress, shear thinning and shear thickening flow behaviors of enzyme liquefied corn stover slurries can be analyzed using a wide-gap rheometry setup with relative measuring geometries to mimic the conditions that may exist in a mixing vessel of a bioreactor while applying controlled and precise levels of strain.     

毕赤酵母发酵产S-腺苷蛋氨酸的甘油-甲醇混合流加策略

在毕赤酵母发酵生产S-腺苷蛋氨酸(SAM)的诱导阶段,以不同甘油-甲醇比例的甘油-甲醇混合培养基进行诱导培养,结果表明以10%(w/v)甘油含量的甘油-甲醇混合培养基进行诱导培养时最有利于SAM的表达,SAM产量达6.09 g/L,比0%甘油含量条件下的SAM产量提高了20.4%。对诱导方式进行优化,先以100%甲醇诱导24 h,然后再连续流加10%(w/v)甘油含量的甘油-甲醇混合培养基,SAM产量可达7.94 g/L,在此基础上,进一步改进诱导方式,SAM产量得到进一步的提高,达到9.80 g/L。     

搅拌式生物反应器悬浮培养水母雪莲细胞的研究

应用 2L通气搅拌式生物反应器一步批式培养水母雪莲细胞。采用倾斜式搅拌桨代替透平桨 ,研究了搅拌转速、通气量和接种量对细胞生长和黄酮合成的影响 ,发现在 75r min、70 0～1000L min和 4.0～ 5.0gDCW L接种量下细胞生长和黄酮合成比较好。经过 12d培养细胞干重达 13.8gDCW L ,黄酮产量 416mg L ,黄酮含量占细胞干重的 30%。水母雪莲细胞生长及黄酮合成的进程表明 ,黄酮积累与细胞生长呈正相关。对细胞聚集体分布的研究发现 ,流变压力使细胞聚集体分裂 ,使反应器中细胞生长受到影响 ,黄酮产量较摇瓶中降低     

VERO细胞生物反应器放大培养初探

目的:研究用生物反应器放大进行Vero细胞微载体培养,实现生物反应器之间Veto细胞放大培养.方法:5L微载体生物反应器以10g/L微载体浓度培养Vero细胞,96h时经漂洗、消化、接种于30L微载体生物反应器,实现放大后的30L微载体生物反应器细胞怏速增殖,期间对不同时期的微载体细胞进行细胞计数、细胞代谢分析和形态观察.结果:5L生物反应器细胞经过96h灌注培养,平均细胞密度达到7.81×10~6cells/mL.5L微载体细胞放大到30L微载体生物反应器,平均细胞收获率为32.3%;放大到30L生物反应器后经过144h培养,细胞密度达到9.19×10~6cells/mL;放大后的细胞代谢途径依然以葡萄糖氧化代谢乳酸为主.结论:生物反应器由5L到30L进行Veto细胞放大培养是可行的.     

A non-rotational, computer-controlled suspension bioreactor for expansion of umbilical cord blood mononuclear cells

The proliferation and differentiation characteristics of umbilical cord blood mononuclear cells were examined in a non-rotational suspension bioreactor with a fishtail mixer. The system consisted of a glass vessel, a mixer that moved vertically, a data acquisition and control system to continuously monitor pH, temperature and dissolved O₂. The bioreactor provided superior expansion of total HSCs and not total cell number, as well as expression of stemness-related genes which followed with increasing in number of colony-forming cells during 14 days of culture compared to T -lask culture. Vertical agitation thus reduces the total cell number, which may be related to increased shear stress, but has no effect on HSC function.     

Limited use of Centritech Lab II Centrifuge in perfusion culture of rCHO cells for the production of recombinant antibody

Perfusion cultures of recombinant Chinese hamster ovary cells, producing recombinant antibody against the S surface antigen of Hepatitis B virus, were carried out in continuous and intermittent mode using a Centritech Lab II Centrifuge. In the continuous perfusion process, despite the absence of shear stress from the pump head, long-term operation was not possible because of continuously repeated exposure to oxygen limitation and low temperature, as well as shear stress from centrifugal force. In the intermittent perfusion processes, the frequency of cell-passage through the centrifuge was substantially reduced, compared with the continuous perfusion mode; however, the degree of reduction could not guarantee stable long-term operation. Although various operating parameters were applied in the intermittent perfusion cultures, high cell densities could not be maintained stably. In a single bioreactor culture system, a specific cell that is returned from the centrifuge to the bioreactor could be transferred from the bioreactor to the centrifuge again in the next cycle. These repetitive damages, caused by shear stress from the pump head and centrifugal force, as well as exposure to suboptimal conditions such as oxygen limitation and low temperature below 37 degrees C, were more serious at higher perfusion rates. Subsequently, damaged cells and dead cells were continuously accumulated in the bioreactor. Culture temperature shift from 37 to 33 degrees C increased antibody concentrations but showed inhibitory effects on cell growth. The negative effects of lowering culture temperature on cell growth overwhelmed the positive effects on antibody production. To protect cells from shear stress, Pluronic F-68 was 2-fold concentrated in the culture medium; nevertheless, a significantly higher concentration of Pluronic F-68 (2 g/L) may have inhibitory effects on cell growth.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Comparison of neomycin production from Streptomyces fradiae cultivation using soybean oil as the sole carbon source in an air-lift bioreactor and a stirred-tank reactor

Streptomyces fradiae was cultivated in both an air-lift bioreactor and a jar-fermentor with various agitation rates from 200 to 800 rpm to investigate differences in neomycin production between the two reactors. Final neomycin concentrations in the jar-fermentor operated at 600 rpm and the air-lift bioreactor were 3.19 and 1.39 g/l, respectively. On the other hand, levels of soybean oil consumption in the two reactors were 25.9 and 9.4 g/l, respectively. Shear stress due to mechanical agitation caused changes in the morphology of mycelia and influenced neomycin production. The morphological changes of the mycelia in the jar-fermentor caused the viscosity of the culture broth to decrease by half, and soybean oil consumption and fatty acid uptake rate to increase 3- and 1.8-fold, respectively, in comparison with those of the air-lift bioreactor. The product yield coefficient determined from the level of soybean oil consumption in the air-lift bioreactor was similar to that of the jar-fermentor at 600 rpm, but the neomycin yield was less than one-half. In the case of the jar-fermentor, the yield increased with increasing agitation rate and was maximum at 600 rpm. To maximize neomycin production in S. fradiae cultures using soybean oil as sole carbon source, it was necessary to provide a degree of shear stress to the mycelia and to optimize liquid mixing. In an air-lift bioreactor, the soybean oil consumption may be suppressed due to a low degree of liquid mixing.     

Flow behaviour of a POSS biopolymer solution

A non-biodegradable polyhedral oligomeric silsesquioxane (POSS) nanocomposite biopolymer has been developed for fabrication of medical devices and for tissue engineering human organs. The polymer in solution, containing 2 wt% of POSS, has been synthesized, characterized and investigated to determine its key rheological properties. Thus, the variation of shear stress and viscosity as a function of shear rate has been determined at ambient temperature to estimate yield stress and the index of pseudoplasticity, respectively. The temperature dependence of viscosity and the effect of ageing on the viscosity of the polymer have also been investigated. Results are compared with those of a conventional polycarbonate urethane (PCU) polymer solution. The POSS-PCU polymer solution shows near-Newtonian behaviour in the shear rate range to 1000 s(-1), having an apparent viscosity of approximately 3000 mPa s and a pseudoplasticity index of 0.90, decreasing slightly as the polymer solution is aged over 9 months. The temperature dependence of viscosity of the POSS polymer is extremely low and does not change with ageing but the yield strength increases from 2.7 Pa to 8.3 Pa.     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor

It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle‐enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·10⁸ L^?1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sⁿ at shear rate about 0.05 s^?1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 μm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5‐fold.     

Submarine pollination in the marine angiosperm Zostera marina (Zosteraceae). II. Pollen transport in flow fields and capture by stigmas

Flow chamber observations of the filamentous pollen of Zostera marina L. (Potamogetonales) revealed that pollen rotated and moved toward inflorescences where they were captured by stigmas. The mechanics of this abiotic pollination process were examined and found to be related to the flow environment around emergent flowers. The translational movement of pollen was imparted by the advection of the fluid (e.g., pollen kinetic energy, K, ranged from 0.8 x 10-14 to 2.4 x 10-14 J, and the average K of the fluid was _ 0.7 x 10-14 J), while the rotational motion was imparted by the fluid shear stress (tau) within the velocity gradient (e.g., pollen shear stress, sigmat = omegamu where omega is the rotational velocity and mu is the dynamic viscosity, ranged from 3.4 x 10-4 to 26 x 10-4 Pa, and the average fluid shear stress was tau _ 10 x 10-4 Pa; Ackerman, 1997, American Journal of Botany 84: 1099-1109). These results indicate that there is a greater potential for pollination by filamentous pollen relative to spherical pollen. Functionally, while spherical pollen needs to be directly upstream from stigmas to be captured, filamentous pollen need only be in the vicinity of inflorescences and flowers to be captured by stigmas. Thus, in addition to direct interception on stigmas, filamentous pollen can be captured while they rotate past flowers or when they are redirected through the velocity gradient towards flowers. Filamentous pollen is an adaptation to submarine pollination in seagrasses.     

Accurate prediction of wall shear stress in a stented artery: newtonian versus non-newtonian models

A significant amount of evidence linking wall shear stress to neointimal hyperplasia has been reported in the literature. As a result, numerical and experimental models have been created to study the influence of stent design on wall shear stress. Traditionally, blood has been assumed to behave as a Newtonian fluid, but recently that assumption has been challenged. The use of a linear model; however, can reduce computational cost, and allow the use of Newtonian fluids (e.g., glycerine and water) instead of a blood analog fluid in an experimental setup. Therefore, it is of interest whether a linear model can be used to accurately predict the wall shear stress caused by a non-Newtonian fluid such as blood within a stented arterial segment. The present work compares the resulting wall shear stress obtained using two linear and one nonlinear model under the same flow waveform. All numerical models are fully three-dimensional, transient, and incorporate a realistic stent geometry. It is shown that traditional linear models (based on blood's lowest viscosity limit, 3.5 Pa s) underestimate the wall shear stress within a stented arterial segment, which can lead to an overestimation of the risk of restenosis. The second linear model, which uses a characteristic viscosity (based on an average strain rate, 4.7 Pa s), results in higher wall shear stress levels, but which are still substantially below those of the nonlinear model. It is therefore shown that nonlinear models result in more accurate predictions of wall shear stress within a stented arterial segment.     

Effect of shear stress on activity of cellular enzyme in animal cell

The effect of shear stress on the activity of cellular enzyme in an animal cell was discussed by using a flow channel. The activity of lactate dehydrogenase (LDH) in cells exposed to a shear stress of 0.5 Pa for 12 h was about 4-fold greater than in the cells without exposure to shear stress. The relative LDH activity was correlated with the dissipation energy density of the flowing medium. A good correlation was obtained and it was found that the dependency of cellular enzyme activity on the shear stress and the exposure time was related to the transmission of the energy from the flowing medium to attached cells.List of Symbols b m width of flow channel - E J/m² dissipation energy density - h m distance between two plates - Q m³/s volume flow rate - t s exposure time - u m/s velocity of medium - y m distance from wall attached cells - Pa s viscosity - Pa shear stress