The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Automatic control of the extra-corporal bypass: system analysis, modelling and evaluation of different control modes.

Automatic control of the blood gas parameters during extracorporeal circulation has the potential to improve the quality of this procedure and to relieve the personnel from a time consuming task. This paper describes a model of the underlying system for a standard clinical set-up and pinpoints the major difficulties which are the variations of the process gains and the blood- and gas-flow dependent dead times and time constants. Scheduled PI-controllers both for the arterial oxygen as well as for the carbon dioxide partial pressure were designed. Scheduling was based on the blood flow rate. These controllers were tested in a simulation environment. The control systems remained stable under all tested operating condition, but if the blood flow rate was changed abruptly rather large load errors occurred. The performance was improved markedly by adding a feed-forward control path which directly influences the actuating signals based on the actual blood flow rate and the hemoglobin contents, variables which are measured anyway. The major conclusion of this study is to use such direct feed-forward compensation even if more sophisticated control algorithms are used.     

Some factors affecting oxygen uptake by red blood cells in the pulmonary capillaries

In this study we investigate the equations governing the transport of oxygen in pulmonary capillaries. We use a mathematical model consisting of a red blood cell completely surrounded by plasma within a cylindrical pulmonary capillary. This model takes account of convection and diffusion of oxygen through plasma, diffusion of oxygen through the red blood cell, and the reaction between oxygen and haemoglobin molecules. The velocity field within the plasma is calculated by solving the slow flow equations. We investigate the effect on the solution of the governing equations of: (i) mixed-venous blood oxygen partial pressure (the initial conditions); (ii) alveolar gas oxygen partial pressure (the boundary conditions); (iii) neglecting the convection term; and (iv) assuming an instantaneous reaction between the oxygen and haemoglobin molecules. It is found that: (a) equilibrium is reached much more rapidly for high values of mixed-venous blood and alveolar gas oxygen partial pressure; (b) the convection term has a negligible effect on the time taken to reach a prescribed degree of equilibrium; and (c) an instantaneous reaction may be assumed. Explanations are given for each of these results.     

An airlift pump device for low pressure perfusion storage of the isolated heart

A portable apparatus for the continuous hypothermic perfusion of the isolated heart is described. The system has been used successfully to store pig and baboon hearts for periods of up to 48 hr, and to store human donor hearts for periods of 7 to 17 hr before being transplanted. The perfusate is both oxygenated and circulated by gas flow from a pressurized oxygen cylinder, using the air-lift pump principle. The apparatus has no moving parts and requires no electrical energy supply; malfunction is, therefore, extremely unlikely. A regulator has been incorporated which can be adjusted to increase or decrease the myocardial perfusion pressure. The system and environmental variables which can affect flow and pressure within the apparatus are discussed. The storage time allowed by this system will enable transportation of donor hearts between most of the world's major cities.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Characterization and improvement of oxygen transfer in pilot plant external air-lift bioreactor for mycelial biomass production

The oxygen transfer dynamics in a pilot plant external air-lift bioreactor (EALB) during the cultivation of mycelial biomass were characterized with respect to hydrodynamic parameters of gas holdup (), oxygen transfer coefficient (K_La) and superficial gas velocity (U _g), and dissolved oxygen (DO). An increased flow rate of air supply was required to meet the increased oxygen demand with mycelial biomass growth. Consequently, an increase in air flow rate led to an increase in , K_La and the DO level. The enhancement of oxygen transfer rate in the cultivated broth system, however, was limited with highly increased viscosity of the mycelial broth. An increase in air flow rate from 1.25 to 2.00 v/v/m resulted in a low increment of oxygen transfer. The newly designed pilot plant EALB with two air spargers significantly improved processing reliability, aeration rate and K_La. The pilot plant EALB process, operated under a top pressure from 0 to 1.0 bars, also demonstrated a significant improvement of oxygenation efficiency by more than 20% in DO and K_La. The performance of the two sparger EALB process under top pressure demonstrated an efficient and economical aerobic system with fast mycelial growth and high biomass productivity in mycelial biomass production and wastewater treatment.     

Modelling inert gas exchange in tissue and mixed-venous blood return to the lungs

Inert gas exchange in tissue has been almost exclusively modelled by using an ordinary differential equation. The mathematical model that is used to derive this ordinary differential equation assumes that the partial pressure of an inert gas (which is proportional to the content of that gas) is a function only of time. This mathematical model does not allow for spatial variations in inert gas partial pressure. This model is also dependent only on the ratio of blood flow to tissue volume, and so does not take account of the shape of the body compartment or of the density of the capillaries that supply blood to this tissue. The partial pressure of a given inert gas in mixed-venous blood flowing back to the lungs is calculated from this ordinary differential equation. In this study, we write down the partial differential equations that allow for spatial as well as temporal variations in inert gas partial pressure in tissue. We then solve these partial differential equations and compare them to the solution of the ordinary differential equations described above. It is found that the solution of the ordinary differential equation is very different from the solution of the partial differential equation, and so the ordinary differential equation should not be used if an accurate calculation of inert gas transport to tissue is required. Further, the solution of the PDE is dependent on the shape of the body compartment and on the density of the capillaries that supply blood to this tissue. As a result, techniques that are based on the ordinary differential equation to calculate the mixed-venous blood partial pressure may be in error.     

Design and performance characteristics of a continuous multistage tower fermentor

The design of a continuous multistage tower fermentor is described. The fermentor consists of five stages separated by perforated plates. Each stage includes mechanical mixing provided by two disc turbine impellers and has its own impeller shaft with bearing assembly and flexible coupling that enables the operation of an arbitrary number of stages. The normal operation of this system enables the co-current flow of gas and liquid, but the system can function countercurrently as well. The purpose of this study was to examine the hydrodynamic performance, i.e., the pressure gradient along the tower, the mixing time, gas holdup, the residence lime distribution of the continuous phase, the value of the backflow coefficient, and the oxygen transfer rate under conditions usually used during fermentations. From the interrelations between parameters influencing the proper performance of this system, an optimal design of plate geometry for processes requiring high oxygen transfer rate was formulated.     

A novel split-cylinder airlift reactor for fed-batch cultures

Conventional airlift reactors are not adequate to carry out variable volume processes since it is not possible to achieve a proper liquid circulation in these reactors until the liquid height is higher than that of the downcomer. To carry out processes of variable volume, the use of a split-cylinder airlift reactor is proposed, in the interior of which two multi-perforated vertical baffles are installed in order to provide several points of communication between the reactor riser and downcomer. This improves the liquid circulation and mixing at any liquid volume. In fed-batch cultures, it is important to know how liquid height affects the hydrodynamic characteristics and the volumetric oxygen transfer coefficient since this impacts on the kinetic behavior of any fermentation. Thus, in the present work, the effect of the liquid height on the mixing time, the overall gas hold-up, and the volumetric oxygen transfer coefficient of the proposed airlift reactor were determined. The mixing time was increased and the volumetric oxygen transfer coefficient decreased due to the increase of the liquid height in the reactor in all the superficial gas velocities tested, which corresponded to a pseudohomogeneous flow regime. The experimental values of the mixing time and the mass-transfer coefficient were properly described through correlations in which the U_GR/H_L ratio was used as the independent variable. Thus, this variable might be used to describe the hydrodynamic behavior and the oxygen transfer coefficient of airlift reactors when such reactors are used in processes where the liquid volume changes with time. However, these correlations are useful for the particular device and for the particular operating conditions at which they were obtained. These empirical correlations are useful to understand some factors that influence the mixing time and volumetric oxygen transfer coefficient, but such correlations do not have a sufficient predictive potential for a satisfactory reactor design. The overall gas hold-up values were not significantly affected when the liquid height was lower than the downcomer height. However, the values decreased abruptly when the reactor was operated with liquid heights over the downcomer height, especially at high superficial gas velocities.     

Arterial hypoxia does not affect subchondral pressure and regional blood flow

The influence of arterial hypoxia on bone marrow pressure, regional blood flow and oxygen and carbon dioxide tensions was investigated by simultaneous and continuous measurements in the femoral condyles of 8 rabbits. Arterial hypoxia was obtained by hypoventilation. The subchondral gas tensions and regional blood flow were measured by a previously described technique based on mass spectrometry. Arterial hypoxia caused a significant decrease in subchondral oxygen tension and an increase in subchondral carbon dioxide tension. There was no significant change in bone marrow pressure and regional blood flow.     

A mathematical model of electron transfer within the mitochondrial respiratory cytochromes

A simple mathematical model of electron flow along the mitochondrial respiratory cytochrome assembly and the transfer of electrons to molecular oxygen is presented. First, an expression for the current-voltage relationship for a biological oxygen electrode is derived, and from this the relationship between oxygen consumption rate and oxygen partial pressure is determined. An independent relationship between mitochondrial oxygen partial pressure and oxygen supply rate is then derived. By eliminating oxygen partial pressure from these two expressions, we may obtain a relationship between oxygen supply rate and oxygen consumption rate. This model is then used to investigate the effects of tissue dysoxia, uncoupling of oxidative phosphorylation, increased cellular diffusional resistance and inhomogeneities in oxygen supply on oxygen consumption. It is concluded that each of the above contribute in varying degrees to the phenomenon of "pathological oxygen supply dependency".     

The influence of pressure on the growth of methanotrophic bacteria

The growth rate and the maximum cell concentration of methanotrophic bacteria are limited by the transfer of methane and oxygen to the culture fluid. The operation under moderate pressure results in an increase in driving force for the mass transfer of both nutrients and, therefore, in a large increase in the attainable biomass concentration. Our laboratory pressure fermenter with a volume of 12 litres operates under a system pressure of up to 0.5 MPa. In this reactor a maximum productivity of 6 g biomass/lh is achieved. However, operating under moderate system pressure and exhaust gas recycling has also disadvantages because the concentrations of the gas phase components may inhibit the growth process. From the results of the laboratory fermenter we have developed kinetic models of the influence of dissolved oxygen and carbon dioxide on the specific growth rate of the methanotrophic strain GB 25. These models are the basis for processing under increased system pressure and exhaust gas recycling.     

Gas Exchange of Algae: II. Effects of Oxygen, Helium, and Argon on the Photosynthesis of Chlorella pyrenoidosa

Changes in the oxygen partial pressure of air over the range of 8 to 258 mm of Hg did not adversely affect the photosynthetic capacity of Chlorella pyrenoidosa. Gas exchange and growth measurements remained constant for 3-week periods and were similar to air controls (oxygen pressure of 160 mm of Hg). Oxygen partial pressures of 532 and 745 mm of Hg had an adverse effect on algal metabolism. Carbon dioxide consumption was 24% lower in the gas mixture containing oxygen at a pressure 532 mm of Hg than in the air control, and the growth rate was slightly reduced. Oxygen at a partial pressure of 745 mm of Hg decreased the photosynthetic rate 39% and the growth rate 37% over the corresponding rates in air. The lowered metabolic rates remained constant during 14 days of measurements, and the effect was reversible after this time. Substitution of helium or argon for the nitrogen in air had no effect on oxygen production, carbon dioxide consumption, or growth rate for 3-week periods. All measurements were made at a total pressure of 760 mm of Hg, and all gas mixtures were enriched with 2% carbon dioxide. Thus, the physiological functioning and reliability of a photosynthetic gas exchanger should not be adversely affected by: (i) oxygen partial pressures ranging from 8 to 258 mm of Hg; (ii) the use of pure oxygen at reduced total pressure (155 to 258 mm of Hg) unless pressure per se affects photosynthesis, or (iii) the inclusion of helium or argon in the gas environment (up to a partial pressure of 595 mm of Hg).     

The application of fluidics technology for organ preservation

A prototype design of a portable, pulsatile, perfusion preservation device based on a novel application of fluidics technology was tested to evaluate its ability to oxygenate preservation solution and to examine the relationship between organ resistance, perfusion pressure, and perfusion flow characteristics. The effects of organ resistance on pulse rate, perfusion pressure, and perfusion flow were modeled. Interstitial PO2 in canine hearts stored at 4 degrees C for 12 hours in the fluidics device (n = 5) and in static hypothermic storage (n = 5) was also compared. Increasing outflow resistance did not have an effect on operating frequency of the fluidics actuator. Perfusion pressure rose as outflow resistance was increased, and the flow of preservation solution decreased proportionately. The PO2 of the preservation solution increased to 300 mm Hg in two hours and reached a plateau that exceeded 400 mm Hg within six hours. The aortic flow profile during pulsatile perfusion resembled a square wave function with a mean pulse duration of 0.30 +/- 0.05 seconds. Oxygen delivery by the fluidics perfusion device exceeded the oxygen requirements of the hypothermically preserved organs at all resistance levels. Initial interstitial PO2 in the hearts of both groups was greater than 150 mm Hg. In perfused hearts, PO2 declined 30% by the 12th hour, whereas complete depletion of oxygen was noted in the static storage group within six hours. The fluidics organ perfusion/transport apparatus weighs less than 18 kg, uses no electrical power, and can operate continuously for 10 to 12 hours expending 780 L of oxygen.     

The Diffusion of Oxygen, Carbon Dioxide, and Inert Gas in Flowing Blood

Measurements were made of exchange rates of oxygen, carbon dioxide, and krypton-85 with blood at 37.5°C. Gas transfer took place across a 1 mil silicone rubber membrane. The blood was in a rotating disk boundary layer flow, and the controlling resistance to transfer was the concentration boundary layer. Measured rates were compared with rates predicted from the equation of convective diffusion using velocities derived from the Navier-Stokes equations and diffusivities calculated from the theory for conduction in a heterogeneous medium. The measured absorption rate of krypton-85 was closely predicted by this model. Significant deposition of material onto the membrane surface, resulting in an increased transfer resistance, occurred in one experiment with blood previously used in a nonmembrane type artificial lung. The desorption rate of oxygen from blood at low P_o2¹ was up to four times the corresponding transfer rate of inert gas. This effect is described somewhat conservatively by a local equilibrium form of the convective diffusion equation. The carbon dioxide transfer rate in blood near venous conditions was about twice that of inert gas, a rate significantly greater than predicted by the local equilibrium theory. It should be possible to apply these theoretical methods to predict exchange rates with blood flowing in systems of other geometries.     

吸氧对椎管内麻醉下剖宫产术后疼痛的影响

目的:评价麻醉前和术中持续吸氧对椎管内麻醉下剖宫产术后疼痛的效果。方法:选择ASAI-II级择期行剖宫产手术的初产妇100例,将其随机分为面罩吸氧组和空气吸入组(对照组)。吸氧组于术前30 min及术中通过面罩全程给氧,吸入氧浓度为60%,空气组则不给予特殊处理。检测和比较两组产妇不同时点的心率、血压及SpO2的变化,手术时间,视觉模拟评分(VAS),新生儿Apgar评分,胎儿氧饱和度,新生儿脐动静脉血气,产妇血气以及术后24 h内恶心呕吐的发生率。结果:两组产妇各时间点心率、血压、SpO2、手术时间及新生儿Apgar评分、胎儿氧饱和度比较均无显著性差异(P0.05)。吸氧组术后6 h、12 h、24 h的VAS评分分别为(4.07±0.10)、(2.13±0.12)和(0.42±0.08),均明显低于对照组的(6.10±0.11)、(4.02±0.13)及(1.10±0.22)(P0.05)。吸氧组新生儿脐动静脉血气、产妇血气氧分压均显著高于对照组(P0.05),术后24h内恶心呕吐的发生率显著低于对照组(P0.05)。结论:麻醉前和术中持续吸氧能显著减轻椎管内麻醉下剖宫产术后疼痛,同时有效降低术后恶心呕吐的发生率。     

Network Scale Modeling of Lymph Transport and Its Effective Pumping Parameters

The lymphatic system is an open-ended network of vessels that run in parallel to the blood circulation system. These vessels are present in almost all of the tissues of the body to remove excess fluid. Similar to blood vessels, lymphatic vessels are found in branched arrangements. Due to the complexity of experiments on lymphatic networks and the difficulty to control the important functional parameters in these setups, computational modeling becomes an effective and essential means of understanding lymphatic network pumping dynamics. Here we aimed to determine the effect of pumping coordination in branched network structures on the regulation of lymph flow. Lymphatic vessel networks were created by building upon our previous lumped-parameter model of lymphangions in series. In our network model, each vessel is itself divided into multiple lymphangions by lymphatic valves that help maintain forward flow. Vessel junctions are modeled by equating the pressures and balancing mass flows. Our results demonstrated that a 1.5 s rest-period between contractions optimizes the flow rate. A time delay between contractions of lymphangions at the junction of branches provided an advantage over synchronous pumping, but additional time delays within individual vessels only increased the flow rate for adverse pressure differences greater than 10.5 cmH₂O. Additionally, we quantified the pumping capability of the system under increasing levels of steady transmural pressure and outflow pressure for different network sizes. We observed that peak flow rates normally occurred under transmural pressures between 2 to 4 cmH₂O (for multiple pressure differences and network sizes). Networks with 10 lymphangions per vessel had the highest pumping capability under a wide range of adverse pressure differences. For favorable pressure differences, pumping was more efficient with fewer lymphangions. These findings are valuable for translating experimental measurements from the single lymphangion level to tissue and organ scales.     

Short duration hyperbaric oxygen treatment effects blood flow in rats: pilot observations

Hyperbaric oxygen (HBO) treatment has been found to improve healing in living tissues, especially those poor in oxygen. The effects of HBO have also been tested in rat experiments. However, oxygen partial pressure in rat's arterial blood is normally about twice that in humans. Disregarding this, a human HBO protocol has been applied in previous rat experiments with HBO. Laser Doppler flowmetry (LDF) is a non-invasive means for measuring blood flow. Using LDF, we measured the blood perfusion rate in rats receiving HBO, according to a modified protocol, in a region of healing soft tissue with bone defect. The results indicate that, in rats, shorter HBO treatment with high O2 pressure can significantly improve the blood flow of healing tissues. In this study, an elevated blood perfusion rate was still evident 2 weeks after the ending of HBO therapy, which indicates improved revascularization in the wound area. A short HBO protocol would save time and effort in future HBO experiments on rats.     

呼吸机无创通气治疗急性心源性肺水肿临床研究

目的探讨大型多功能呼吸机及双相气道正压通气呼吸机治疗急性心源性肺水肿的临床价值。方法将48例急性心源性肺水肿患者分为无创通气治疗组（24例）和对照组（24例）,观察治疗前、后1h两组的血气分析及相关的症状、体征及病情缓解分值并进行统计学配对比较处理。结果治疗前各匹配组分析P〉0.05,说明两组基础病情具有可比性;治疗后1 h动脉血氧饱和度、血氧分压、呼吸频率、心率及缓解积分各组比较P〈0.01。结论表明治疗组较对照组的心肺功能改善更明显,由此表明该方法有极高的临床应用价值。     

A Mathematical Model of the Controlled Plant of the Réspiratory System

Ability to predict the dynamic response of oxygen, carbon dioxide tensions, and pH in blood and tissues to abrupt changes in ventilation is important in the mathematical modeling of the respiratory system. In this study, the controlled plant (the amount and distribution of O₂ and CO₂) of the respiratory system is modeled. Although the body tissues are divided into a finite number of “compartments” (three tissue groups), in contrast to earlier models, the blood and tissue gas tensions within each compartment are considered to be continuously distributed in time and in one spatial coordinate. The mass conservation equations for oxygen and carbon dioxide involved in the blood-tissue gas exchange are described by a set of partial differential equations which take into account convection of O₂ and CO₂ caused by the flow of blood as well as diffusion due to local tension gradients. Nonlinear algebraic equations for the dissociation curves, which take into account the Haldane and Bohr effects in blood, are used to obtain the relationships between concentrations and partial pressures. Time-variable delays caused by the arterial and venous transport of the respiratory gases are also included. The model so constructed successfully reproduced actual O₂ and CO₂ tensions in arterial blood, and in muscle venous and mixed venous blood when ventilation was abruptly changed.