Microgravity tissue engineering

Summary Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter × 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.     

旋转磁场对小麦种子萌发及幼苗生长的影响

以“淮麦19”小麦种子为研究对象,采用磁场强度为11 mT的旋转磁场进行处理,设定不同的时间梯度(10、20、30、60 min),以未经磁场处理的为对照,研究了不同处理时间下旋转磁场对小麦种子发芽指标及幼苗生理特性的影响。结果表明：旋转磁场处理对小麦种子萌发具有促进作用,不同处理时间下,种子的发芽势、发芽率、发芽指数均有显著提高,随着时间的增加呈现先上升后下降的趋势,以处理30 min为最高,分别比对照提高了8.24％、7.37％、11.24％。经旋转磁场处理后,小麦根系发达,根长、根数均显著高于对照;幼苗生长加快,株高除处理60 min外,其他处理均显著高于对照,以处理30 min为最大,高出对照11.13％;叶片数除处理10 min外,其他处理均高于对照,以处理30 min为最大,比对照高出38.89％,且差异显著。旋转磁场处理显著增加了小麦幼苗的物质积累,不同处理时间表现出不同的效果,对幼苗生物积累量影响最佳的是处理20 min。这说明旋转磁场处理提高了小麦种子的萌发能力,加快了小麦幼苗的生长速度。     

对—氯代苯胺（PCA）在填充床生物反应器中的降解作用

以氯代苯胺（ＰＣＡ）为选择基质,用驯化技术从降解对二氯苯（ｐＤＣＢ）的富集培养物中得到了以同化ＰＣＡ为唯一碳源和氮源的混合微生物．将这种固定在填充床反应器中的微生物用于ＰＣＡ的降解作用研究中．在该反应器里,ＰＣＡ的生物降解遵循Ｌｏｇｉｓｔｉｃ方程ｑ＝ｑｍａｘ／（１＋ｅα－βＵｖ）．由方程求出了主要的动力学常数,Ｋｓ（半速率常数）和ｑｍａｘ（最大比基质降解速率）．于ＰＣＡ降解的同时,释放氯离子到培养基中．在水力停留时间３ｈ,进水ＰＣＡ浓度为３６０ｍｇ·Ｌ－１情况下,基质的体积降解率达到１２５ｍｇ·Ｌ－１·ｈ－１;基质的百分去除率为９１％．     

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.     

Perfusion bioreactors for the production of recombinant proteins in insect cells

Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.     

Characterization of agitation environments in 250 ml spinner vessel, 3 L,and 20 L reactor vessels used for animal cell microcarrier culture

Three dimensional particle tracking velocimetry (3-D PTV) was used to characterize the flow fields in the impeller region of three microcarrier reactor vessels. Three typical cell culture bioreactors were chosen: 250 ml small-scale spinner vessels, 3 L bench-scale reactor, and 20 L medium-scale reactor. Conditions studied correspond to the actual operating conditions in industrial setting and were determined based on the current scale-up paradigm: the Kolmogorov eddy length criterion. In this paper we present characterization of hydrodynamics on the basis of flow structures produced because of agitation. Flow structures were determined from 3-D mean velocity results obtained using 3-D PTV. Although the impellers used in 3 L and 20 L reactors were almost identical, the flow structures produced in the two reactors differed considerably. Results indicate that near geometric scale up does not necessarily amount to scale-up of flow patterns and indicates that intensity as well as distribution of energy may vary considerably during such a scale-up.     

Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells

Production of a TGF receptor with high density baculovirus infected Sf-9 cells (7×10⁶cells ml^-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGF receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO₂ partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO₂ at this concentration has been assessed at smaller scale for TGF receptor and -gal expression, in controlled atmosphere incubators.Abbreviations CST cumulative sparging time (s) - dpi days post-infection (d) - CO₂/O₂ diffusivities ratio (=0.784 at 27 °C) - DO % saturation of dissolved oxygen (%) - hpi hours post-infection (h) - He Henry coefficient (He_{CO 2}=32.1×10^-3M atm^-1, He_{O 2}=1.23×10^-3 M atm^-1 at 27 °C) - k_La volumetric liquid-side mass transfer coefficient (h^-1) - LDH Lactate dehydrogenase - MOI multiplicity of infection (pfu cell^-1) - N agitation speed (rpm) - OTR oxygen transfer rate (mole O₂ ml^-1 h^-1) - OUR oxygen uptake rate (mole O₂ ml^-1 h^-1) - p partial pressure (atm) - P total pressure (atm) - pfu plaque forming units - q specific consumption or production rate (mole cell^-1 h^-1) - Q_{H, OUT} headspace outlet gas flow rate - Q_S,NOM nominal volumetric sparged gas flow rate (92 ml s^-1 at bioreactor conditions) - R ideal gas constant (82.05 ml atm mole^-1 K^-1) - SR_{V L} molar sparging rate per unit liquid volume (mole ml^-1 h^-1) - SSR specific sparging rate (mole cell^-1 h^-1) - T temperature (C or K) - V_L culture volume (ml) - VVD volume of feed per volume of culture per day (d^-1) - X cell concentration (cell ml^-1) - Y yield coefficient Indexes CO₂, O₂ related to CO₂ or O₂, respectively - glc glucose - H headspace gas phase or gas/liquid interface - L liquid phase - lact lactate - S sparged phase or gas/liquid interface - V viable