Loss of lipid to plastic tubing

14C-labeled oleic acid and (3)H-labeled monoether in a bile salt solution were perfused through three types of plastic tubing. Large proportions of lipid were lost to the walls of silicone rubber and polyvinyl chloride tubes. The major portion of the lipid lost was recoverable only when chloroform-methanol was perfused through the tubings. On the other hand, very little lipid was lost to the wall of polyethylene tubing. Polyethylene tubing should therefore be used in perfusion studies involving lipid-soluble compounds.     

Oxygen demand and supply in cell culture

Summary The oxygen demand of animal cells in microcarrier culture can be determined using a dissolved oxygen electrode. Continuous monitoring of the oxygen demand by human diploid fibroblasts shows it to be a good indicator of cell number. Furthermore, the oxygen demand of animal cells in large cultures can be supplied using membrane oxygenation using silicone rubber tubing. This method avoids the problems encountered with sparging the culture.     

Evaluation of silicone tubing toxicity using tobacco BY2 culture

Summary Silicone tubing is frequently used for gas exchange in cell culture systems, due to its biocompatibility and high permeability to CO₂ and O₂. In cell culture chambers, medium pH and oxygen levels are often maintained by gas exchange through a coil of silicone tubing. Culture medium is recirculated between the gas exchanger and the culture chamber which contains a suspension of cells. We report that the type of agent used for silicone vulcanization (peroxide or platinum) can markedly affect its biocompatibility, and that tobacco cell culture represents a particularly sensitive indicator of tubing cytotoxicity. Under the conditions studied (cell suspension maintained with forward-reverse flow and stirring), peroxide-cured silicone tubing was toxic to the tobacco BY2 cell culture, in contrast to the platinum-cured silicone tubing that was completely biocompatible. Upon further investigation by mass spectrometry, it was determined that a component with a molecular mass of 288 Da, possibly a tetrachlorinated biphenyl, was present in culture medium in contact with peroxide-cured tubing but not in medium in contact with platinum-cured tubing. Additional curing of peroxide-cured tubing resulted in cell morphology and viability comparable to controls. These data suggest that improperly cured silicone tubing can release catalytic byproducts which can be toxic to plant cells, and that the BY2 tobacco cells represent a suitable model system for studies of materials biocompatibility.     

Use of oxygen-permeable silicone rubber pouches for growing mass cultures of bacteria

Growth of most bacteria often involves the use of expensive incubated shaker systems. In this report, oxygen-permeable silicone rubber pouches, with oxygen permeability over 100 times higher than other polymers, were employed for growing bacterial cultures. With little, if any, agitation oxygen-permeable silicone rubber pouches produced bacterial growth rates equivalent to growth rates obtained in shaker flasks. The silicone rubber pouch described has a glass cuvette integrated into its design that permits readings of bacterial density without opening the pouch. One can sterilize and store powdered bacterial culture medium in silicone rubber pouches ; therefore, bacterial cultures can be initiated by simply adding water and bacteria.     

Use of a silicone tubing sensor to control methanol concentration during fed batch fermentation of Pichia pastoris

A silicone tubing sensor controlled a constant methanol concentration in a fermenter up to 72 hours without the need for on-line gas chromatography or complex feeding schemes based on dissolved oxygen spikes. Methanol concentration was controlled up to 1.0% (v/v) with control around a given set point of ± 0.24%. The length of tubing, airflow through the tubing, pump speed and medium formulation had no effect on the control of methanol concentration.     

Oxygen transfer in a pulse bioreactor

Oxygen transfer in a novel pulse bioreactor has been evaluated. The agitator consists of a series of alternately fixed and movable parallel plates mounted so that the movable plates vibrate at 30 Hz causing a pulsating fluid motion. Pure oxygen, at pressures up to 5 atm, diffuses through silicone rubber tubing that also vibrates at 30 or 60 Hz. The main feature of this bioreactor is high oxygen transfer with low shear to prevent damage to fragile animal cell membranes. We estimate that sufficient oxygen can be supplied to support over 10(8) cells/mL of human diploid foreskin cells growing on microcarriers. (c) 1993 John Wiley & Sons, Inc.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Silicone-tubing aerated bioreactors for somatic embryo production

Bioreactors equipped with silicone tubings for bubble free oxygen supply are suitable for culture of embryogenic cell suspensions. The advantages of bubble free aeration systems over various devices for dispersion of air bubbles are the lack of foam formation and the possibilities of precise control of the desired oxygen set point. The specification of silicone tubing (length, diameter, wall thickness) has to be adapted according to the amount of embryogenic biomass to be produced in the bioreactor. Cell suspensions of Euphorbia pulcherrima were cultured --2 l bioreactor at 60% pO₂, supplied by a silicone tubing system of 155 cm length, 4.0 mm diameter and 0.4 mm wall thickness. The oxygen concentration decreased when the packed cell volume exceeded 14% (=3.7 g l^-1 cell dry weight), indicating the upper limit of oxygen supply by the silicone tubing. Mathematical considerations for membrane aerated bioreactors are presented with the intention of enabling a more precise definition for the configuration of silicone tube systems in different bioreactor types.     

Survival of Legionella pneumophila in a model hot water distribution system

A virulent strain of Legionella pneumophila was inoculated into an enclosed system supplied with unsterilized water from a domestic hot water supply. Growth of bacteria was monitored over 10 weeks. An increase in the number of organisms other than legionellas occurred but few amoebae were observed and none could be cultured. Viable counts of L. pneumophila in the circulation fluid decreased slightly. However, particles of debris which accumulated in the apparatus and which were stained by the indirect fluorescent antibody technique were found to be almost totally composed of L. pneumophila. On dismantling the apparatus Legionella was isolated in moderately high numbers from several different types of surfaces, particularly natural rubber and silicone.     

A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - SSPM Selby Sitka proliferation medium - SSMM Selby Sitka maturation medium The following was presented at the NERC TBLG '95 Meeting as the Bioreactor Workshop     

Development of an automated plant culture system

An apparatus was constructed that could be used to grow plant tissues, organs, and whole plantlets under sterile conditions. This system accommodated independent or multiple concurrent growth of cultures. Growth of plants either equalled or exceeded that observed using the manual transfer procedure. The automated plant culture system (APCS) consists of silicone tubing, 2 impeller pumps, 2 glass medium reservoir bottles, a 3-way stainless steel valve, a plant culture chamber, and an interface module containing relay boards. Control of the APCS is through interfacing with a microcomputer (e.g. Apple IIe or Atari 400). The computer controlled medium introduction, evacuation, and replenishment in a sterile environment. The APCS was inexpensively constructed and provides a labor-saving, long-term method to culture plants in vitro.Mention of a trade name or proprietary product in the paper does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

A comparison of oxygenation methods fro high-density perfusion culture of animal cells

A perfusion culture system was developed to investigate the oxygenation of high-density hybridoma cell cultures. The culture system was composed of a stirred-tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble ( approximately 5 mm in diameter), micron-sized bubble ( approximately 80 mum in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron-sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble-free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high-density animal cell cultures, provided that the culture medium is supplemented with Pluronic F-68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long-term cultivation were found to be more problematic than simple bubble oxygenation of high-density cell culture. The micron-sized bubble oxygenation method is highly recommended for high-density animal cell cultures, provided that Pluronic F-68 is supplemented into the culture medium. (c) 1993 John Wiley & Sons, Inc.     

The Use of Polarography in the Assay of Oxygen Diffusing from Roots in Anaerobic Media

A “polarographic” technique by which the rate of outward diffusion of oxygen from plant roots may be measured, is described in detail. Oxygen has been found to diffuse from the roots of all of the ten species of bog-plants so far tested, and the oxygen diffusion rates (ODR) of eight of these species show the existence of interspecific differences. Model roots made from silicone rubber tubing give “polarographs” identical with those obtained around the living roots. Suberization accompanying root maturation is thought to account for a. the association of oxygen diffusion with the apical regions of roots, and b. the lowered diffusion rates associated with winter dormancy. This localisation of root oxygen diffusion may be of survival value in reduced soils. Experiments suggest that aerenchyma can function as an oxygen reservoir in the plant if the stomata were closed for long periods. Calculations of expected ODR based on the internal gas concentrations of a model system give readings very similar to the ODR obtained experimentally using the silicone rubber tubing models. Further calculations, based on reported internal gas concentrations for Menyanthes, rice, Spartina, and Schoenus, also show a close similarity between real and expected values. It is concluded that in these species at least, there is negligible resistance in certain regions of the root to the passage of oxygen from the root intercellular spaces to the surrounding medium. Results have shown that root bases are relatively impermeable to oxygen, and it is suggested that oxygen intake into roots in the basal regions reported by other workers, probably occurs through basally-borne laterals. One important function of diffusing oxygen from the root is probably the supply of an external source of oxygen to the root meristem.     

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO_2gas). Because the cells are consuming oxygen supplied by diffusion, a difference between pO_2gas and that experienced by the cells (pO_2cell) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen‐permeable silicone rubber membranes can be used to control pO_2cell during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene‐propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte‐like increases with decreasing pO₂ only when using oxygen‐permeable silicone membrane‐based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO_2cell being equal to pO_2gas at the tissue‐membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO₂ distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO_2cell, and these results suggest they are a valuable tool for investigating pO₂ effects in many applications, such as stem cell differentiation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The use of soluble steroids within inflatable breast prostheses.

A cortisone derivative (Solumedrol, Upjohn) has been added to the saline used to fill inflatable breast prostheses. In the experimental laboratory this drug passed slowly through the silicone rubber bag into a surrounding solvent. Clinically, this use has been evaluated in 100 cases of augmentation mammaplasty, with no evidence of wound complications attributable to the steroid. The influence of significant capsule formation (or "hardness") was less than 5 percent in this series.     

Development of a single-pass ceramic matrix bioreactor for large-scale mammalian cell culture

A single-pass, plug-flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via sllicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side-by-side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single-pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale-up. (c) 1992 John Wiley & Sons, Inc.     

Influence of Osmolarity and the Presence of an Osmoprotectant on Lactococcus lactis Growth and Bacteriocin Production

Growth inhibition of Lactococcus lactis provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium. Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.     

Growth and differentiation of epidermal cells from the rainbow trout established as explants and maintained in various media

Growth and a number of differentiated characteristics of cultured epidermal cells from the rainbow trout Oncorhynchus mykiss were compared using two commercially available serum–free media, a dermal substrate/serum free kit and a serum–containing medium which had been previously optimized for epidermal cell culture. Each medium supported short term growth over 15 days. Only the medium supplied for dermal substrate culture supported longer growth periods. This medium was supplied for use with a collagen/stromal substrate but gave good cultures even without the substrate. Differentiation, measured by examining mucous cells, cytokeratins, epidermal growth factor receptor, gap junction status and ultrastructure showed that serum–free media gave quantitatively and qualitatively superior expression and short term retention of differentiation over serum–containing medium. Epithelial cell growth with expression of differentiated characteristics can be maintained in primary culture in serum–free medium for at least as long as in serum–containing medium. This provides a useful technique for use when serum presence in medium is undesirable or proves toxic to the specialized cell type under investigation.     

Antibody production by a hybridoma cell line at high cell density is limited by two independent mechanisms

Our previous attempt to model the stationary phase of production-scale hollow-fiber bioreactors using a scaled-down micro hollow-fiber bioreactor resulted in a predicted antibody production rate that was three- to fourfold lower than the actual value (Gramer and Poeschl, 2000). Medium limitations were suspected as the reason for the discrepancy. In this study, various increases in medium feed rate were implemented in the micro bioreactor by increasing the diameter of the silicone tubing that houses the hollow fibers. Because larger diameter tubing may induce oxygen limitations, we also explored the effect of medium recirculation to enhance oxygenation. Antibody production in the micro bioreactor increased both as a result of increased medium supply and due to medium recirculation. However, these parameters increased antibody production through two independent mechanisms. The increased medium supply resulted in a higher cell-specific antibody production rate, but not a higher viable cell density. Medium circulation resulted in the support of a higher viable cell density, but had little effect on the cell-specific secretion rate. The two mechanisms of enhanced antibody production were additive, demonstrating that simultaneous parameters can limit antibody production by this cell line in a hollow-fiber system. When the medium feed and circulation rates were increased to a volumetrically proportional scale, scale-up predictions from the micro bioreactor matched the actual data from the production-scale system to within 15%. These data demonstrate the usefulness of the micro bioreactor for characterizing cell growth and limiting mechanisms at high cell densities.