Optimum fiber spacing in a hollow fiber bioreactor

A high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 x 10(8) viable cells/mL in the extracapillary space-corresponding to an overall reactor density of 7 x 10(7) cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later stages.     

Human lymphocyte PEEK-WC hollow fiber membrane bioreactor

In this study we developed a PEEK-WC hollow fiber (HF) membrane bioreactor for the maintenance of human peripheral lymphocytes as model system for the in vitro investigation of disease pathogenesis, chemical effects and individual drug sensitivity. Peripheral lymphocytes isolated from donor's human buffy coat were cultured in the shell compartment of the PEEK-WC-HF bioreactor and stimulated with PHA 5microg/mL for the first 48h of culture to enhance cytokine production and cell proliferation. Thereafter, cells were cultured in the presence of Hypericum perforatum (St. John's wort) in order to induce cytochrome P450s enzymes, CYP2E, involved in the biotransformation of endogenous molecules and exogenous compounds. The metabolic activity of cells with respect to glucose consumption and oxygen uptake was maintained for all the culture time without the addition of mitogen. Two cytokines IL-2 and IL-10, which are specific pattern of lymphocytes T helper 1 and T helper 2, respectively, were produced in the bioreactor up to 14 days of culture. Lymphocytes were also able to biotransform acetaminophen through the formation of the main metabolite paracetamidofenil-beta-glucuronide, which is the product of glucuronidation reaction, as a result of the Hypericum perforatum administration that induced the catalytic activity of the CYP2E1. These results demonstrated the usefulness of the bioreactor as the support system that reproduces physiological parameters such as a constant perfusion of medium, nutrients and oxygen maintaining the in vitro integrity of lymphocyte viability and functions.     

Effect of harvesting protocol on performance of a hollow fiber bioreactor.

In this study, a bioreactor subject to Starling flow in closed shell batch harvest mode was compared to two forms of additional forced extracapillary (EC) space convection including EC circulation and EC cycling. Despite the presence of Starling flow as the dominant EC convection mechanism in the batch harvest system, the bioreactor start up was fairly good. However, the antibody productivity of the batch harvest system fell off rapidly after day 20 resulting in only 4.5 g of antibody produced. EC circulation with flow parallel to the fibers had a slightly better start up than the batch harvest. However, the antibody productivity also dropped after day 20 with EC circulation, resulting in only 7.5 g of antibody produced. EC cycling with flow both parallel and perpendicular to the fibers resulted in a start up similar to that of EC circulation. However, in contrast to the other two systems, antibody productivity in the EC cycling system was stable over the 60-day experiment resulting in the production of 23 g of antibody. These results demonstrate the importance of inducing the proper flow distribution in the EC space to allow consistent and stable production in hollow fiber bioreactors.     

Plant cell immobilization in a dual hollow fiber bioreactor

Summary Lithospermum erythrorhizon was immobilized in a dual hollow fiber bioreactor (DHFBR) to maintain high cell density and to operate continuously. The cells grew well and its dry biomass density was 325 g/L of the void volume for the cell growth. Volumetric and specific productivities of phenolics were 221 mg/L.day and 0.68 mg/g.dry wt.day, respectively, which are 58 and 2 times of those of shake flask cultures.     

Ethanol production in a hollow fiber bioreactor using Saccharomyces cerevisiae

Summary A new approach for continuous production of ethanol was developed using a Hollow fiber fermentor (HFF). Saccharomyces cerevisiae cells were packed into the shell-side of a hollow fiber module. Using 100 g/l glucose in the feed gave an optimum ethanol productivity, based on total HFF volume, of 40 g ethanol/l/h at a dilution rate of 3.0 h^-1. Under these conditions, glucose utilization was 30%. However, at 85% glucose utilization the productivity was 10 g ethanol/l/h. This compares to batch fermentor productivity of 2.1 g ethanol/l/h at 100% glucose utilization.     

Continuous production of acrylamide byBrevibacterium sp. immobilized in a dual hollow fiber bioreactor

Summary Acrylamide was continuously produced from acrylonitrile usingBrevibacterium sp. CHl grown and immobilized in a dual hollow fiber bioreactor of 8.0 cm³. The biomass reached as high as 200 gm/L of the space available for the cell growth. The volumetric productivity of the reactor was 88 gm/L. h and the conversion of acrylonitrile varied with acrylonitrile concentration, pH and feed rate.     

Protein-free human-human hybridoma cultures in an intercalated-spiral alternate-dead-ended hollow fiber bioreactor

Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.(c) John Wiley & Sons, Inc.     

Perfluorocarbon facilitated O2 transport in a hepatic hollow fiber bioreactor

A mathematical model describing O₂ transport in a hepatic hollow fiber (HF) bioreactor supplemented with perfluorocarbons (PFCs) in the circulating cell culture media was developed to explore the potential of PFCs in properly oxygenating a bioartificial liver assist device (BLAD). The 2‐dimensional model is based on the geometry of a commercial HF bioreactor operated under steady‐state conditions. The O₂ transport model considers fluid motion of a homogeneous mixture of cell culture media and PFCs, and mass transport of dissolved O₂ in a single HF. Each HF consists of three distinct regions: (1) the lumen (conducts the homogeneous mixture of cell culture media and PFCs), (2) the membrane (physically separates the lumen from the extracapillary space (ECS), and (3) the ECS (hepatic cells reside in this compartment). In a single HF, dissolved O₂ is predominantly transported in the lumen via convection in the axial direction and via diffusion in the radial direction through the membrane and ECS. The resulting transport equations are solved using the finite element method. The calculated O₂ transfer flux showed that supplementation of the cell culture media with PFCs can significantly enhance O₂ transport to the ECS of the HF when compared with a control with no PFC supplementation. Moreover, the O₂ distribution and subsequent analysis of ECS zonation demonstrate that limited in vivo‐like O₂ gradients can be recapitulated with proper selection of the operational settings of the HF bioreactor. Taken together, this model can also be used to optimize the operating conditions for future BLAD development that aim to fully recapitulate the liver's varied functions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A novel alginate hollow fiber bioreactor process for cellular therapy applications

Gel‐matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet‐like clusters to treat Type 1 diabetes, the need for large‐scale production can be anticipated. The throughput of the commonly used nozzle‐based devices for cell encapsulation is limited by the rate of droplet formation to ～0.5 L/h. This work describes a novel process for larger‐scale batch immobilization of mammalian cells in alginate‐filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intra‐capillary space medium flow was negligible, (2) extra‐capillary alginate gelling was complete and (3) 83 ± 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate‐forming cell line that grew from mostly single cells to pancreatic islet‐sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small‐scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate‐immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel‐matrix assisted cultures for cellular therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

An intercalated-spiral wound hollow fiber bioreactor for the culture of mammalian cells

Summary A dual circuit bioreactor is developed; it consists of two intercalated-spiral sets of hollow fibers with alternated dead-ends. The construction is relatively simple, but guarantees uniform distribution of the arterial and venous circuits. Data are presented to illustrate the ability of the bioreactor to culture a human lymphoblastoid B-cell line.     

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Summary Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×10⁶ cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×10⁸ cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.     

Evaluation of a hepatocyte-entrapment hollow fiber bioreactor: a potential bioartificial liver

We have developed a hepatocyte entrapment hollow fiber bioreactor for potential use as a bioartificial liver. Hepatocytes were entrapped in collagen gel inside the lumen of the hollow fibers. Medium was perfused through the intraluminal region after contraction of the hepatocyte-entrapment gel. Another medium stream, comparable to the patient's blood during clinical application, passed through the extracapillary space. Viability of hepatocytes remained high after 5 days as judged by the rate of oxygen uptake and viability staining. Urea and albumin synthetic activities were also sustained. Transmission electron microscopic examination demonstrated normal ultrastructural integrity of hepatocytes in such a bioreactor. With its sort-term, extracorporeal support of acute liver failure, the current bioreactor warrants further investigation. (c) 1993 John Wiley & Sons, Inc.     

Design and performance study of a novel immobilized hollow fiber membrane bioreactor

A dual-layer coaxial hollow fiber (DLHF) bioreactor for cell immobilization developed to overcome nutrients transport limitation is presented. Cells were contained in the annular space between two coaxial hollow fibers, and nutrients were supplied by a forced convective transport from the shell side through the annular space to the lumen side. With judicious selection of the membrane materials, a low operating transmembrane pressure of 50 kPa, and using E. coli as the model organism, a high cell density of 10(11) cells/mL annular space volume and a high cell viability of (up to 80%) were obtained.     

Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors, and in an industrial scale hollow fiber bioreactor system

In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.     

A novel multi-coaxial hollow fiber bioreactor for adherent cell types. Part 1: hydrodynamic studies.

A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described. It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments. Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC). The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC. Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)). These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers. Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor. Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments.     

A radial flow hollow fiber bioreactor for the large-scale culture of mammalian cells

A radial flow hollow fiber bioreactor has been developed that maximizes the utilization of fiber surface for cell growth while eliminating nutrient and metabolic gradients inherent in conventional hollow fiber cartridges. The reactor consists of a central flow distributor tube surrounded by an annular bed of hollow fibers. The central flow distributor tube ensures an axially uniform radial convective flow of nutrients across the fiber bed. Cells attach and proliferate on the outer surface of the fibers. The fibers are pretreated with polylysine to facilitate cell attachment and long-term maintenance of tissuelike densities of cell mass. A mixture of air and CO(2) is fed through the tube side of the hollow fibers, ensuring direct oxygenation of the cells and maintenance of pH. Spent medium diffuses across the cell layer into the tube side of the fibers and is convected away along with the spent gas stream. The bioreactor was run as a recycle reactor to permit maximum utilization of nutrient medium. A bioreactor with a membrane surface area of 1150 cm(2) was developed and H1 cells were grown to a density of 7.3 x 10(6) cells/cm(2).     

Modeling analysis of an intercalated-spiral alternate-dead-ended hollow fiber bioreactor for mammalian cell cultures

The model analysis of a bioreactor with two intercalated-spiral sets of hollow fibers with alternated dead ends is presented. Design equations are derived based on a model with two jumped fibers and an approximation of the fluid mechanics with a small fiber radius-to-length ratio. The performance of the bioreactor is simulated with and without cell growth utilizing a segregated radial flow model in the extracapillary space. The pressure modulus, the wall Peclet number, the Thiele modulus, and the Monod constant are used as model parameters. The results can be used to assess the proper choice of fiber permeability, fiber length, fiber spacing, and flow velocity.     

The design of stirred reactors with hollow fiber catalysts for Michaelis-Menten kinetics

A method of calculating the conversion in Rony's hollow fiber reactor is outlined. It, is assumed that, the kinetics are of Michaelis-Menten type and that diffusion within the hollow fiber, as well as through its wall, should be taken into account. The normalization of the Thiele modulus suffices to unify the treatment of internal diffusion and the pseudosteady state hypothesis is found to be valid under almost all conditions.     

Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate-limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.