A vortex-bowl disk atomizer system for the production of alginate beads in a 1500-liter fermentor

Using a model system, a concept for the immobilization of microbial cultures within alginate beads directly in a 1500-L fermentor with a height to diameter ratio of 1.85 is described. The system is comprised of a 60-cm diameter bowl fixed to the top of an agitation shaft, where calcium-ion-rich media is continuously recirculated from the bulk solution to the bowl. The rotation of the shaft and bowl creates a climbing film (vortex) of solution. An atomizing disk centrally recessed within the bowl sprays an alginate solution into the climbing film where the droplets harden into beads. The effect of heat treatment on the alginate solution on resulting bead properties was examined. The sterilization operation did not appear to have a major effect on the alginate bead mechanical properties of firmness and elasticity which was much more a function of alginate concentration. Beads of various sizes were produced by the unit. The system was characterized by the dimensionless numbers Reomega = (omega x rho x D(2))/mu and ReQ = (Q x rho)/(mu x D). At Reomega and ReQ values less than 500 and 0.15, respectively, the mechanism was direct drop. Parent droplets followed by satellite droplets were observed. When either the flow rate or speed was increased, filaments formed predominantly, which was unwanted in this system because filament breakdown into smaller droplets does not occur due to the proximity of the disk to the climbing film in the bowl. This system could be applied to the immobilization of microorganisms, as well as plant or animal cell cultures, and for other sizes or fermentors. The overflow from the bowl carries the gellified beads into the bulk solution where immobilized cells could act upon the fermentation media.     

Growth of anchorage dependent mammalian cells on glycine-derivatized polystyrene in suspension culture

Summary Glycine-derivatized polystyrene beads were prepared and used as microcarriers to grow normal cells of human embryonic kidney, rhesus monkey kidney, and human foreskin fibroblasts in suspension cultures. Growth of the cells on polystyrene beads derivatized with other amino acids, peptides, and carboxylic acids also was investigated.     

A new culture system for the cultivation of mammalian cells for the production of several biologically active substances

A maximum cell density of 8×10⁶ viable human fibroblast cells/ml was obtained on microcarriers at a perfusion rate of 0.6 mL/min — a rate which maintained a quasi-steady state. The maximum tPA production was approximately 1.2 g/ml and obtained when the cell density was relatively constant during the later periods of cultivation. Specific UTP and tPA production rates had a correlation factor of 0.85, and cell growth to oxygen consumption had an 0.92 correlation factor. ATP generation was strongly correlated to glutamine consumption, with a lower correlation to oxygen utilization.     

Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture

A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day(-1). The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day(-1). Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.     

Design and in vitro and in vivo evaluation of mucoadhesive microcapsules of glipizide for oral controlled release: A technical note

Conclussion  Thus, large spherical microcapsules with a coat consisting of alginate and a mucoadhesive polymer (sodium CMC, methylcellulose, Carbopol, or HPMC) could be prepared by an orifice-ionic gelation process. The microcapsules exhibited good mucoadhesive properties in an in vitro test. Glipizide release from these mucoadhesive microcapsules was slow and extended over longer periods of time and depended on composition of the coat. Drug release was diffusion controlled and followed zero-order kinetics after a lag, period of 1 hour. In the in vivo evaluation, alginate-Carbopol microcapsules could sustain the hypoglycemic effect of glipizide over a 14-hour period. These mucoadhesive microcapsules are, thus, suitable for oral controlled release of glipizide.     

Cell aggregate suspension culture for large-scale production of biomolecules

Summary A method is described for the rapid reversible conversion of a number of continuous cell lines from anchorage-dependent growth to growth as aggregates of cells in suspension culture. Employing this technique, an inoculum of three 75-cm² flasks of BALB/c SV3T3 cells was grown to 60 liters of aggregate suspension in 14 days. This yielded 120 ml of packed cells or 9.1×10¹⁰ cells. Similar results were obtained for other cell lines. Biomolecules such as migration-inhibition factor (MIF) and plasminogen activator were produced from these cultures.     

Dynamics of recombinant protein production by mammalian cells in immobilized perfusion culture

In spite of the generally stable nature of immobilized perfusion culture, its profile of target protein production frequently shows variations. This might be explained by the drift in the metabolism of cultured cells. To address this issue, we performed a set of four Opticell bioreactor cultures producing recombinant anticogulant protein PCGFX. All the cultures lasted 40-50 days with the oxygen consumption rate (OCR) mostly around 10 μmol min⁻¹; nevertheless, glucose and lactate metabolism was fluctuated with a parallel fluctuation in the recombinant protein productivity (RPP). The mean productivity of recombinant PCGFX was determined to be about 1.0 mg day⁻¹ for all the cultures. The statistical analysis revealed a significant correlation between the lactate production rate (LPR) and RPP in two cultures. A significant correlation was further found between average OCR and RPP in another culture where OCR was exceptionally lowered under serum-free conditions. No parameter significantly correlated with RPP in the remaining one culture; thus, the overt drift of RPP resulted, at least partly, from that of the cell metabolic activity and the present data should be helpful to explore a strategy for maximizing productivity.     

Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells

Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10–20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4–8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate cculture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6–10 mg/l/day was maintained for 28 days.     

Serum proteome profiling for diagnostics of ovarian cancer using ClinProt magnetic technique and MALDI-TOF mass spectrometry

Using ClinProt magnetic beads with reverse-phase (MB-HIC 8 and HB-HIC 18), weak cation exchange (MB-WCX) and metal affinity (MB-IMAC Cu) surfaces fractions of peptides and proteins were isolated from human sera for their profiling by MALDI-TOF mass spectrometry. Proteome profiling of sera from basically healthy women (47 subjects, average age 49) and from women with verified ovarian cancer (stages 1-IV, 47 patients, average age 51) by means of MB-WCX beads allowed to generate the best diagnostic models based on Genetic Algorithm and Supervised Neural Network classifiers; these models demonstrated 100% sensitivity and specificity during analysis of the test set. Introduction of additional sera from patients with colorectal cancer (19) and ulcerous colitis (5) to the statistical model confirmed 100% ovarian cancer recognition. Statistical analysis of mass-spectrometry peak areas included to the diagnostic classifiers showed 3 peaks characteristic for ovarian cancer and 4 peak areas exhibiting changes associated with both ovarian and colorectal cancer.     

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (q(O2) and q(CO2), respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate q(O2), q(CO2) and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), q(O2) and q(CO2) ranges were 5.14-5.77 and 5.31-6.36 pmol/cell day, respectively, resulting in RQ values of 0.98-1.14. Changes to DO had a slight reducing effect on respiration rates with q(O2) and q(CO2) values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with q(O2) and q(CO2) values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective q(O2) and q(CO2) values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in q(O2) and q(CO2) values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real-time q(O2) and q(CO2) estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing.     

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).     

A technique for determining the shear sensitivity of mammalian cells in suspension culture

Summary A murine hybridoma was subjected to constant shear rates within a couette viscometer for periods of 15 hours. Shear effects on cells were determined by live cell counts, cell viability and by the release of the cytoplasmic enzyme lactate dehydrogenase into the culture medium. Cell damage was observed at a shear rate of 870s^-1 but not at 420s^-1.     

Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma.

The advantage of using proteins and peptides as biomarkers is that they can be found readily in blood, urine, and other biological fluids. Such sample types are easily obtained and represent a potentially rich palette of biologically informative molecules. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a key tool for rapidly interrogating such sample types. The goal of clinical proteomics is to harness the power of this tool for identifying novel, condition-specific protein fingerprints that may, in turn, lead to the elucidation and use of diseasespecific biomarkers that may be used to diagnose disease as well as to evaluate disease severity, disease progression, and intervention efficacy. Here we have evaluated a simple, affordable bench-top MALDI-TOF mass spectrometer to generate protein profiles from human plasma samples of asthma patients and healthy individuals. We achieve this profiling by using C8-functionalized magnetic beads that enrich a specific subset of plasma proteins based on their absorption by this resin. This step is followed by elution, transfer onto a prestructured sample support (AnchorChip technology), and analysis in a bench-top MALDI-TOF mass spectrometer (OmniFLEX) with AutoXecute acquisition control which enables automated operation with reproducible results. Resulting spectra are compiled and analyzed through the pattern recognition component of ClinProTools software. This approach in combination with ClinProTools software permits the investigator to rapidly scan for potential biomarker peptides/proteins in human plasma. The reproducibility of plasma profiles within and between days has been evaluated. The results show that the novel and facile approach with manual magnetic-bead sample preparation and a low-cost bench-top MALDI-TOF mass spectrometer is suitable for preliminary biomarker discovery studies.     

Optimization of use of UEA-1 magnetic beads for endothelial cell isolation

Most endothelial cells (EC) in the body belong to the microvasculature. Isolation and subsequent culture of these microvessel EC contributes greatly to our understanding of the heterogeneity and vascular specificity that exist between one organ site and another. However, a major obstacle is the overgrowth of contaminating cells (fibroblasts, pericytes, smooth-muscle cells) in cultures. Since 1990 the use of magnetic beads in combination with either a lectin, Ulex europaeus agglutinin-1 (UEA-1), or a monoclonal antibody has represented a powerful tool for the isolation/purification of microvessel EC. In the former case, operative conditions remain to be optimized to obtain pure cultures of EC.We have performed studies to optimize conditions of use for magnetic beads coated with UEA-1. Incubating beads with cells, the influences are studied of time, temperature, cell concentration, and number of beads per target cell for two cell types, human umbilical vein EC (HUVEC) and skin fibroblasts (HSF), either isolated or mixed. The effect of the last parameter was also checked on the behavior of cells undergoing proliferation after isolation. Results, expressed as isolation efficiency (from 40% to 90%) allowed us to select a 15-min incubation time at 4°C with rotary agitation, an optimal concentration of 4 x 10⁵ cells/ml, and an optimal cell:bead ration of 1:3. From a mixed cell population and in these conditions, even very low HUVEC:HSF proportions of 2.5:97.5 allowed us to obtain a pure HUVEC population in subsequent culture.Abbreviations UEA-1 Ulex europaeus agglutinin-1 - EC endothelial cells - HUVEC human umbilical vein endothelial cells - HSF human skin fibroblasts - MPC magnetic particle concentrator - IE isolation efficiency     

A cell-detaching reactor for inoculation of anchorage-dependent CHD and Vero cells between stepwise-expanded bioreactors

A cell-detaching reactor was developed to collect cells growing on microcarriers for inoculation between stepwise-expanded bioreactors. It consisted of a trypsinization zone and a separation zone, which were separated by a 200-mesh stainless steel screen. The screen allowed the cells only to pass through to the next bioreactor, after the cells have been trypsinized and detached from microcarriers. The operating feasibility of the cell-detaching reactor was tested with anchorage-dependent recombinant Chinese hamster ovary (rCHO) and African green monkey kidney (Vero) cells. rCHO and Vero cells were first cultured in a small microcarrier bioreactor, and then inoculated via the cell-detaching reactor into either a packed-bed bioreactor (for rCHO cells) or a larger microcarrier bioreactor (for Vero cells). For rCHO cells, the cell density reached 1.3 × 10⁷ cells/ml in the perfusion culture, and Vero cells reached 1.3 × 10⁶ cells/ml in the batch culture.     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

Harvesting and concentration of human influenza A virus produced in serum-free mammalian cell culture for the production of vaccines

A process scheme for the harvesting and concentration of cell culture-derived human influenza A virus is presented. The scheme comprises two static filtration steps, chemical inactivation by beta-propiolactone and cross-flow ultrafiltration. Human influenza A virus A/PR/8/34 (H1N1) was produced in roller bottles with serum-free medium using MDCK cells as a host. Cultivations resulted in specific hemagglutination (HA) activities of 393 HAU (100 microL)(-1) and turbidities of 0.479 OD measured as the extinction of light at 700 nm (mean values are presented). The concentrations of soluble protein and DNA in the harvests were 72 microg/mL and 5.73 microg/mL, respectively. An average product yield of 79% based on HA activity was achieved after clarification by depth (85%) and microfiltration (93%). The turbidities of cell culture supernatants were reduced to 2% of their initial value. Concentration with 750 kDa hollow-fiber modules by a factor of 20 resulted in 97% recovery of the product when operated at a constant flux of 28 L/(m(2) h) and a wall shear rate of 9,500 s(-1). The amount of protein and DNA could be reduced to 16% and 33% of their initial amount, respectively. An overall product yield of 77% was achieved. Clarified supernatants and concentrates were further analyzed by non-reducing SDS-PAGE and agarose gel electrophoresis. Particle volume distributions of concentrates were obtained by dynamic light scattering analysis. From the results it can be concluded that the suggested process scheme is well suited for the harvesting and concentration of cell culture-derived influenza A virus.     

Action of 50 Hz magnetic fields on cyclic AMP and intercellular communication in monolayers and spheroids of mammalian cells

To investigate the influence of physiological parameters such as cell density and three-dimensional cell contact on the biological action of a 2 mT/50 Hz magnetic field, mouse fibroblasts were exposed as monolayers and as multicellular spheroids. Changes in cyclic AMP content of cells and alterations in gap junction-mediated intercellular communication were measured immediately after 5 min of exposure to the field. In monolayers of intermediate cell density (1 × 10⁵ cells/cm²), the field treatment caused an increase in cAMP to 121% of the control level, whereas, at 3 × 10⁵ cells/cm² (near confluence), a decrease to 88% of the unexposed cells was observed. Furthermore, field exposure stimulated gap-junction communication to 160% of the control level as determined by Lucifer yellow dye exchange. In spheroids, alterations in the radial profile of cellular cAMP were observed that were due both to field-induced local cAMP changes and to increased gap-junction permeability for this second messenger, the latter causing radial cAMP gradients to be flattened. The results indicate a strong dependence of field action on physiological parameters of the system exposed. © 1995 Wiley-Liss, Inc.     

A mathematical model for the design of fibrin microcapsules with skin cells

The use of fibrin in tissue engineering has greatly increased over the last 10 years. The aim of this research was to develop a mathematical model to relate the microcapsule-size and cell-load to growth and oxygen depletion. Keratinocytes were isolated from rat skins and microencapsulated dropping fibrinogen and thrombin solutions. The cell growth was measured with MTT-assay and confirmed using histochemical technique. The oxygen was evaluated using a Clark sensor. It was found that Fick–Monod model explained the cell growth for the first 48 h, but overestimated the same thereafter. It was necessary to add a logistic equation to reach valid results. In relation to the preferred implant alternative, when considering large initial cell loads, the possibility to implant small loads of fast-growing cells arises from the simulations. In relation to the microcapsule size, it was found that a critical diameter could be established from which cell growth velocity is about the same.