Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Determination of dissolved oxygen in photosynthetic systems by nitroxide spin-probe broadening

Concentrations of dissolved oxygen were monitored by following the width of the midfield line of the electron spin resonance spectrum of a nitroxide spin-probe. Measurements of peak-to-trough widths of first derivative spectra yielded accurate data over a high range of O2 concentrations (up to 5mM). Continuous traces of second harmonic line heights yielded similar results and proved to be advantageous for kinetic measurements, but were nonlinear and less sensitive at O2 levels above 2 mM. Photosynthetic oxygen evolution and respiratory oxygen uptake in the cyanobacterium Agmenellum quadruplicatum were thus examined over a broad range of oxygen concentrations. Upon prolonged (greater than 1 min) illumination, effects of photooxidative damage to both photosynthesis and respiration were demonstrated in the same experimental system. With the addition of an impermeable paramagnetic broadening agent, rapid transients in intracellular concentrations of dissolved O2 also could be measured.     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.     

Growth kinetics of vitis vinifera cell suspension cultures: I. Shake flask cultures

Vitis vinifera cell suspension cultures carried out in shake flasks were closely examined for biomass growth and cell division in relation to carbohydrate, NH(4), NO(3)PO(4), and dissolved oxygen (DO)consumption. After inoculation, the oxygen uptake rate of the cultures measured on-tine was observed to increase continuously to a maximum value of 3.8 mmol O(2)L(-1)h(-1) at day 7 when cell division ceased and dissolved oxygen reached its lowest level of 17% air saturation. During this first phase of growth, the specific oxygen uptake rate remained constant at approximately 0.6 mmol 02 O(2) g(-1) dw h(-1)or approximately 2.2 mumol O(2), (10(6) cells)(-1) h(-1) whereas dry biomass concentration increased exponentially from 1.5 to 6.0 g dw L(-1). Thereafter, dry biomass concentration increased linearly to approximately 14 g dw L(-1) at day 14 following nitrate and carbohydrate uptake. During this second phase of growth, the biomass wet-to-dry weight ratio was found to increase in an inverse relationship with the estimated osmotic pressure of the culture medium. This corresponded to inflection points in the dry and wet biomass concentration and packed cell volume curves. Furthermore, growth and nutrient uptake results suggest that extracellular ammonium or phosphate ion availability may limit cell division. These findings indicate that cell division and biomass production of plant cell cultures may not always be completely associated, which suggests important new avenues to improve their productivity. (c) 1995 John Wiley & Sons, Inc.     

Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture

Oxygen transport is a major limitation in large-scale mammalian cell culture. The effects of the dissolved oxygen concentration (DO; from 0.1 to 100% saturation with air) on Sp2/0-derived mouse hybridomas were investigated using continuous culture. The steady-state concentration of viable cells increased with decreasing DO until a critical dissolved oxygen concentration of 0.5% of air saturation was reached. The cell concentration declined at lower DO because of incomplete glutamine oxidation, and the specific lactate production from glucose increased to offset the reduced energy production from glutamine. Cell viability increased as the DO was decreased; the viability continued to increase even when the DO was reduced below 0.5%. The specific oxygen uptake rate was essentially constant for DO greater than or equal to 10% of air saturation and then decreased with decreasing DO. The P/O ratio (ATP molecules produced per O atom consumed) appears to change from 2 to 3 between 10 and 0.5% DO. The specific ATP production rate calculated using this assumption decreases only slightly with decreasing DO. The optimum DO of 50% for antibody production is different than the optimum (approximately 0.5% DO) for cell growth.     

On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors

O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor. O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance. Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h. Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values. A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation. This protocol can be directly applied for high-throughput measurements.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. II. Ionically adsorbed cells

Continuous culture studies of Azotobacter vinelandii cells immobilized by ionic adsorption to Cellex E anion exchange resin were conducted under oxygen-limited conditions for comparison to free-cell cultures. Immobilization had little effect upon the specific respiration and sucrose consumption rates as compared to free cells. However, maxima in specific nitrogen fixation rate and nitrogenase activity as a function of dissolved oxygen occurred at a C(O(2) ) value of approximately 0.005 mM as opposed to 0.02 mM for free cells. Further, in contrast to free-cell culture, most of the fixed nitrogen appeared in the medium rather than within intact cells. There were strong indications that reproduction of bound cells often resulted in cell lysis accounting for the fixed nitrogen content in solution.     

Oxygen supply without gas-liquid film resistance to xanthomonas campestris cultivation

Alternative methods of oxygen supply are of crucial importance, especially in viscous fermentations and shear-sensitive fermentations. A method of oxygen supply that completely eliminates the gas-liquid transport resistance has been presented. The method involves a need-based liquid-phase decomposition of hydrogen peroxide to provide the necessary oxygen. When Xanthomonas campestris was cultivated (viscous cultivation) using this method of oxygen supply, dissolved oxygen (DO) levels were maintained above the setpoint of 50% throughout the cultivation, whereas the conventional cultivation was able to meet culture oxygen demand only for about 6 h in a 72-h fermentation. Furthermore, the maximum specific growth rate and xanthan yields in the novel cultivation were 89% and 169%, respectively, of those obtained in conventional cultivation. A mathematical model was also developed to simulate and predict results in fermentations employing the presented methodology. In addition, studies with HOCl pretreatments indicated that monofunctional catalase may be responsible for the decomposition of H2O2 supplied externally to cells; HOCl pretreatments also increased the tolerance of cells to H2O2. The decomposition kinetics of externally supplied H2O2 was Michaelis-Menten in nature with vmax = 1.196 x 10(-6) M s-1 and Km = 0.21 mM. The catalase concentration was estimated to be 3.4 x 10(-10) mol/g of cells. Copyright 1998 John Wiley & Sons, Inc.     

Kinetics and distribution of alcohol oxidising activity in Acholeplasma and Mycoplasma species

Alcohol metabolism by Acholeplasma and Mycoplasma cell suspensions was determined using changes in dissolved oxygen tension to monitor oxygen uptake. All seven Acholeplasma test species oxidised ethanol and (where tested) propanol, butanol and pentanol. The rate of oxidation, at any particular substrate concentration, decreased with increasing alcohol molecular mass. Amongst 20 Mycoplasma species tested, M. agalactiae, M. bovis, M. dispar, M. gallisepticum, M. pneumoniae and M. ovipneumoniae oxidised ethanol. Propanol was also oxidised by M. dispar and isopropanol by M. agalactiae, M. bovis and M. ovipneumoniae. Isopropanol was oxidised at particularly high rates (V(max)100 nmol O(2) taken up min(-1) mg cell protein(-1)) and with a relatively high affinity (K(m) value<2 mM); oxygen uptake was consistent with oxidation to acetone. The significance of alcohol oxidation is unclear, as it would not be predicted to lead to ATP synthesis.     

Hydrogel-perfluorocarbon composite scaffold promotes oxygen transport to immobilized cells

Cell encapsulation provides cells a three-dimensional structure to mimic physiological conditions and improve cell signaling, proliferation, and tissue organization as compared to monolayer culture. Encapsulation devices often encounter poor mass transport, especially for oxygen, where critical dissolved levels must be met to ensure both cell survival and functionality. To enhance oxygen transport, we utilized perfluorocarbon (PFC) oxygen vectors, specifically perfluorooctyl bromide (PFOB) immobilized in an alginate matrix. Metabolic activity of HepG2 liver cells encapsulated in 1% alginate/10% PFOB composite system was 47-104% higher than alginate systems lacking PFOB. A cubic model was developed to understand the oxygen transport mechanism in the alginate/PFOB composite system. The theoretical flux enhancement in alginate systems containing 10% PFOB was 18% higher than in alginate-only systems. Oxygen uptake rates (OURs) of HepG2 cells were enhanced with 10% PFOB addition under both 20% and 5% O2 boundary conditions, by 8% and 15%, respectively. Model predictions were qualitatively and quantitatively verified with direct experimental OUR measurements using both a perfusion reactor and oxygen sensing plate, demonstrating a greater OUR enhancement under physiological O2 boundary conditions (i.e., 5% O2). Inclusion of PFCs in an encapsulation matrix is a useful strategy for overcoming oxygen limitations and ensuring cell viability and functionality both for large devices (>1 mm) and over extended time periods. Although our results specifically indicate positive enhancements in metabolic activity using the model HepG2 liver system encapsulated in alginate, PFCs could be useful for improving/stabilizing oxygen supply in a wide range of cell types and hydrogels.     

Visible-light promoted degradation of the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT): a kinetic study

Visible-light photo-irradiation of the commercial phenolic antioxidants (PhAs) butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), in the presence of vitamin B2 (riboflavin, Rf), in methanolic solutions and under aerobic conditions, results in the photo-oxidation of the PhAs. The synthetic dye photosensitiser Rose Bengal was also employed for auxiliary experiments. With concentrations of riboflavin and PhAs of ca. 0.02 mM and < 1 mM, respectively, the excited triplet state of the vitamin (3Rf*) is quenched by BHT in a competitive fashion with dissolved ground state triplet oxygen. From the quenching of 3Rf*, the semireduced form of the pigment is generated through an electron transfer process from BHT, with the subsequent production of superoxide anion radical (O2*-) by reaction with dissolved molecular oxygen. In parallel, the species singlet molecular oxygen, O2(1delta(g)), is also generated. Both reactive oxygen species produce the photodegradation of BHT. In the case of BHA, the lack of any effect exerted by superoxide dismutase drives out a significant participation of a O2(*-)-mediated mechanism. BHA mainly interacts with O2(1delta(g)) and exhibits a desirable property as an antioxidant--a relatively high capacity for O2(1delta(g)) de-activation and a low photodegradation efficiency by the oxidative species. Electrochemical determinations support the proposed photodegradative mechanism.     

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     

Physiological and morphological observations on Thiovulum sp.

Cell suspensions of Thiovulum sp., collected from enrichment cultures, were grown, maintained, and harvested for periods up to 7 months. In open-flow cultures run with aerated seawater, a continuous supply of hydrogen sulfide was provided by diffusion through a semipermeable membrane from either a live culture of Desulfovibrio esturaii, neutralized sodium sulfide, or a N2-H2S gas mixture. Attempts to grow Thiovulum in pure culture failed despite variation in concentrations of dissolved oxygen and hydrogen sulfide in stratified as well as in completely mixed systems. Uptake of 14CO2 and some organic compounds by purified cell suspensions was measured, and values were corrected for the activity of heterotrophic as well as autotrophic contaminants as determined in control experiments. Cell populations exhibited maximum uptake activities during formation of the characteristic veils. Substantial uptake of CO2 in air-saturated seawater was coincident with an optimal concentration of hydrogen sulfide of about 1 mM. Glutamate and a selection of vitamins (B12M biotin, and thiamine) did not significantly affect the uptake of CO2. No substantial uptake of carbon from acetate, glutamate, mannitol, and Casamino Acids was found. Within the range of error indicated, the data are consistent with acceptance of a chemolithotrophic nature of Thiovulum.     

Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture

A novel method for increasing dissolved oxygen concentration in culture media has been developed. It involves adding hydrogen peroxide (H2O2) to the medium which is then decomposed to oxygen and water by catalase. Some factors affecting oxygenation of culture were investigated. Maximal oxygen concentration occurred in 50 ml of the medium containing 0-2 g wet mycelium and 0.2% glucose at pH 5.0. A new apparatus for automated addition of H2O2 to the bioreactor to keep the dissolved oxygen concentration constant over the range 1-100% +/- 2% was tested. A significant increase (over sixfold) of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable (30% +/- 2%).     

Online detection of feed demand in high cell density cultures of Escherichia coli by measurement of changes in dissolved oxygen transients in complex media

A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli. The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption. In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation. In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction. Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process. The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome.     

Estimation of heat production by cultured cells in suspension using semi-automated flow microcalorimetry

Modifications to a heat conduction flow microcalorimeter are described which allow registration of heat production by cells cultured in suspension. LS cells produced 34 +/- 3 pW per cell. Over an 8.5 h period, cell numbers increased by 9% and heat production per cell by 18%. Oxygen consumption per cell was 0.244 +/- 0.02 mumol min-1 per 10(8) cells and the enthalpy change was -836 kJ/mol O2. An automated pumping system allowed sequential registration of heat production by untreated cells and those exposed to a metabolic inhibitor. The results showed that 0.1 mM 2,4-dinitrophenol caused a greater increase in power (+65% at 1.5 h) than in oxygen consumption (+36%). The opposite occurred in the case of cells treated with 1 mM potassium cyanide, heat dissipation being depressed (-48%) slightly less than oxygen uptake (-52%). The results illustrate the potential of careful calorimetric determinations in studying metabolic events in the growth and division of cells in culture.     

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.     

Effect of dissolved oxygen and Eh and Bacteroides fragilis during continuous culture.

Bacteroides fragilis subsp. fragilis was maintained in a chemostat modified for anaerobic conditions to test the effects of dissolved oxygen and Eh on growth. Using a defined medium containing glucose and a dilution rate of 0.16 h -1, a stable population of 3 X 10(9) colony-forming units/ml was present. At this steady state, the pH was 5.6, the Eh was -50 mV, and the dissolved oxygen concentration was 0% atmospheric saturation. The Eh was then adjusted to +300 mV by adding potassium ferricyanide while oxygen was excluded; in this system there were no demonstrable changes from the steady state in viable cells, pH, glucose concentration, or volatile fatty acid production. In other experiments oxygen was introduced into the original steady state at a dissolved oxygen concentration of 10% atmospheric saturation for a period of 6 to 8 h. During O2 exposure, the viable cell count decreased at a rate comparable to the theoretical washout rate for a static bacterial culture. Similar results were obtained with a dissolved oxygen concentration of 25 and 100%. Other effects of O2 exposure included an increase in Eh from -50 to +250 mV, a decrease in glucose consumption, and a decrease in volatile fatty acid production. These results suggest that dissolved oxygen has a bacteriostatic effect on B. fragilis in continuous culture, which may be independent of changes in Eh alone.     

Continuous exposure of airway epithelial cells to hydrogen peroxide: protection by KGF

Reactive oxygen species (ROS) increase permeability in the airway epithelium. Extended periods of oxidant exposure may be experienced by those suffering from chronic inflammation of the lungs, receiving supplemental oxygen, or living in areas with high levels of air pollution. We studied the effects of long-term, continuous exposure to hydrogen peroxide (H(2)O(2)) on the trans-epithelial electrical resistance (TER) across cultured monolayers of a transformed cell line of human bronchial epithelial cells, 16HBE14o- (16HBE). A TER perfusion system was employed to continuously monitor the TER without disturbing the tissue model. The TER decreased in a dose-dependent manner with increasing concentrations of H(2)O(2) (0.1, 0.5, and 1.0 mM), regardless of pre-incubation conditions. Cell cultures pre-treated with 50 ng/ml keratinocyte growth factor (KGF) showed a significant delay in oxidant-induced TER decreases caused by 0.1 mM H(2)O(2). Exposure to 0.1 mM H(2)O(2) for 350 min led to disruption of tight junction proteins, ZO-1 and occludin, but KGF treatment prevented this damage. The recovery of epithelial barrier function after exposure to oxidants was also studied. Tissue models exposed to 0.5 mM H(2)O(2) for 25 min showed complete recovery of TER after 20 h, independent of culture pre-treatment. In contrast, KGF pre-incubation enhanced the recovery of 16HBE cultures exposed for 50 min to 0.5 mM H(2)O(2).     

Atmospheric stability in cell culture vessels.

We have investigated that atmospheric stability in polystyrene and glass cell culture vessels by measuring the dissolved O2 and CO2 in the media of both seeded and unseeded culture vessels incubated at 37 degrees C. There was no diffusion of either O2 or CO2 through glass vessels. At low partial pressures of oxygen (PO2), oxygen diffused into the polystyrene flasks at a rate of 1 to 2 mmHg per 24 hr, and at high PO2, oxygen diffused slowly out of polystyrene flasks. CO2 diffused out of polystyrene flasks with a half-time of 260 hr resulting in a considerable elevation in pH. In seeded polystyrene flasks with the PO2 less than or equal to room air, cellular oxygen consumption was masked by the inward diffusion of oxygen. In addition, the fall in pH due to metabolic CO2 and organic acid production during cell growth in polystyrene flasks was buffered by the diffusion of CO2 out of the vessels.