A completely noninvasive method of dissolved oxygen monitoring in disposable small‐scale cell culture vessels based on diffusion through permeable vessel walls

Disposable cell culture vessels are extensively used at small scales for process optimization and validation, but they lack monitoring capabilities. Optical sensors that can be easily adapted for use in small‐scale vessels are commercially available for pH, dissolved oxygen (DO), and dissolved carbon dioxide (DCO₂). However, their use has been limited due to the contamination and compatibility issues. We have developed a novel solution to these problems for DO monitoring. Oxygen diffusion through permeable vessel wall can be exploited for noninvasive monitoring. An optical oxygen sensor can be placed outside the oxygen permeable vessel wall thereby allowing oxygen diffusing through the vessel wall to be detected by the sensor. This way the sensor stays separate from the cell culture and there are no concerns about contaminants or leachants. Here we implement this method for two cell culture devices: polystyrene‐made T‐75 tissue culture flask and fluorinated ethylene propylene (FEP)‐made Vuelife^® cell culture bag. Additionally, mammalian and microbial cell cultures were performed in Vuelife^® cell culture bags, proving that a sensor placed outside can be used to track changes in cell cultures. This approach toward noninvasive monitoring will help in integrating cell culture vessels with sensors in a seamless manner. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:172–177, 2014     

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

Overcoming challenges in WAVE Bioreactors without feedback controls for pH and dissolved oxygen

The biopharmaceutical industry is increasing its use of the WAVE Bioreactor for culturing cells. Although this disposable bioreactor can be equipped to provide real-time pH and dissolved oxygen (DO) monitoring and control, our goal was to develop a process for culturing CHO cells in this system without relying on pH and DO feedback controls. After identifying challenges in culturing cells without controlling for pH and DO in the WAVE Bioreactor, we characterized O(2) and CO(2) transfer in the system. From these cell-free studies, we identified rock rate and rock angle as key parameters affecting O(2) transfer. We also identified the concentration of CO(2) in the incoming gas and the rate of gas flow into the headspace as key parameters affecting CO(2) transfer--and therefore pH--in the disposable culture chamber. Using a full-factorial design to evaluate the rock rate, rock angle, and gas flow rate defined for this WAVE Bioreactor process, we found comparable cell growth and pH profiles in the ranges tested for these three parameters in two CHO cell lines. This process supported cell growth, and maintained pH and DO within our desired range--pH 6.8-7.2 and DO exceeding 20% of air saturation--for six CHO cell lines, and it also demonstrated comparable cell growth and viability with the stirred-tank bioreactor process with online pH and DO control. By eliminating the use of pH and DO probes, this process provides a simple and more cost-effective method for culturing cells in the WAVE Bioreactor.     

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

Small-scale upstream bioprocess development often occurs in flasks and multi-well plates. These culturing platforms are often not equipped to accurately monitor and control critical process parameters; thus they may not yield conditions representative of manufacturing. In response, we and others have developed optical sensors that enable small-scale process monitoring. Here we have compared two parameters critical to control in industrial cell culture, pH and dissolved oxygen (DO), measured with our optical sensors versus industrially accepted electrochemical probes. For both optical sensors, agreement with the corresponding electrochemical probe was excellent. The Pearson Correlations between the optical sensors and electrochemical probes were 98.7% and 99.7%, for DO and pH, respectively. Also, we have compared optical pH sensor performance in regular (320 mOsm/kg) and high-osmolality (450 mOsm/kg) cell culture media to simulate the increase in osmolality in pH-controlled cultures. Over a pH range of 6.38-7.98 the average difference in pH readings in the two media was 0.04 pH units. In summary, we have demonstrated that these optical sensors agree well with standard electrochemical probes. The accuracy of the optical probes demonstrates their ability to detect potential parameter drift that could have significant impact on growth, production kinetics, and protein product quality. We have also shown that an increase in osmolality that could result from controlling pH or operating the reactor in fed-batch mode has an insignificant impact on the functionality of the pH patches.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development

Increasing the throughput and efficiency of cell culture process development has become increasingly important to rapidly screen and optimize cell culture media and process parameters. This study describes the application of a miniaturized bioreactor system as a scaled-down model for cell culture process development using a CHO cell line expressing a recombinant protein. The microbioreactor system (M24) provides non-invasive online monitoring and control capability for process parameters such as pH, dissolved oxygen (DO), and temperature at the individual well level. A systematic evaluation of the M24 for cell culture process applications was successfully completed. Several challenges were initially identified. These included uneven gas distribution in the wells due to system design and lot to lot variability, foaming issues caused by sparging required for active DO control, and pH control limitation under conditions of minimal dissolved CO2. A high degree of variability was found which was addressed by changes in the system design. The foaming issue was resolved by addition of anti-foam, reduction of sparge rate, and elimination of DO control. The pH control limitation was overcome by a single manual liquid base addition. Intra-well reproducibility, as indicated by measurements of process parameters, cell growth, metabolite profiles, protein titer, protein quality, and scale-equivalency between the M24 and 2 L bioreactor cultures were very good. This evaluation has shown feasibility of utilizing the M24 as a scale-down tool for cell culture application development under industrially relevant process conditions.     

Comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers

Optimization of culture conditions such as the dissolved O₂ (DO) concentration, temperature and pH was attempted regarding both cell growth and the production of tissue plasminogen activator (TPA) in a microcarrier cell culture of human embryo lung cells. The growth rate was suppressed at a DO concentration below 30% saturation. From the pH range 7.2–7.6, both the specific growth rate and maximal cell concentration decreased. At a lower temperature than 37°C, although both the specific growth rate and the maximal cell concentration decreased, the cell concentration was maintained for a longer time during the production period, high TPA productivity being maintained. As the optimal conditions for culture growth, a DO concentration of 30% saturation or over, temperature of 37°C and pH of 7.4 are recommended. However, for TPA production after cell culture growth, the DO concentration should be in the range 20–30% O₂ saturation, and the temperature and pH should be lowered to 33°C and 6.8, respectively.     

Advanced online monitoring of cell culture off‐gas using proton transfer reaction mass spectrometry

Mass spectrometry has been frequently applied to monitor the O₂ and CO₂ content in the off‐gas of animal cell culture fermentations. In contrast to classical mass spectrometry the proton transfer reaction mass spectrometry (PTR‐MS) provides additional information of volatile organic compounds by application of a soft ionization technology. Hence, the spectra show less fragments and can more accurately assigned to particular compounds. In order to discriminate between compounds of non‐metabolic and metabolic origin cell free experiments and fed‐batch cultivations with a recombinant CHO cell line were conducted. As a result, in total eight volatiles showing high relevance to individual cultivation or cultivation conditions could be identified. Among the detected compounds methanethiol, with a mass‐to‐charge ratio of 49, qualifies as a key candidate in process monitoring due to its strong connectivity to lactate formation. Moreover, the versatile and complex data sets acquired by PTR MS provide a valuable resource for statistical modeling to predict non direct measurable parameters. Hence, partial least square regression was applied to the complete spectra of volatiles measured and important cell culture parameters such as viable cell density estimated (R² = 0.86). As a whole, the results of this study clearly show that PTR‐MS provides a powerful tool to improve bioprocess‐monitoring for mammalian cell culture. Thus, specific volatiles emitted by cells and measured online by the PTR‐MS and complex variables gained through statistical modeling will contribute to a deeper process understanding in the future and open promising perspectives to bioprocess control. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:496–504, 2014     

Potential strategies for process control and monitoring of stabilization of dairy wastewaters in batch aerobic treatment systems

This research was conducted to study the relationships between pH, dissolved oxygen (DO), and oxidation–reduction potential (ORP) during low-intensity aeration of dairy wastewaters and to determine potential strategies for monitoring and/or control of this treatment process. The results of this study ascertained that, close to the detection limit of commercially available DO probes (0.1 mg/l), DO is a poor indicator of the oxidation–reduction status of the dairy wastewater during this treatment processes. All the three parameters (ORP, DO, and pH) displayed features defining stabilization of the wastewater and hence all three can be used singly or in combination to monitor and/or to control this treatment process. The study also established strong linear relations between ORP and the log of DO; manifest in the high-correlation coefficients of 0.98 and 0.95 at the aeration rates of 0.067 and 0.034-l [air] l⁻¹ [manure] min⁻¹, respectively. The latter observation confirms the higher sensitivity of ORP over DO at very low-oxygen levels; a fact which indicates the superiority of ORP in the monitoring and control of oxidation–reduction status of the wastewater close to DO detection limit. Finally, both total volatile solids (TVS) and chemical oxygen demand (COD), which are common measures of wastewater stabilization; correlated well with pH, DO, and ORP during the entire treatment process. However, because the measurements of DO are erratic close to the DO detection limit, and because ORP and pH measurements are much more consistent than the DO in the entire range of treatment, these two parameters will be more suitable for monitoring and control especially of extended aeration treatments.     

Effects of acute hypoxic stress on biochemical parameters,immune regulation and metabolic capacity of the blood in genetically improved farmed tilapia (GIFT,Oreochromis niloticus)

Dissolved oxygen (DO) in the culture water is an important environmental factor in fish farming. This study tested whether genetically improved farmed tilapia (GIFT, Oreochromis niloticus) challenged with a hypoxic episode would show inhibited growth, increased metabolic stress and a reduced immune response. GIFT subjected to 72 hr of semi‐lethal hypoxia (determined by linear regression as DO = 0.64 mg/L) showed changes in blood biochemistry, energy metabolism and related immune responses. In the early stages of stress, GIFT showed increased anaerobic respiration and increased lactic acid accumulation. Compared with a control group, white and red blood cell counts, hematocrit and blood hemoglobin concentration in GIFT exposed to 4 hr of semi‐lethal hypoxic stress were not significantly changed. However, the levels of glutamic‐pyruvic transaminase, glucose, cholesterol and cortisol were significantly higher under hypoxic stress at 2 and 4 hr. In the later stages of stress, GIFT displayed increased oxygen utilization and increased aerobic respiration associated with increased red blood cell count, hematocrit and hemoglobin concentration. However, the white blood cell count, serum lysozyme and complement C3 activities began to decline, associated with increased mortality. Reverting to a normal DO environment (5.0 mg/L) for an additional 72 hr, GIFT showed strong recovery, with respiratory metabolic enzymes, immune indicators and associated energy metabolites restored to near normal levels. Our observations contribute to a better understanding of the mechanisms of energy and immune regulation in fish, and will help reduce the damage caused by hypoxic stress during culture.     

Dual perfluorocarbon method to noninvasively monitor dissolved oxygen concentration in tissue engineered constructs in vitro and in vivo

Noninvasive in vivo monitoring of tissue implants provides important correlations between construct function and the observed physiologic effects. As oxygen is a key parameter affecting cell and tissue function, we established a monitoring method that utilizes ¹⁹F nuclear magnetic resonance (NMR) spectroscopy, with perfluorocarbons (PFCs) as oxygen concentration markers, to noninvasively monitor dissolved oxygen concentration (DO) in tissue engineered implants. Specifically, we developed a dual PFC method capable of simultaneously measuring DO within a tissue construct and its surrounding environment, as the latter varies among animals and with physiologic conditions. In vitro studies using an NMR‐compatible bioreactor demonstrated the feasibility of this method to monitor the DO within alginate beads containing metabolically active murine insulinoma βTC‐tet cells, relative to the DO in the culture medium, under perfusion and static conditions. The DO profiles obtained under static conditions were supported by mathematical simulations of the system. In vivo, the dual PFC method was successful in tracking the oxygenation state of entrapped βTC‐tet cells and the surrounding peritoneal DO over 16 days in normal mice. DO measurements correlated well with the extent of cell growth and host cell attachment examined postexplantation. The peritoneal oxygen environment was found to be variable and hypoxic, and significantly lower in the presence of metabolically active cells. The significance of the dual PFC system in providing critical DO measurements for entrapped cells and other tissue constructs, in vitro and in vivo, is discussed. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758–766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 452–462, 2013     

Biomanufacturing process analytical technology (PAT) application for downstream processing: Using dissolved oxygen as an indicator of product quality for a protein refolding reaction

Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small‐scale bioreactor (3 L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse‐phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (k_La) is used for scale‐up of the aeration parameters to manufacturing scale tanks (2,000 L, 15,000 L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to “actively manage process variability using a knowledge‐based approach.” Biotechnol. Bioeng. 2009; 104: 340–351 © 2009 Wiley Periodicals, Inc.     

A case study in converting disposable process scouting devices into disposable bioreactors as a future bioprocessing tool

In this study, we perform mass transfer characterization (k_La) on a novel mechanically driven/stirred Process Scouting Device, PSD, (SuperSpinner D 1000®, SSD) and demonstrate that this novel device can be viewed as disposable bioreactor. Using patch‐based optical sensors, we were able to monitor critical cell culture environmental conditions such as dissolved oxygen (DO) and pH in SSD for comparison to a 1 L standard spinner (SS) flask. We also coupled these mass transfer studies with mixing time studies where we observed relative high mixing times (5.2 min) that are typically observed in production scale bioreactors. Decreasing the mixing time 3.5‐fold resulted in 30% increase in k_La (from 2.3 to 3.0 h^?1) and minimum DO level increased from 0% to 20% for our model hybridoma cell line. Finally, maximum viable cell density and protein titer stayed within ±20% of historical data, from our standard 5 L stirred bioreactor (Biostat®) operated under active DO control. Biotechnol. Bioeng. 2012; 109: 2790–2797. © 2012 Wiley Periodicals, Inc.     

Effect of temperature,pH, dissolved oxygen,and hydrolysate on the formation of triple light chain antibodies in cell culture

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB–drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed‐batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large‐scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ～50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Bioprocess considerations for T-cell therapy: Investigating the impact of agitation,dissolved oxygen,and pH on T-cell expansion and differentiation

Adoptive T-cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single-use 24-well microbioreactor (micro-Matrix) was assessed for its use as a high-throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion-mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 10⁶ cells/ml. The distribution of CD8+ central memory T cells (T_CM), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T-cell manufacturing process.     

Monitoring pH and dissolved oxygen in mammalian cell culture using optical sensors

Here, we have studied two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO₂) and pH, measured with fluorescent sensors thus allowing the study of the metabolic state of cells in culture without removing or damaging cells during cultivation. Two cell lines, namely, NS0 and CHO were batch-grown in 24-well plates at different serum concentrations with the sensors implemented in the bottom of each well. The data showed a good relationship between the dO₂ and pH data obtained from fluorescent probes and the growth and death characteristics of cells. The method has provided a high throughput on-line multi-parametric analysis of mammalian cell cellular activity.     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014