Oxygen uptake rates in cultured rat hepatocytes

One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large-scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long-term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 +/- 0.57 pmol/sec/mug DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 +/- 1.5 pmol/sec/mug DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage-dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. (c) 1992 John Wiley & Sons, Inc.     

Characterization and modulation of fibroblast/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks

Various assays of different complexity are used in research on angiogenesis in health and disease. The results of these assays increasingly impact the field of tissue engineering because preformed microvascular networks may connect and conduct to the vascular system of the host, thereby helping us to support the survival of implanted cells and tissue constructs. An interesting model that supports the formation of EC (endothelial cells) tubular structures in vitro is based on co-culturing them with fibroblasts. Our initial multilayer approach was recently transferred into a three-dimensional spheroid model using HUVEC (human umbilical vein endothelial cells) as model cells. The aim of the present study is to further characterize, extend and validate this fibroblast/EC spheroid co-culture system. We have evaluated the model with a maximum size of 600-650 μm attained on day 3 from inoculation of 4×104 fibroblasts with 1×104 EC. Cell count and spheroid diameter significantly decreased as a function of time, but the EC network that developed over a period of 14 days in culture was clearly visible and viable, and central cell death was excluded. We successfully included HMVEC (human microvascular endothelial cells) of dermal origin in the system and replaced FBS (fetal bovine serum) with human AB serum, which positively impacted the EC network formation at optimized concentrations. The need for exogenous growth factors [VEGF (vascular endothelial growth factor), EGF (epithelial growth factor), bFGF (basic fibroblast growth factor) and IGF-1 (insulin-like growth factor-1)] routinely added to classical EC media was also assessed. The behaviour of both fibroblasts and EC in response to a combination of these exogenous growth factors differed critically in fibroblast/EC spheroid co-cultures compared with the same cells in the multilayer approach. VEGF was the most relevant exogenous factor for EC network formation in fibroblast/EC multilayers, but was ineffective in the spheroid system. IGF-1 was found, in general, to be dispensable; however, while it had a negative impact on EC networking in the presence of bFGF and EGF in the multilayer, it did not in the spheroid approach. We conclude that the critical determinants of EC network formation and cell survival are not universal, but have to be specifically optimized for each culture model.     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Estimation of rates of oxygen uptake and carbon dioxide evolution of animal cell culture using material and energy balances

Material and degree of reductance balance equations are used to estimate the rates of oxygen uptake and carbon dioxide evolution of animal cell cultures. Lumped compositions, molecular weight and reductance degree of cellular protein, monoclonal antibody, biomass and amino acid consumption (excluding glutamine and alanine) are found to be relatively constant for different hybridoma cell lines and may be used as regularities. The calculated rates of oxygen uptake and carbon dioxide evolution agree well with experimental values of several different cultures reported in the literature. This simple method gives the same results as calculated on the basis of a detailed metabolic reaction network. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gas‐permeable membranes and co‐culture with fibroblasts enable high‐density hepatocyte culture as multilayered liver tissues

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Specific oxygen, ammonia, and nitrate uptake rates of a biological nutrient removal process treating elevated salinity wastewater

Anaerobic/anoxic/aerobic systems inoculated without and with NaCl acclimated cultures, i.e., Models A and B, respectively, were fed with a synthetic wastewater at various salinity levels. After achieving a steady state, the systems were shocked with 70 g/l NaCl for four consecutive days before returning to pre-shock conditions. At the steady-state, the specific oxygen uptake rates (SOURs) increased with an increase of sodium chloride concentration (from 5.40 to 9.72 mg O₂/g mixed liquor suspended solids (MLSS)-h at 0–30 g/l NaCl for Model A and from 6.84 to 17.64 mg O₂/g MLSS-h at 5–30 g/l NaCl for Model B). In contrast, the specific ammonia uptake rate (SAUR) and specific nitrate uptake rate (SNUR) decreased with increasing chloride concentration (from 4.76 to 2.14 mg NH₃–N/g MLSS-h and 2.50 to 1.22 NO3–N/g MLSS-h, for Model A, and from 3.84 to 2.71 mg NH₃–N/g MLSS-hr and 2.54 to 1.82 mg NO₃–N/g MLSS-hr, for Model B). During the shocked period, the SOUR in most scenarios increased whereas the SAUR and SNUR tended to decrease. The impact of the chloride shock on nitrifiers was more obvious than on denitrifiers; however, after a certain recovery period, the activities of both nitrifiers and denitrifiers in terms of SAUR and SNUR were approximately the same as those prior to shock.     

Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell^?1 s^?1, and 5 and 35 amol cell^?1 s^?1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.     

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.     

Correlation of O2 uptake rate and mitochondrial activity in carrot cell cultures exposed to laminar fluid shear

Measuring uncoupled oxygen uptake rate (OUR) could provide a convenient method for quantifying changes in the metabolic activity of plant cultures caused by hydrodynamic shear. Experiments on Daucus carota (carrot) cells were performed in a novel O₂-permeable Couette viscometer at varying levels of laminar shear (6 to 100 N m^–2). When the uncoupled OUR of the cells was compared with mitochondrial activity (determined by 2,3,5-triphenyl tetrazolium chloride assay), a significant correlation was observed (R=0.91 by linear regression).     

杂交瘤细胞培养中摄氧速率(OUR)的在线检测及其与细胞生长及代谢的关系

在批式及灌流培养条件下研究了杂交瘤细胞在无血清培养基中的生长、代谢情况与氧消耗的关系。应用动力学方法在线进行OUR的检测,同时离线取样检测其他参数。结果发现OUR与谷氨酰胺的消耗、抗体的生成及活细胞密度间有明显的相关关系,进一步的分析还发现在对数生长期,OUR与活细胞密度间具有良好的线性关系,qOUR(0.103±0.028)×10^-12mol/cell/h,可以通过它来进行细胞密度的在线检测。并通过以ΔOUR=0时刻作为灌流调整点进行连续灌流培养的初步实验验证了OUR作为培养过程反馈控制参数的可能性。     

Determination of in vivo oxygen uptake and carbon dioxide evolution rates from off-gas measurements under highly dynamic conditions

In vivo kinetics of Saccharomyces cerevisiae are studied, in a time window of 150 s, by analyzing the response of O(2) and CO(2) in the fermentor off-gas after perturbation of chemostat cultures by metabolite pulses. Here, a new mathematical method is presented for the estimation of the in vivo oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) directly from the off-gas data in such perturbation experiments. The mathematical construction allows effective elimination of delay and distortion in the off-gas measurement signal under highly dynamic conditions. A black box model for the fermentor off-gas system is first obtained by system identification, followed by the construction of an optimal linear filter, based on the identified off-gas model. The method is applied to glucose and ethanol pulses performed on chemostat cultures of S. cerevisiae. The estimated OUR is shown to be consistent with the independent dissolved oxygen measurement. The estimated in vivo OUR and CER provide valuable insights into the complex dynamic behavior of yeast and are essential for the establishment and validation of in vivo kinetic models of primary metabolism.     

Optimization of the cell seeding density and modeling of cell growth and metabolism using the modified Gompertz model for microencapsulated animal cell culture

Cell microencapsulation is one of the promising strategies for the in vitro production of proteins or in vivo delivery of therapeutic products. In order to design and fabricate the optimized microencapsulated cell system, the Gompertz model was applied and modified to describe the growth and metabolism of microencapsulated cell, including substrate consumption and product formation. The Gompertz model successfully described the cell growth kinetics and the modified Gompertz models fitted the substrate consumption and product formation well. It was demonstrated that the optimal initial cell seeding density was about 4-5 x 10(6) cells/mL of microcapsule, in terms of the maximum specific growth rate, the glucose consumption potential and the product formation potential calculated by the Gompertz and modified Gompertz models. Modeling of cell growth and metabolism in microcapsules provides a guideline for optimizing the culture of microencapsulated cells.     

Inhibition of uncoupling protein 2 by genipin reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Uncoupling protein 2 (UCP2) was reported to be involved in insulin-glucose homeostasis, based on well established event that inhibition of UCP2 stimulates insulin secretion in pancreatic β-cells. However, the role of UCP2 on insulin-stimulated glucose uptake in adipose tissue, which is an indispensable process in insulin-glucose homeostasis, remains unknown. In this study, UCP2 was inhibited by genipin in 3T3-L1 adipocytes, which increased mitochondrial membrane potential, intracellular ATP level and production of reactive oxygen species (ROS). Importantly, insulin-stimulated glucose uptake in 3T3-L1 adipocytes was largely impaired in the presence of genipin, and recovered by CCCP, a mitochondrial uncoupler. Furthermore, genipin leaded to suppression of insulin signal transduction through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). These results suggest that mitochondrial uncoupling in adipocytes positively regulates insulin-stimulated glucose uptake in adipocytes, and UCP2 may play an important role in insulin resistance.     

Analysis of noise and bias in fermentation oxygen uptake rate data

The calculation of many derived fermentation variables such as the respiratory quotient (RQ) and mass transfer coefficient (K(L)a) requires the differences between the molar percentages of oxygen and carbon dioxide in the fermentor inlet and exit gas, called the %OUR and %CER. Noise and bias in %CER data is of order that in the exit gas carbon dioxide analysis. However, the relative amount of noise in the %OUR is one to two orders of magnitude greater than the noise in the raw oxygen analyses because the %OUR is calculated as a small difference between two large quantities. The noise in the %OUR is white with a Gaussian amplitude probability distribution of absolute standard deviation 0.0145. A chi-square filter of the %OUR data is shown to considerably improve the quality of the calculated RQ and K(L)a for a fermentation of Streptomyces clavuligerus. (c) 1992 John Wiley & Sons, Inc.     

Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli

A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.     

The use of oxygen uptake rate to monitor discovery of microbial and enzymatic biocatalysts

Arising from the requirement for discovery of novel biocatalysts with unusual properties, a process was developed which uniquely combines aspects of continuous culture with the measurement of oxygen uptake. This adaptation of the chemostat can be used to facilitate the isolation of a number of microorganisms with desirable properties, particularly those with useful metabolic capabilities and/or enzymes. The technique was also used to provide feedback on the metabolic status of a microbial population and increase the feed flow rate (i.e., dilution rate) thereby enabling the isolation of microorganisms with enhanced 1,3‐propanediol dehydrogenase activity. The use of oxygen uptake as an indicator of cellular activity enables indirect measurement of substrate utilization and provides a real‐time online assessment of the status of microbial enrichment or evolutionary processes and provides an opportunity, through the use of feedback systems, to control these processes. To demonstrate the utility of the technique, oxygen uptake rate (OUR) was compared with a range of conventional analytical techniques that are typically used to monitor enrichment/evolutionary processes and showed good correlation. Further validation was demonstrated by monitoring a characterizable microbial population shift using OUR. The population change was confirmed using off‐line analytical techniques that are traditionally used to determine microbial activity. OUR was then used to monitor the enrichment of microorganisms capable of using a solvent (1‐methyl‐2‐pyrrolidinone) as the sole source of carbon for energy and biomass formation from a heterogeneous microbial population. After purification the microorganisms taken from the enrichment process were able to completely utilize 1 g L^?1 1‐methyl‐2‐pyrrolidinone within 24 h demonstrating that the technique had correctly indicated the enriched population was capable of growth on 1‐methyl‐2‐pyrrolidinone. The technique improves on conventional microbial enrichment that utilizes continuous culture by providing a real‐time assessment of the enrichment process and the opportunity to use the OUR output for automated control and variation of one or more growth parameters. Biotechnol. Bioeng. 2009;102: 673‐683. © 2008 Wiley Periodicals, Inc.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element