Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads

Our overall objective is to develop a cell culture analogue bioreactor (CCA) that can be used together with a corresponding physiologically based pharmacokinetic model (PBPK) to evaluate molecular mechanisms of toxicity. The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal. The CCA bioreactor is a physical replica of the PBPK; where the PBPK specifies organs, the CCA bioreactor contains compartments with a corresponding cell type that mimics some of the characteristic metabolism of that organ. The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium. The CCA bioreactor and the model can be coupled to evaluate the plausibility of the molecular mechanism that is input into the model. This paper focuses on the design, development, and characterization of a CCA bioreactor to be used in naphthalene dose response studies. A CCA bioreactor prototype developed previously is improved upon by culturing the cells on microcarrier beads. Microcarrier beads with cells attached can form packed beds with cell culture medium perfusing the beds. In this study, two packed beds of cells, one with L2 cells (rat lung) and one with H4IIE cells (rat hepatoma), are linked in a physiologically relevant arrangement by a common recirculating cell culture medium. Studies of this CCA bioreactor presented here include mixing profiles, effect of reactor environment on cell viability and intracellular glutathione, naphthalene distribution profile, and initial naphthalene dosing studies. Unlike the prototype system there is no detectable response to naphthalene addition; in a companion paper we show that this discrepancy can be explained by differences in liquid residence times in the organ compartments. The perfusion reactor design is shown to have significant operating improvements over prototype designs.     

The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors

Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome. A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials. A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments. A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK. These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate. The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system. Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells. Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in. (2.54 cm) square silicon chip. With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model. A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices.     

Development of a microscale cell culture analog to probe naphthalene toxicity

Prediction of human response to drugs or chemicals is difficult as a result of the complexity of living organisms. We describe an in vitro model that can realistically and inexpensively study the adsorption, distribution, metabolism, elimination, and potential toxicity (ADMET) of chemicals. A microscale cell culture analog (microCCA) is a physical replica of the physiologically based pharmacokinetics (PBPK) model. Such a microfabricated device consists of a fluidic network of channels to mimic the circulatory system and chambers containing cultured mammalian cells representing key functions of animal "organ" systems. This paper describes the application of a two-cell system, four-chamber microCCA ("lung"-"liver"-"other tissue"-"fat") device for proof-of-concept study using naphthalene as a model toxicant. Naphthalene is converted into reactive metabolites (i.e., 1,2-naphthalenediol and 1,2-naphthoquinone) in the "liver" compartment, which then circulate to the "lung" depleting glutathione (GSH) in lung cells. Such microfabricated in vitro devices are potential human surrogates for testing chemicals and pharmaceutics for toxicity and efficacy.     

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

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

Effects of naphthalene metabolites on cultured cells from eye lens

Naphthalene is toxic to the eye and results in opacification of the lens. To investigate the metabolic events that may be occurring in the lens epithelial cells, a cell line of lens from a transgenic mouse was incubated with various metabolites of naphthalene. Naphthoquinone at 50 microM was toxic to most cells with a depletion of glutathione levels noted within 6 h of incubation. At 10 microM, naphthoquinone caused an increase in specific activity of the enzyme DT-diaphorase. This enzyme is thought to be a defense against quinones since semiquinone formation is thought to be lessened. Naphthalene-1,2-dihydrodiol at 50 microM also caused an increase in the specific activity of the DT-diaphorase, while at 10 microM no apparent change occurred in the cells. Although there was evidence of metabolic alterations in the cells with the metabolites of naphthalene, the protein profile by two-dimensional gel electrophoresis did not change and there was no indication of an increase in carbonyl formation in the soluble proteins of the cells. These experiments indicate that the metabolites of naphthalene can cause alteration in the metabolism of the lens cells but may not cause apparent changes in the major proteins within the lens epithelium.     

Scaling-down biopharmaceutical production processes via a single multi-compartment bioreactor (SMCB)

Biopharmaceutical production processes often use mammalian cells in bioreactors larger than 10,000 L, where gradients of shear stress, substrate, dissolved oxygen and carbon dioxide, and pH are likely to occur. As former tissue cells, producer cell lines such as Chinese hamster ovary (CHO) cells sensitively respond to these mixing heterogeneities, resulting in related scenarios being mimicked in scale-down reactors. However, commonly applied multi-compartment approaches comprising multiple reactors impose a biasing shear stress caused by pumping. The latter can be prevented using the single multi-compartment bioreactor (SMCB) presented here. The exchange area provided by a disc mounted between the upper and lower compartments in a stirred bioreactor was found to be an essential design parameter. Mimicking the mixing power input at a large scale on a small scale allowed the installation of similar mixing times in the SMCB. The particularities of the disc geometry may also be considered, finally leading to a converged decision tree. The work flow identifies a sharply contoured operational field comprising disc designs and power input to install the same mixing times on a large scale in the SMCB without the additional shear stress caused by pumping. The design principle holds true for both nongassed and gassed systems.     

New miniaturized hollow-fiber bioreactor for in vivo like cell culture, cell expansion, and production of cell-derived products

We have developed a miniaturized hollow-fiber bioreactor system for mammalian cell culture with a volume of 1 mL. Cell and medium compartments of the bioreactor are separated by a semipermeable membrane, and oxygenation of the cell compartment is accomplished using an oxygenation membrane. As a result of the geometry of the transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60, and REH were cultivated up to densities of 3.5 x 10(7)/mL without medium change or manipulation of the cells. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with flow rate. Depending on the molecular size cutoff of the membranes used, added growth factors such as GM-CSF, as well as factors secreted from the cells, are retained in the cell compartment for up to 1 week. This new miniaturized hollow-fiber bioreactor offers advantages in tissue engineering by continuous nutrient supply for cells in high density, retention of added or autocrine produced factors, and undisturbed long-term culture in a closed system.     

Conversion of 1-naphthol to naphthoquinone metabolites by rat liver microsomes: demonstration by high-performance liquid chromatography with reductive electrochemical detection

1-Naphthol has recently been shown to be selectively toxic to short-term organ cultures of human colorectal tumor tissue. The mechanism underlying 1-naphthol's selective toxicity is as yet unknown, but may be due to the formation of naphthoquinone metabolites, which are known to be highly toxic to tumor cells. By using high-performance liquid chromatography with reductive electrochemical detection, it has been possible to show that 1-naphthol is converted to naphthoquinone metabolites by rat liver microsomes. At least two metabolic pathways, independent of cytochrome P-450, appear to be involved. Iron-dependent lipid peroxidation appears to be responsible for at least part of the conversion of 1-naphthol to predominantly 1,4-naphthoquinone, and it seems likely that superoxide anion radical generation by NADPH-cytochrome P-450 reductase could also catalyze this conversion. 1-Naphthol therefore seems to be converted to cytotoxic naphthoquinone metabolites by mechanism(s) dependent upon the generation of free radicals in rat liver microsomes. The results also demonstrate the utility of HPLC with reductive electrochemical detection for investigations of quinone metabolite formation and the measurement of quinones of both physiological and environmental interest.     

Compartmental architecture and dynamics of hematopoiesis

BACKGROUND: Blood cell formation is maintained by the replication of hematopoietic stem cells (HSC) that continuously feed downstream "compartments" where amplification and differentiation of cells occurs, giving rise to all blood lineages. Whereas HSC replicate slowly, committed cells replicate faster as they become more differentiated. METHODOLOGY/SIGNIFICANT FINDING: We propose a multi-compartment model of hematopoiesis, designed on the principle of cell flow conservation under stationary conditions. Cells lost from one compartment due to differentiation are replaced by cells from the upstream compartment. We assume that there is a constant relationship between cell input and output in each compartment and fix the single parameter of the model using data available for granulocyte maturation. We predict that approximately 31 mitotic events separate the HSC from the mature cells observed in the circulation. Besides estimating the number of compartments, our model allows us to estimate the size of each compartment, the rate of cell replication within each compartment, the mean time a given cell type contributes to hematopoiesis, the amplification rate in each compartment, as well as the mean time separating stem-cell replication and mature blood-cell formation. CONCLUSIONS: Despite its simplicity, the model agrees with the limited in vivo data available and can make testable predictions. In particular, our prediction of the average lifetime of a PIG-A mutated clone agrees closely with the experimental results available for the PIG-A gene mutation in healthy adults. The present elucidation of the compartment structure and dynamics of hematopoiesis may prove insightful in further understanding a variety of hematopoietic disorders.     

A novel system for evaluation of drug mixtures for potential efficacy in treating multidrug resistant cancers

Multidrug resistant (MDR) cancer is difficult to treat. Chemicals that are effective MDR modulators have never exited clinical trials as FDA approved products due to side effects. It has been hypothesized that using a combination of chemotherapeutics with a mixture of MDR modulators (each with different side effects) may lead to useful treatment strategies. Because the experimental space for combination treatments can be large, this space may be impracticable to explore using animal studies. Here we describe an in vitro system based on microfabrication and cell culture that can potentially be used to explore large experimental spaces efficiently. The Microscale Cell Culture Analog (µCCA) concept mimics the body's response using interconnected compartments that represent various tissues or organs. A µCCA is based on the structure of an appropriate physiologically based pharmacokinetic (PBPK) model and emulates the body's dynamic response to exposure to various drugs and chemicals. For this problem we have chosen a µCCA with living cells representing the liver (HepG2/C3A), bone marrow (MEG‐01), uterine cancer (MES‐SA), and a MDR variant of uterine cancer (MES‐SA/DX‐5). In proof of concept experiments we found in 24 h “acute” exposures and 72 h treatments that the µCCA system predicts combining the chemotherapeutic, doxorubicin, with cyclosporine and nicardipine, as MDR modulators will have greater efficacy than using doxorubicin by itself or with either modulator alone. This combined strategy is selective in inhibiting MES‐SA/DX‐5 cell proliferation and may prove to be advantageous in vivo by specifically targeting MDR cancer with acceptable side‐effects. This cell specific synergy was not observed in traditional 96‐well plate assays. By combining the µCCA with a PBPK model, appropriate drug doses and area under the curve exposure for in vivo trials can be extrapolated directly from the results obtained with this device. This device and approach should be useful in screening potential drug/modulator combinations to determine candidate treatments for MDR cancer. Indeed this approach may be useful for in vitro evaluation of human response to a wide range of exposures to mixtures of chemicals or drugs. Biotechnol. Bioeng. 2009;103: 187–198. © 2008 Wiley Periodicals, Inc.     

Ultrastructural localization of the circulating anodic antigen and the circulating cathodic antigen in the liver of mice infected with Schistosoma mansoni: a sequential study

The present study describes the ultrastructural localization of two important circulating schistosome antigens--the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA)--in livers of mice at various time intervals after infection with Schistosoma mansoni. For the demonstration of these antigens at the electron microscope level use was made of a direct, double immunogold labeling procedure, in which CAA-specific monoclonal antibodies, labeled with 5-nm gold particles, and CCA-specific monoclonal antibodies, labeled with 15-nm gold particles, were used. Both antigens were localized in granules and in inclusion bodies of Kupffer cells and granuloma macrophages and it was found that in these compartments the degree of 5- and 15-nm gold labeling increased with the duration of the infection. Sometimes gold particles were also encountered on the cell surface and in endocytotic vesicles of these cells, in endothelial cells, and in the space of Disse. From these data it was concluded that in the liver CAA and CCA were primarily accumulated in granules and inclusion bodies of Kupffer cells and granuloma macrophages. It is discussed whether at these locations both antigens are degraded by lysosomal enzymes and whether these antigens are complexed with antibodies.     

Notch signaling is not sufficient to define the affinity boundary between dorsal and ventral compartments

The developing limbs of Drosophila are subdivided into distinct cells populations known as compartments. Short-range interaction between cells in adjacent compartments induces expression of signaling molecules at the compartment boundaries. In addition to serving as the sources of long-range signals, compartment boundaries prevent mixing of the adjacent cell populations. One model for boundary formation proposes that affinity differences between compartments are defined autonomously as one aspect of compartment-specific cell identity. An alternative is that the affinity boundary depends on signaling between compartments. Here, we present evidence that the dorsal selector gene apterous plays a role in establishing the dorsoventral affinity boundary that is independent of Notch-mediated signaling between dorsal and ventral cells.     

Polarized secretion of urokinase-type plasminogen activator by epithelial cells.

Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis.     

Chromium(VI) reduction in a membrane bioreactor with immobilized Pseudomonas cells

Chromate reduction was studied in a membrane bioreactor under action of Pseudomonas bacteria immobilized in agar–agar films on the surface of synthetic membrane. Immobilized cells are protected from the excessive toxic action at high chromate concentration that improves cell activity compared with free cells. Almost complete chromate reduction was observed at stepwise introducing of chromate in feed solution allowing maintenance of optimal chromate concentration. Reduction is suppressed by high metabolite concentrations, which reached on the sixth step of chromate adding in studied system. Cell ability to reduce chromate is restored after changing of feed and receiving solutions allowing remediation of Cr(VI)-contaminated water in semi-batch operation of membrane bioreactor.     

Functional symmetry of endomembranes

In higher eukaryotic cells pleiomorphic compartments composed of vacuoles, tubules and vesicles move from the endoplasmic reticulum (ER) and the plasma membrane to the cell center, operating in early biosynthetic trafficking and endocytosis, respectively. Besides transporting cargo to the Golgi apparatus and lysosomes, a major task of these compartments is to promote extensive membrane recycling. The endocytic membrane system is traditionally divided into early (sorting) endosomes, late endosomes and the endocytic recycling compartment (ERC). Recent studies on the intermediate compartment (IC) between the ER and the Golgi apparatus suggest that it also consists of peripheral ("early") and centralized ("late") structures, as well as a third component, designated here as the biosynthetic recycling compartment (BRC). We propose that the ERC and the BRC exist as long-lived "mirror compartments" at the cell center that also share the ability to expand and become mobilized during cell activation. These considerations emphasize the functional symmetry of endomembrane compartments, which provides a basis for the membrane rearrangements taking place during cell division, polarization, and differentiation.     

Incorporation of 3T3-L1 cells to mimic bioaccumulation in a microscale cell culture analog device for toxicity studies

Deficiencies in the early ADMET (absorption, distribution, metabolism, elimination, and toxicity) information on drug candidates extract a significant economic penalty on pharmaceutical firms. We have developed a microscale cell culture analog (microCCA) device that can potentially provide better, faster, and more efficient prediction of human and animal responses to a wide range of chemicals. The system described in this paper is a simple four-chamber microCCA ("lung"-"liver"-"fat"-"other tissue") designed on the basis of a physiologically based pharmacokinetics (PBPK) model of a rat. Cultures of L2, HepG2/C3A, and differentiated 3T3-L1 adipocytes were selected to mimic the key functions of the lung, liver, and fat compartments, respectively. Here, we have demonstrated the application of the microCCA system to study bioaccumulation, distribution, and toxicity of selected compounds. Results from the bioaccumulation study reveal that hydrophobic compounds such as fluoranthene preferentially accumulated in the fat chamber. Only a small amount of fluoranthene was observed in the liver and lung chambers. In addition, the presence of the differentiated 3T3-L1 adipocytes in the microCCA device significantly reduced naphthalene and naphthoquinone-induced glutathione (GSH) depletion. These findings suggest the potential utilization of the microCCA system to assess ADMET characteristics of the compound of interest prior to animal or human trials.     

Scale-up impacts on mass transfer and bioremediation of suspended naphthalene particles in bead mill bioreactors

Scale-up effects on mass transfer and bioremediation of suspended naphthalene particles have been studied in 20 and 58L bead mill bioreactors and compared to data generated earlier with a laboratory scaled bioreactor. The bead mill bioreactor performance with respect to naphthalene mass transfer rate was dependent on the size and loading of the inert particles, as well as the rotational speed of the roller apparatus. The optimum operating conditions were found to be 15mm glass beads at a loading of 50% (total volume of particles/working volume of bioreactor: v/v%) and a bioreactor rotational speed of 50rpm. The highest naphthalene mass transfer coefficients obtained in the large scale system under these optimum conditions (19.6 and 22.4h(-1) for 20 and 58L vessels, respectively) were higher than those determined previously in a 2.5L bead mill bioreactor (0.7h(-1)). The acute toxicity tests indicated that the bioreactor effluent was less toxic than the untreated naphthalene suspension. Biodegradation rates obtained in these large scale bead mill bioreactors under optimum conditions (36-37.4mgL(-1)h(-1)) were higher than those achieved in the control bioreactors of similar sizes (11.4 and 11.6mgL(-1)h(-1)) but were slower than those previously determined in a 2.5L bead mill bioreactor (59-61.5mgL(-1)h(-1)). The limitation of oxygen in the large scale systems and damage of the bacterial cells due to the crushing effects of the large beads are likely contributing factors in the lower observed biodegradation rates. The optimum conditions with respect to naphthalene mass transfer might not necessarily translate to optimum performance with regard to bioremediation.