Differential subcellular localization of hZip1 in adherent and non-adherent cells.

Two human divalent cation transporters of the ZIP family, hZip1 and hZip2, homologous to Irt1 (Arabidopsis thaliana), the first identified member, have been described. They were shown by transfection into K562 cells to be localized at the plasma membrane and to mediate zinc uptake. Here we report a differential subcellular localization of hZip1 according to cell type. By transient expressions of EGFP-hZip1, FLAG-tagged or native hZip1, we observed that hZip1 has a vesicular localization in COS-7 cells or in several epithelial cell lines, corresponding partially to the endoplasmic reticulum. Using anti-hZip1 antibodies, we confirmed the intracellular localization of the endogenous protein in PC-3, a prostate cancer cell line.     

Continuous column culture system for adherent cells

A new continuous column culture system for adherent cells was developed using beads. The beads were packed in a column and an appropriate medium was continuously passed through. The whole system was kept under closed conditions. L cells and C6 cells were cultured by this new system. The number of cells increased linearly up to 16 days and reached a maximum at around 18 days. As the heat production remained constant for 16 days, it can be concluded that cells grown in this system had identical characteristics. The final concentration of cells reached was 1.0 × 10⁸ml^?1. The cells could grow both in the upward and the downward direction. Advantages of this system are: (1) Cells can be recovered in their adherent form on the beads; (2) cells can easily be collected from the column by trypsinization, and (3) cells remaining in the column after trypsinization can grow again.     

Noninvasive quality estimation of adherent mammalian cells for transplantation

The noninvasive quality estimation of adherent mammalian cells for transplantation is reviewed. The quality and heterogeneity of cells should be estimated before transplantation because cultured cells are not homogeneous but heterogeneous. The estimation of cell quality should be performed noninvasively because most protocols of regenerative medicine are autologous cell system. The differentiation level and contamination of other cell lineage could be estimated by two-dimensional cell morphology analysis and tracking using a conventional phase contrast microscope. The noninvasive determination of the laser phase shift of a cell using a phase-shifting laser microscope, which might be more noninvasive, and more useful than the atomic force microscope and digital holographic microscope, was carried out to determine the three-dimensional cell morphology, and the estimation of the cell cycle phase of each adhesive cell and the mean proliferation activity of a cell population. Chemical analysis of the culture supernatant by conventional analytical methods such as ELISA was also useful to estimate the differentiation level of a cell population. Chemical analysis of cell membrane and intracellular components using a probe beam, an infrared beam, and Raman spectroscopy was useful for diagnosing the viability, apoptosis, and differentiation of each adhesive cell.     

High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10⁸ cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.     

哺乳动物细胞灌流培养工艺开发与优化

近年来生物药市场需求量激增,高产量、高质量、低成本的哺乳动物细胞灌流培养工艺顺势成为工业界和学术界普遍关注的热点。文中围绕灌流培养工艺特有的操作环节及工艺优化应着重关注的细节展开论述,综述了近年来在灌流培养工艺开发和优化上取得的进步和提出的策略,以期为哺乳动物细胞灌流培养技术的开发提供参考。     

Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells

This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes.     

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

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

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.     

Model simulation and analysis of perfusion culture of mammalian cells at high cell density.

Rate equations recently proposed by the authors for growth, death, consumption of nutrients, and formation of lactic acid, ammonium, and monoclonal antibody of hybridoma cells are used to simulate and analyze the behavior of perfusion cultures. Model simulations are in good agreement with experimental results from three different cell lines under varied perfusion and cell bleed rates except for cultures with very low viability. Analysis of simulations and experimental results indicates that in perfusion cultures with a complete cell separation cell bleed rate is a key parameter that strongly affects all the process variables, whereas the perfusion rate mainly affects the total and viable cell concentrations and the volumetric productivity of monoclonal antibody. Growth rate, viability, and specific perfusion rate of cells are only a function of the cell bleed rate. This also applies to cultures with partial cell separation in the permeate if the effective cell bleed rate is considered. It is suggested that the (effective) cell bleed rate of a perfusion culture should be carefully chosen and controlled separately from the perfusion rate. In general, a low cell bleed rate that warrants a reasonable cell viability appears to be desirable for the production of antibodies. Furthermore, model simulations indicate the existence of an optimum initial glucose concentration in the feed. For the cell lines considered, the initial glucose concentration used in normal cell culture media is obviously too high. The initial glutamine concentration can also be reduced to a certain extent without significantly impairing the growth and antibody production but considerably reducing the ammonia concentration. The mathematical model can be used to predict these optimum conditions and may also be used for process design.     

Error propagation from prime variables into specific rates and metabolic fluxes for mammalian cells in perfusion culture

Error propagation from prime variables into specific rates and metabolic fluxes was quantified for high‐concentration CHO cell perfusion cultivation. Prime variable errors were first determined from repeated measurements and ranged from 4.8 to 12.2%. Errors in nutrient uptake and metabolite/product formation rates for 5–15% error in prime variables ranged from 8–22%. The specific growth rate, however, was characterized by higher uncertainty as 15% errors in the bioreactor and harvest cell concentration resulted in 37.8% error. Metabolic fluxes were estimated for 12 experimental conditions, each of 10 day duration, during 120‐day perfusion cultivation and were used to determine error propagation from specific rates into metabolic fluxes. Errors of the greater metabolic fluxes (those related to glycolysis, lactate production, TCA cycle and oxidative phosphorylation) were similar in magnitude to those of the related greater specific rates (glucose, lactate, oxygen and CO₂ rates) and were insensitive to errors of the lesser specific rates (amino acid catabolism and biosynthesis rates). Errors of the lesser metabolic fluxes (those related to amino acid metabolism), however, were extremely sensitive to errors of the greater specific rates to the extent that they were no longer representative of cellular metabolism and were much less affected by errors in the lesser specific rates. We show that the relationship between specific rate and metabolic flux error could be accurately described by normalized sensitivity coefficients, which were readily calculated once metabolic fluxes were estimated. Their ease of calculation, along with their ability to accurately describe the specific rate‐metabolic flux error relationship, makes them a necessary component of metabolic flux analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min^–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10⁶ cells ml^–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

Perfusion culture apparatus for suspended mammalian cells

A variety of processes have been proposed for mammalian cell culture in the commercial production of useful substances (e.g., monoclonal antibodies, therapeutic and diagnostics proteins). Among them, the perfusion culture of suspended non-immobilized cells is the most advantageous. Perfusion culture can be classified by the separation process of suspended cells from the culture mixture into three types, namely filtration, gravitational settling and centrifugation. From a commercial point of view, the present situation and technical problems of suspended-cell perfusion culture will be reviewed based on the three types, The recent development of perfusion culture has been carried out mainly on the filtration separation process, but the centrifugation process seems to have a promising future because of operation stability and scale-up feasibility. The reasons will be explained in details.     

Influence of shear stress on adherent mammalian cells during division

Summary The influence of shear stress (0 to 4.5 N m^–2) on morphology and adherence of anchorage-dependent baby hamster kidney (BHK 21 cl3) cells during cell division was investigated by means of a time lapse film. With increasing shear stress level divided cells needed more time for spreading.     

Characteristics of the production of active oxygen species from adherent and non-adherent neutrophils

Our objective was to evaluate the characteristics of the production of AOS from the neutrophils that had adhered to the endothelial cells, fibronectin or polystyrene, using the method of electron paramagnetic resonance (EPR) spin trapping. Neutrophils and endothelial cells were isolated from human venous blood and umbilical veins, respectively. AOS production from neutrophils was not elicited only by adhesion. The stimulation of adherent neutrophils with phorbol myristate acetate (PMA) induced the production of AOS. The production of AOS from adherent neutrophils to endothelial cells, but not to fibronectin or polystyrene, decreased with the interval time between the adhesion and the stimulation by PMA. The amount of AOS produced by the neutrophils adherent to fibronectin or polystyrene was maintained for one hour after stimulation with PMA, whereas that by suspended neutrophils gradually decreased with the time after stimulation. Results indicate that adherent and non-adherent neutrophils exhibit differing time course of AOS production.     

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells.

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.