Effects of pH-variations on the kinetics of growth and energy metabolism in cultured T-lymphoma cells: a microcalorimetric study.

The progression of T-lymphoma cells (CCRF-CEM) growing in suspension has been monitored during long term (12-28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH, p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 10(6) cells per ml). The lowering of p(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmol/l, respectively, did not influence Pcell or the growth pattern. No "crowding effect" was observed for the cells in the investigated concentration range (0.6-1.3) x 10(6) cells per ml.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.     

A new method for on-line measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures

Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liquid phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and constant k(L)a value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured on-line with a very good accuracy of +/-5%, and the specific OUR for a CHO cell line was determined during batch (growth phase) and continuous culture as, respectively, equal to 2. 85x10(-13) and 2.54x10(-13) mol O(2) cell(-1) h(-1). It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the cell concentration itself, especially during transition phases like the end of the growth phase in a batch culture.     

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。     

Xylose metabolism in Debaryomyces hansenii UFV-170. Effect of the specific oxygen uptake rate

The new yeast Debaryomyces hansenii UFV-170 was tested in this work in batch experiments under variable oxygenation conditions. To get additional information on its fermentative metabolism, a stoichiometric network was proposed and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. The yeast metabolism resulted to be practically inactive under strict oxygen-limited conditions (qO2 = 12.0 mmol(O2) C-mol(DM)(-1) h(-1)), as expected by the impossibility of regenerating NADH2+. Significant fractions of the carbon source were addressed to both respiration and biomass growth under excess oxygen levels (qO2 > or = 55.0 mmol(O2) C-mol(DM)(-1) h(-1)), thus affecting xylitol yield (Y(P/S) = 0.41-0.52 g g(-1)). Semi-aerobic conditions (qO2 = 26.8 mmol(O2) C-mol(DM)(-1) h(-1)) were able to ensure the best xylitol production performance (Pmax = 76.6 g L(-1)), minimizing the fractions of the carbon source addressed either to respiration or biomass production and increasing Y(P/S) up to 0.73 g g(-1). An average P/O ratio of about 1.0 mol(ATP) mol(O)(-1) allowed estimation of the main kinetic-bioenergetic parameters of the biosystem. The overall ATP requirements of biomass were found to be particularly high and dependent on the oxygen availability in the medium as well as on the physiological state of the culture. Under semi-aerobic and aerobic conditions, they varied in the ranges 13.5-15.4 and 9.74-10.2 mol(ATP) C-mol(DM)(-1), respectively, whereas during the best semi-aerobic bioconversion they progressively increased from 5.68 to 24.7 mol(ATP) C-mol(DM)(-1). After a starting phase of adaptation to the medium, the cell achieved a phase of decelerated growth during which its excellent xylose-to-xylitol capacity kept almost constant after 112 h up to the end of the run.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Human 293 cell metabolism in low glutamine-supplied culture: interpretation of metabolic changes through metabolic flux analysis

Metabolic flux analysis is a useful tool to analyze cell metabolism. In this study, we report the use of a metabolic model with 34 fluxes to study the 293 cell, in order to improve its growth capacity in a DMEM/F12 medium. A batch, fed-batch with glutamine feeding, fed-batch with essential amino acids, and finally a fed-batch experiment with both essential and nonessential amino acids were compared. The fed-batch with glutamine led to a maximum cell density of 2.4x10(6) cells/ml compared to 1.8x10(6) cells/ml achieved in a batch mode. In this fed-batch with glutamine, it was also found that 2.5 mM ammonia was produced compared to the batch which had a final ammonia concentration of 1 mM. Ammonia was found to be growth inhibiting for this cell line at a concentration starting at 1 mM. During the fed-batch with glutamine, the flux analysis shows that a majority of amino acid fluxes and Kreb's cycle fluxes, except for glutamine flux, are decreased. This observation led to the conclusion that the main nutrient used is glutamine and that during the batch there is an overflow in the Kreb's cycle. Thus, a fed-batch with glutamine permits a better utilization of this nutrient. A fed-batch with essential amino acid without glutamine was also assayed in order to reduce ammonia production. The maximum cell density was increased further to 3x10(6) cells/ml and ammonia production was reduced below 1 mM. Flux analysis shows that the cells could adapt to a medium with low glutamine by increasing the amino acid fluxes toward the Kreb's cycle. Adding nonessential amino acids during this feeding strategy did not improve growth further and the nonessential amino acids accumulated in the medium.     

Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.     

谷氨酰胺和天冬酰胺对CHO细胞生长、代谢及抗体表达的影响

以离心换液的批培养为例,通过设计谷氨酰胺和天冬酰胺不同的添加方式来考察两者对CHO细胞生长,代谢及产物表达的影响。结果表明：基础培养基中谷氨酰胺和天冬酰胺不能简单地相互替换,缺失谷氨酰胺或天冬酰胺的基础培养基均不能支持dhfr-CHO细胞的正常生长和产物表达,仅谷氨酰胺和天冬酰胺的浓度同时达到4mmol/L,才能满足细胞生长所需。另外,代谢副产物氨的生成仅与谷氨酰胺和天冬酰胺的加和线性相关,与两者添加比例无关。但适当提高天冬酰胺与谷氨酰胺的比例可提高抗体表达水平,同时减少乳酸的生成。因此,为培养基开发与优化过程中谷氨酰胺和天冬酰胺的添加策略提供了依据,为建立高效的流加培养过程奠定了基础。     

On-line gas analysis in animal cell cultivation: II. Methods for oxygen uptake rate estimation and its application to controlled feeding of glutamine

Different methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge of K(L)a, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, a new method of aeration was designed. Oxygen and CO(2) transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100-fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O(2), CO(2), Ar, and N(2). The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on-line methods for OUR measurement were tested during the cultivation of animal cells. The new method allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. (c) 1995 John Wiley & Sons, Inc.     

Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production

An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD-18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed-batch culture using a serum-free, glutamine-free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On-line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 x 10(9) cells/L and a final IVC of 1.6 x 10(12) cells-h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed-batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783-792, 1997.     

Rabies virus production in high vero cell density cultures on macroporous microcarriers

The purpose of the study was to investigate the rabies virus multiplication in Vero cell cultures performed on porous microcarriers, MCs (cellulose-Cytopore and gelatin-Cultispher G), which provide higher available surface area compared with solid (nonporous) MCs (DEAE-Cytodex 1). In a set of experiments performed at the same MC concentration (MCs per milliliter), cell densities regularly obtained in porous MC cultures were comparable, but almost twice as high as those in solid MC cultures. In addition, 41.1 +/- 3.9-, 35.2 +/- 2-, and 19.6 +/- 5.8-fold increases in cell concentration, relative to the initial cell number, along with maximum rabies virus titers of 6.3 +/- 0.3 x 10(4), 5 +/- 0.1 x 10(4), and 4.3 +/- 0.2 x 10(4) FFD(50)/mL were observed in Cytopore, Cultispher G, and Cytodex 1 MC cultures, respectively. When higher concentrations of MCs were employed, lower performances of virus production and MC-cell occupation (cells per MC or cells per square millimeter) were observed. Cell attachment to MCs was shown to be faster for Cytopore MCs and Cytodex 1 MCs than for Cultispher G MCs. Concerning the kinetics of cell multiplication on MCs, exponential cell growth, at similar specific cell growth rates, took place on Cytopore, Cultispher G, and Cytodex 1 MCs. In addition, cell densities as high as 2.1 +/- 0.2 x 10(6) cells/mL on Cytopore MCs, 1.8 +/- 0.1 x 10(6) cells/mL on Cultispher G MCs, and 1 +/- 0.3 x 10(6) cells/mL on Cytodex 1 MCs were regularly obtained in batch cultures. Optical as well as scanning and transmission electron microscopy studies carried out to analyze MC structure, MC cell occupation, and cell permissivity to virus infection demonstrated that there was uniform cell distribution in the external and internal areas of the MCs, suggesting an efficiency of virus synthesis. Our results indicate the usefulness of these supports for rabies virus antigen production, as well as possibilities for further optimization.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

葡萄糖对重组CHO细胞生长代谢及EPO表达的影响

在CHO细胞批培养中 ,葡萄糖浓度从8.9增加到49.6mmol/L ,最大活细胞密度没有明显的差异 ,乳酸对葡萄糖的得率系数首先随着葡萄糖浓度的增加而增加 ,葡萄糖浓度达到 17.9mmol/L后 ,乳酸对葡萄糖的得率系数基本上维持恒定。在本实验中 ,葡萄糖浓度对谷氨酰胺代谢没有明显的影响。EPO的累积浓度首先随着起始葡萄糖浓度的增加 ( 8.9～ 17.9mmol L)而增加 ,进而又随着葡萄糖浓度的增加 (17.9～ 49.6mmol L)而下降 ,表明存在一最适浓度 ,在此浓度下重组CHO细胞的EPO表达最大。     

Oxygen uptake rate regulation during cell growth phase for improving avermectin B<Subscript>1a</Subscript> batch fermentation on a pilot scale (2 m<Superscript>3</Superscript>)

Avermectin B_1a batch fermentation of Streptomyces avermitilis in a 2 m³ fermentor was investigated by oxygen uptake rate (OUR) regulation during cell growth phase. OUR was controlled by adjusting of aeration and agitation. Result showed that OUR strongly affected cell growth and antibiotics production. Avermectin B_1a biosynthesis could be effectively enhanced when OUR was stably regulated at an appropriate level in batch fermentation of S. avermitilis. Avermectin B_1a yield reached 5568 ± 111 mg/l by controlling maximal OUR between 15 and 20 mmol/l/h during cell growth phase, which was increased by 21.8% compared with the control (maximal OUR above 20 mmol/l/h). The stimulation effect on avermectin B_1a production could be attributed to the improved supply of propionic acid and acetic acid, the precursors of avermectin B_1a, in the cells. Hence, this OUR control method during cell growth phase may be a simple and applicable way to improve industrial production of avermectin.     

Specific oxygen uptake rate variations during batch fermentation of Bacillus thuringiensis subspecies kurstaki HD-1

The specific oxygen uptake rate (q(O)2, respiration rate) of Bacillus thuringiensis subsp. kurstaki HD-1 was very high at inoculation and was found to decrease essentially monotonically throughout both vegetative growth phase and transition phase under different batch culture conditions. Average q(O)2 values decreased from 8-10 mmol/g h at 1 h after inoculation to less than 2 mmol/g h by the time growth ended. The results are shown to be consistent with the few previous reports on q(O)2 in B. thuringiensis in the literature but also novel in that this pattern of monotonic decline has not been described previously. Both pH control and EDTA in low concentration shortened the vegetative growth phase and reduced the 10 h biomass concentration. Using plots of q(O)2 versus specific growth rate, mu, biomass yield based on the oxygen used for growth, was calculated for transition phase to be 0.041-0.047 g/mmol, consistent with literature values. The same plot also showed that the presence of EDTA resulted in an atypical q(O)2-mu trajectory and apparently much higher biomass yield from the oxygen consumed.     

Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported K(s) vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NH(x)/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures. (c) 1994 John Wiley & Sons, Inc.     

锌诱导的蛋白合成增加可抵抗链佐霉素所致的胰岛细胞损伤

本工作在离体细胞水平观察ZnGl_2对链佐霉素(STZ)诱发的胰岛β细胞损伤的保护作用,并分析其可能的作用机制。结果如下:向培养的胰岛细胞中加入生理盐水和STZ(3mmol/L),孵育12h后,活细胞数由实验前的70万个/ml降至43.93±1.16万个/ml;将ZnCl_2(0.25、0.5、1.0mmol/L)和相同剂量的STZ一同加入细胞,可不同程度地缓解STZ对胰岛的破坏作用,在含不同浓度ZnCl_2的培养液中活细胞数分别恢复至47.39±0.88,58.06±2.29,67.72±1.48万个/ml,与STZ破坏组相比,分别具有显著差异,并呈量效关系。在给予ZnCl_2(1.0mmol/L)的同时,向细胞中加入蛋白合成抑制剂亚胺环已酮(100μg/ml),可翻转ZnCl_2的作用,活细胞数由63.17±2.15万个/ml,又重新减至45.77±0.76万个/ml。单独加亚胺环己酮对活细胞数目无明显影响。用~3H-亮氨酸掺入实验观察ZnCl_2对胰岛细胞蛋白合成的影响发现,单独给予ZnCl_2(1.0mmol/L)仅使蛋白合成轻微增加,与盐水对照组无显著差异;在给ZnCl_2的同时加入STZ,则蛋白合成明显增多,保护组与STZ破坏组比较,差异显著。上述结果表明,增加细胞内蛋白合成,以加强细胞自身对外来损伤的修复力,可能是ZnCl_2保护胰岛细胞的机制之一。     

Metabolism of PER.C6 cells cultivated under fed-batch conditions at low glucose and glutamine levels

This is the first study to examine PER.C6 cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6(TM) cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced q(Gln) and q(NH(4)(+)) by approximately 30%, q(Ala) 85%, and q(NEAA) 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced q(Glc) about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y(O(2)/Glc) of approximately 6 mol/mol. However, despite no increase in q(Gln), an increased ammonium ion accumulation and Y(NH(4)(+)/Gln) were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6 cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The q(Gln) value was again reduced and, despite an increased q(NH(4)(+)) compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6 cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6 cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism.