Growth limiting factors influencing high density culture of insect cells in Grace's medium

Summary Growth limiting factors influencing the high density cultivation of insect cells were studied. A stationary phase in Grace's medium was found to be due to the deprivation of the active form of FBS components. Various compounds were added in the early stationary phase to observe the recovery of cell growth. With the addition of yeastolate, final cell densities were 4-fold and 3.5-fold higher in monolayer and suspension cultures, respectively. Pluronic F-68 increases the specific growth rate and the growth yield of the cell as well as protects the cell from the shear damage.     

High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10⁷ cells/ml, when the specific perfusion rate was set to 2.3 vol day^-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.     

High cell density culture of Yarrowia lipolytica using a one-step feeding process

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.     

High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor

A simple feeding strategy was developed and successfully employed for nutritional control in a 2-L fed-batch culture of hybridoma cells. A previously developed stoichiometric model for animal cell growth was used to design a supplemental medium for feeding. Undialyzed fetal bovine serum and trace metals (Fe(2+), SeO(3) (2-), Li(+), Zn(2+), and Cu(2+)) were fed to the cells periodically in addition to the automatic feeding of other nutrients in the supplemental medium. In this study, the maximum viable cell density was increased from 6.3 x 10(6) to 1.7 x 10(7) cells/mL, and the culture span was extended from 340 to 550 hours. The final monoclonal antibody titer achieved was 2400 mg/L. The specific production rates for ammonia and lactate were further reduced from 0.0045 and 0.0048 in our previous fed-batch experiments to 0.0028 and 0.0036 mmol/10(9) cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional batch culture.     

Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies

In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end-products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL-1606) was cultivated in T-flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed-batch culture and by 26-fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed-batch culture and by 50-fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five- and tenfold respectively, compared with conventional batch culture. (c) 1994 John Wiley & Sons, Inc.     

Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed-batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 x 10(6) cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium

A strategy for fed-batch cultivation of t-PA producing recombinant CHO cells is presented, based on the substitution of glucose and glutamine for slowly metabolized nutrients and in a rational design of the medium. Media for the batch and fed stages were based on the cell specific amino acid requirements, which allowed a more accurate determination of the initiation of the fed stage and the frequency of nutrient addition from then on. Salt concentration was also reduced in both media to avoid an increase in osmolality. As a consequence of this rational design, most amino acid did not accumulate significantly during the fed stage, as usually occurs when their supply is not based on cell requirements; also, lower amounts of by-products were obtained when osmolality level was kept low, that altogether increased viability, longevity and t-PA production when compared with a reference batch culture. Alternating glucose and galactose during the fed stage, allowed lactate detoxification of the cells through their own metabolism. This allowed an increase in cell growth and cell viability with respect to a fed-batch culture in which only glucose was used in the fed stage.     

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.     

苏云金杆菌补料高密度培养的研究

研究了苏云金杆菌的补米高密度培养。系统探讨了补料方式、补料成分和补料时间对发酵水平的影响。结果表明,利用同样物料,补料高密度培养比分批培养的晶体含量提高４２．２％,效价提高３６．４％。     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

High density cell culture ofRhodotorula glutinis using oxygen-enriched air

Summary Fed-batch culture ofRhodotorula glutinis, an oleaginous yeast, was carried out to obtain high biomass concentration. With air, final concentration of biomass was 100–110 g/L, whereas with oxygen-enriched air, 185 g/L was obtained. Obligate aerobic metabolism ofR. glutinis under oxygen limitation and low oxygen requirement under lipidaccumulating condition were found to be advantageous for the high density culture of this microbe.     

Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium

Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.     

High density culture of anchorage-dependent animal cells by polyurethane foam packed-bed culture systems

Summary Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of polyurethane foam (PUF) and grew to a high cell density (1.1 × 10⁸ cells/cm³ PUF and 4.2 × 10⁷ cells/cm³ PUF, respectively) in a PUF-plates packed-bed culture system. This density of Vero cells was twice that obtained previously with a PUF-particles packed-bed culture system. A maximum cell density of 6.7 × 10⁷ cells/cm³ culture vessel volume was obtained in a PUF-disc packed-bed culture of Vero cells. From the cell density of CHO-K1, growing in a monolayer on the surface of PUF and a petri dish, per bulk volume of PUF, we estimated that a surface area to volume ratio of PUF plates effective for cell growth was about 109 cm²/cm³.Offprint requests to: K. Funatsu     

Primary culture of insect midgut cells

This protocol describes the preparation of primary cell cultures from Lepidopteran midgut. These cultures have been used to identify factors that control midgut growth and differentiation, cell responses to these factors, effects of toxins on midgut growth, and the regulation of cell physiology. The protocol is divided into (1) procedures for cell collection, (2) composition of the culture, and (3) assay methods used for cell health, proliferation, and differentiation. Collection and setup require 4–6 h. Once established, a culture can survive several months at 25°C, be kept a year or longer at 4°C, or be frozen for indefinite storage.     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.