Showing papers in "Cytotechnology in 1996"

Engineering challenges in high density cell culture systems

Abstract: High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO2, a significant accumulation of dissolved CO2 is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.

CO(2) in large-scale and high-density CHO cell perfusion culture.

Abstract: Productivity in a CHO perfusion culture reactor was maximized when pCO2 was maintained in the range of 30–76 mm Hg. Higher levels of pCO2 (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO2 production rates for CHO cells in perfusion culture at 5.55×10-17 mol cell-1 sec-1 and 5.36×10-17 mol cell-1 sec-1 respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (kLa+kAA) for oxygen and carbon dioxide were 0.07264 min-1 and 0.002962 min-1 respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×107 cells/mL, the low CO2 removal efficiency would limit culture density to only 2.4×106 cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO2 transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2–3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO2 levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO2) for a culture at 107 cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.

Baculovirus-insect cell interactions

Abstract: Baculovirus interactions with host cells range from the physical interactions that occur during viral binding and entry, to the complex and subtle mechanisms that reculate host gene expression and modify and regulate cellular and organismal physiology and defenses. Fundamental studies of baculovirus biochemistry and molecular biology have yielded many interesting and important discoveries on the mechanisms of these virus-host interactions. Information from such studies has also resulted in exciting new strategies for environmentally sound insect pest control, and in the development and improvement of a valuable eukaryotic expression vector system. In addition a number of important and valuable model biological systems have emerged from studies of baculoviruses. These include robust systems for studies of eukaryotic transcription, viral DNA replication, membrane fusion, and apoptosis. Because functions have been identified for only a small number of baculovirus genes, we can expect many exciting new discoveries in the future and an unfolding of the complex and intricate relationship between baculoviruses and insect cells.

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production.

Abstract: Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K( L ) a and of mixing (via pH measurements) in bioreactors up to 8 m(3) at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO(2) has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.

Production of monoclonal antibodies and development of an ELISA for solamargine.

Abstract: The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml−1 to 11.5 nmol ml−1 of solamargine.

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

Abstract: 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 μmax and \(q_{O_2 }\) 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.

Development and characterization of insect cell lines

Abstract: With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the midgut, it may not be possible to obtain cell lines from these tissues from all insect species due to terminal differentiation and other factors. Also, researchers have desired cell lines from certain species, such as the honey bee, for which no success has been obtained. As in the early days of tissue culture, it is difficult to discern why negative results occur. However, as more is learned about the physiology and nutrition of various insects and tissues, we may get clues which will help solve these questions. The remaining chapters in this book will provide the reader with exciting uses for insect cell culture. As I mentioned earlier, the baculovirus expression vector system has provided a stimulus to the field of insect cell culture not seen previously.

Relationship of LRP-human major vault protein to in vitro and clinical resistance to anticancer drugs

Abstract: Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cell lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor forin vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs.

Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells.

Abstract: Production of a TGFβ receptor with high density baculovirus infected Sf-9 cells (7×106cells ml-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGFβ receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO2 partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO2 at this concentration has been assessed at smaller scale for TGFβ receptor and β-gal expression, in controlled atmosphere incubators.

Super-CHO-A cell line capable of autocrine growth under fully defined protein-free conditions

Abstract: Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6) cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6) cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Insect cell cultivation: growth and kinetics.

Abstract: The complete strategy for maximizing the yield of recombinant proteins from insect cell culture must include an optimization of the culture conditions during the growth phase as well as during the subsequent infection phase. The growth of host cells like Spodoptera frugiperda (Sf9 and Sf21) and Trichoplusia ni (BTI-Tn-5Bl-4) to cell densities of ca. 10 × 10E6 cells mlO1 in batch cultures has so far been achieved. Already today some groups have reported even higher viable cell concentrations (>10 × 10E6 cells mlO1) using nutrient feeding strategies. There will be further improvements in this area. However, probably more important will be the characterization of the optimal physiological state that the cells? at high densities? have to be in at the time of infection so as to maintain the same (or reach even higher) specific productivities than in low-density infections.

The prophylactic use of antibiotics in cell culture.

Abstract: This article describes the historical development of the prophylactic use of antibiotics in cell culture as well as their effects on cells. The influence of antibiotics on cell morphology, cellular degeneration and cell death and cellular function is summarized. Cellular DNA as well as protein synthesis are affected which can lead to interference with, or even changes in, metabolic processes. Such effects must be considered in cell culture research. As antibiotics are used in multifold ways, the otherwise standardized conditions in cell culture are no longer comparable. The prophylactic use of antibiotics is rejected for scientific reasons.

Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ.

Abstract: The CHO cell line has achieved considerable commercial importance as a vehicle for the production of human therapeutic proteins, but is known to lack a functional copy of the gene coding for α2,6-sialyltransferase (EC 2.4.99.1). The cDNA for rat α2,6-ST was expressed in a recombinant CHO cell line making interferon-γ, using a novel in vitro amplification vector. The enzyme was expressed efficiently, and resulted in up to 60% of the total sialic acids on interferon-γ being linked in the α2,6-conformation. This sialic acid linkage distribution was more akin to that seen in natural human glycoproteins. In the most successful cell clones, expression of α2,6-sialyltransferase improved the overall level of sialylation by up to 56%, and had no adverse effects on cell growth, IFN-γ productivity or other aspects of IFN-γ glycosylation. These experiments demonstrate how the glycosylation machinery of rodent cells can be genetically manipulated to replicate human tissues.

Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression.

Abstract: Foreign protein production levels in two recombinant Chinese hamster ovary (CHO) cell lines were compared in cells transfected with different expression vectors. One vector pNL1 contained the gene for neomycin resistance (neo r ) and thelacZ gene which codes for intracellular β-galactosidase, with both genes controlled by the constitutive simian virus (SV40) promoter. The other vector CDβG contained the amplifiabledhfr gene andlacZ gene, controlled by the constitutive SV40 and cytomegalovirus (CMV) promoters, respectively. Cell growth and β-galactosidase expression were compared quantitatively after cells were selected in different concentrations of the neomycin analog G418 and methotrexate, respectively. A 62% reduction in growth rate occurred in recombinant CHO cells in which thelacZ anddhfr genes were highly amplified and expressed. In contrast, the combined effects of the unamplifiedneo r gene andlacZ gene expression on the growth kinetics were small. Any metabolic burden caused bylacZ gene expression, which was evaluated separately from the effect ofneo r gene expression, must be negligible, as higher expression of β-galactosidase (1.5×10−6 units/cell) occurred in unamplified cells compared to the cells in whichlacZ was amplified by thedhfr-containing vector (3×10−7 units/cell). Thus, the main factor causing severe growth reduction (“metabolic burden”) in cells containing the amplifieddhfr gene system was not overexpression of β-galactosidase butdhfr andlacZ gene co-amplification anddhfr gene expression.

Large scale transient expression with COS cells.

Abstract: We demonstrate here that transient expression with COS cells can be performed at the one litre scale for a period of more than 10 days. Cells grown in T225 flasks were transfected by electroporation, transferred into spinners, and then grown either in suspension or on microcarriers. A daily medium change significantly extented culture life and production time, compared with standard protocols.

Production of monoclonal antibodies and ELISA for thebaine and codeine.

Abstract: The ratio of hapten and bovine serum albumin in antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. Monoclonal antibodies against thebaine and codeine were produced by hybridoma fused with the sprenocytes immunized with thebaine- and codeine-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. No cross-reaction of anti-thebaine antibody against morphine was observed. Very small cross-reaction appeared in codeine (0.004%). The cross-reaction of anti-codeine antibody against morphine and thebaine was 2.97 and 5.98%, respectively. The full measuring range of the assay extends from 60 pg mL to 1 ng mL for thebaine and 1 ng mL to 100 ng mL for codeine.

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker.

Abstract: A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and β-cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H* were adapted to enable them to grow serum-free in the absence of cholesterol and β-cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×106 cells ml−1 producing 76.6 mg l−1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.

Ammonium ion transport-a cause of cell death.

Abstract: Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na+K+2Cl--cotransporter in both hybridoma and myeloma cells. Further, the Na+/H+ exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH inf4 sup+ for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na+/H+ exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.

Economics of baculovirus-insect cell production systems

Abstract: Commercial production of pharmaceutical proteins in baculovirus-insect cell systems is already a reality, and has therefore not been discussed in detail here. Cost-efficacywill depend on the productivity of the protein in culture, the dose, and the quantities required. According to the model described here, cost-effective production of baculoviruses for use in agriculture should also be feasible, assuming the commercial availability of a low-cost medium, together with a baculovirus with high productivity in cell culture, which is effective at a field application rate of 1012 PIB ha-1 or lower. All of these criteria appear to be achievable, given fairly modest advances over currently available technology. Given the relatively high fixed costs associated with production of baculoviruses on an agricultural scale in bioreactors however, profitability will depend on the scale of production. A substantial market opportunity (perhaps in the order of 1 million hectares) would be necessary in order to exploit the economies of scale achievable with baculovirus—insect cell production systems.

Regulation of cell growth by IRF-1 in BHK-21 cells.

Abstract: Most cell lines that are used for the production of recombinant proteins proliferate spontaneously at a high rate. In many types of cultivation systems these cells still keep growing after having reached the desired cell density. Further proliferation in batch cultures leads to cell death as a consequence of nutrient and oxygen depletion as well as to accumulation of lactate and toxic products. Consequently, in many technical processes, the surplus of cells is removed. We have established cell lines in which proliferation is controlled by a physiological regulator, IRF-1. IRF-1 (Interferon Regulatory Factor 1) is a transcriptional activator and acts as a tumor suppressor. Constitutive overexpression of recombinant IRF-1 leads to inhibition of cell growth. The extent of this growth arrest depends on the intracellular concentration of active IRF-1. To allow IRF-1 expression in various mammalian cells a system for conditional IRF-1 activation has been established. A fusion protein composed of IRF-1 and the hormone binding domain of the human estrogen receptor, was used. This system allows to control gradually the growth of several mammalian cell lines by adjusting the intracellular concentration of active IRF-1 via estradiol in the medium. We have evaluated BHK-21 cells with respect to IRF-1 mediated cell growth inhibition and expression of two secreted proteins. Whereas the productivity of proliferation inhibited cells with respect to constitutively transcribed IgG genes is reduced, productivity of another secreted protein which is controlled by an IRF-1 inducible promoter is strongly enhanced under these conditions.

Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages.

Abstract: A novel type of succinoyl trehalose lipid (STL-1) prepared from n-hexadecane-culture ofRhodococcus erythropolis SD-74 markedly inhibited the growth of a human monocytoid leukemic cell line, U937, and induced its morphological alteration along a monocyte-macrophage lineage. STL-1 markedly increased differentiation-associated characteristics in macrophage, such as nitroblue tetrazolium reducing ability, appearance of Fc receptor, phagocytic activities in U937. Furthermore, U937 cells, which were activated with STL-1 exhibited cytotoxic activity against human lung carcinoma cell line A549. However, STL-1 did not affect growth of a normal human fetal lung cell line TIG-1. The individual components of STL-1, neither sugar moiety nor fatty acids in the free form, were effective at inducing the differentiation of U937 cell. From these results, we concluded that STL-1 has low cytotoxicity against normal human cells and the ester molecule itself is responsible for the activity of inducing differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophage which results in the stimulation of the production of some cytotoxic substances.

Insect cell physiology.

Abstract: Viruses from the genus Baculovirus, are attractive as pesticides and as vectors for producing foreign proteins in insect cells. because insect cells are relatively easy to cultivate, faithfully perform some of the post-translational modifications, allow for complex formation to take place between the two proteins coexpressed in cultures infected with two hybrid viruses, and often can produce large quantities of proteins, baculovirus technology has found its way to a number of undustrial applications for expression of a variety of proteins (see review Murhammer, 1991; O’Reilly et al., 1992). In addition to proteins, production of baculovirus itself as insecticide represents a major potential for use of insect cell cultures (Wood, 1995). The final product yield whether a protein or the virus, is influenced by several factors such as the cell line (eg. Lynn & Hink, 1980; McIntosh & Grasela, 1984; Hink et al., 1991; Wickham et al., 1992) and virus type (Fraser, 1989), passage number of virus (Wickham et al., 1991), medium composition (eg. Cho et al., 1989; Hink et al., 1991), dissolved oxygen concentration (eg. Scott et al., 1992; Wang et al., 1993), time and the multiplicity of infection (MOI) (Licari & Bailey, 1991; Bedard et al., 1994) nature of protein (Hink et al., 1991), cell density (Wickham et al., 1992) and stage of growth and metabolism (eg. Caron et al., 1990; Lindsay et al., 1992; Reuveny et al., 1993). Despite significant advance in the genetics of Baculovirus/insect cell expression system, our understanding of cellular physiology during preand post infection is relatively limited. An expanded understanding of metabolic features of insect cells will prove extremely useful to the bioprocess and biochemical engineers who work towards improving insect cell culture productivity. Several recent articles have addressed consumption of carbohydrate and amino acid and formation of the metabolic by-products in insect cell cultures. Those studies, reviewed first in this article, have significantly enhanced the metabolic knowledge of insect cells. Substantially more insights, however, may be obtained by a regirous analysis of primary metabolic pathways. Our recent work (Ferrance et al., 1993), has demonstrated the feasibility of metabolic flow analysis for insect cell growth in a serum-free medium. This manusscript also provides a summary of metabolic pathway analysis and highlights the assumptions, describes the lessons learned, and ourlines the future work in a more critical fashion than is available in ferrance et al. (1993). Finally, future research directions critical for attaining detailed metabolic insights are outlined in the last section of this manuscript.

CHO cell growth and recombinant interferon-γ production: Effects of BSA, Pluronic and lipids

Abstract: The role of bovine serum albumin in mammalian cell cultures and the possibility of its substitution by other components in a serum-free medium has been investigated. In this study, BSA was shown to be important for growth and product formation in CHO cells expressing recombinant human interferon-γ. There were indications that its stimulating growth effect was dependent on the source of BSA used and probably was related to the purification procedure used for the production of the desired albumin fraction. Cell growth did not occur in the absence of BSA but at low concentration (1 mg ml−1) it was stimulated by the addition of a combination of a commercial lipid mixture plus Pluronic F68. However, under the latter conditions IFN-γ production was adversely effected. The importance of individual lipid components was investigated using a statistical approach based on a Plackett-Burman design. Linoleic acid was identified as a positive variable for cell growth while cholesterol was identified as a negative variable for both cell growth and IFN-γ production. When a combination of linoleic acid plus Pluronic F68 was included in the formulation of low BSA medium, cell growth was similar to that at high BSA concentration (5 mg ml−1) but the IFN-γ concentration was significantly reduced (ca. 45%).

Parvovirus diagnostics and vaccine production in insect cells

Abstract: Baculoviruses have been successfully used to express large amounts of heterologous proteins for various applications (Luckow & Summers, 1988; Luckow, 1991). Recombinant baculoviruses can be produced on a large scale in insect cells. Insect cell cultures offer an attractive alternative to traditional systems for manufacturing of pharmaceuticals, biologicals and diagnostics products. High level expression of heterologous genes makes the baculovirus expression system specially suitable for those cases where large amounts of protein are needed, for instance, production of subunit vaccines or diagnostic reagents. These new generation vaccines will help to obviate the risks associated to the production and use of live-modified viruses in classical vaccines. The aim of this review is to summarize the use of recombinant baculoviruses for the production of vaccines and diagnostic reagents useful for parvoviruses (Table 1). Also, possible ways to improve the levels and the quality of the expression are presented. Finally, an study about the genetic stability of the system is shown.

Shear sensitivity of insect cells

Abstract: While insect cells can be easily damaged in bioreactors as a result of hydrodynamic forces, it is also relatively easy to prevent this damage. Of several possible damage mechanisms, the best understood and preventable is the attachment of cells to gas-liquid interfaces and the subjection of these attached cells to the hydro-dynamic forces and/or physical forces associated with these interfaces. For example, cells attached to gas bubbles in a bioreactor can be transported into the foam layer where they are physically removed from the cell suspension, or they can be killed when the gas bubble they are attached to ruptures at the medium-air interface at the top of the bioreactor. The easiest method to prevent this damage is through the use of specific surface active compounds, such as Pluronic F-68 or Methocel E-50 which prevent the cells from attaching to the gas-medium interface.

Cell suicide in starving hybridoma culture : survival-signal effect of some amino acids

Abstract: Two mouse hybridoma cell lines cultured in different basal media withthe iron-rich protein-free supplement were subjected to deliberatestarvation by inoculation into media diluted with saline to 50% or less.In the diluted media the growth was markedly suppressed and a largefraction of cells died by apoptosis. The cells could be rescued fromapoptotic death by individual additions of amino acids, such as glycine,L-alanine, L-serine, L-threonine, L-proline, L-asparagine, L-glutamine,L-histidine, D-serine, β-alanine or taurine. Amino acids withhydrophobic or charged side chains were without effect. The apoptosispreventing activity manifested itself even in extremely diluted media,down to 10% of the standard medium. The activity of L-alanine in theprotection of cells starving in 20% medium was shown also in semicontinuousculture. In the presence of 2 mM L-alanine the steady-state viable cell density more than doubled, with respect to control, andthe apoptotic index dropped from 37% in the control to 16%. It wasconcluded that the apoptosis-preventing amino acids acted as signalmolecules, rather than nutrients, and that the signal had a character ofa survival factor. The specificity of present results, obtained with twodifferent hybridomas, supports our view (Franěk and Chladkova-Sramkova, 1995) that the membranetransport macromolecules themselves may play the role of therecognition elements in a signal transduction pathway controlling thesurvival of hybridoma cells.

Bcl-2 inhibits apoptosis and extends recombinant protein production in cells infected with Sindbis viral vectors.

Abstract: Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2 cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h-1). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neo cells, which were all dead by three days post-infection. Though infected AT3-neo cells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2 cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2 cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.