Anti-apoptotic genes, bag-1 and bcl-2, enabled hybridoma cells to survive under treatment for arresting cell cycle

Hybridoma 2E3-O cells were transfected with bcl-2 alone or with bcl-2 and bag-1 in combination. The bcl-2/bag-1 transfectant survived maintaining viability above 75% for almost 5 days when the cells were treated with excess (30 mM) thymidine for arresting cell cycle, whereas the mock transfectant survived for only 2 days, and the bcl-2 alone transfectant lived for 4 days. Owing to this extended viable culture period, the bcl-2/bag-1 transfectant produced twofold amount of antibody in comparison with the mock transfectant in non-proliferating state prepared by the excess thymidine treatment. When their proliferation was arrested by serum limitation, the bcl-2/bag-1 transfectant and the bcl-2 alone transfectant survived for 3 days maintaining viability above 75% while the mock transfectant survived only 1 day. The bcl-2/bag-1 transfectans produced the antibody at the rate three times as high as the bcl-2 alone transfectant and the mock transfectant in non-proliferating state established by serum limitation. Such genetic engineering of hybridoma cells for improving survival in the non-proliferating state will be useful for using nutrients in culture medium efficiently to produce antibody, since nutrients could be diverted from cell proliferation to antibody production in such non-proliferating viable cell culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Overexpression of bcl-2, apoptosis suppressing gene: Prolonged viable culture period of hybridoma and enhanced antibody production

Human bcl-2 DNA was introduced into mouse hybridoma 2E3 cells and expressed at a high level by using BCMGSneo vector, which reportedly amplifies as multiple copies in the cells independently of their chromosomes. The high expression of bcl-2 in BCMGSneo-bcl-2 transfectants was confirmed by western blotting. In batch cultures, the overexpression of bcl-2 raised the maximum viable cell density by 45%, delayed the initiation of apoptosis by 2 days, and prolonged the viable culture period by 4 days. The delayed initiation of apoptosis was detected by emergence of the ladder pattern on DNA electrophoresis and increase of the dead cell number. The bcl-2 transfectants produced lgG(1) fourfold per batch culture in comparison with 2E3 cells transfected with BCMGSneo but not with bcl-2: a little less than twofold due to the improved survival of the cells and more than twofold due to the enhanced lgG(1) production rate per cell of the bcl-2 transfectants. The method to engineer hybridoma cells genetically with bcl-2 using BCMGSneo vector for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. (c) 1995 John Wiley & Sons, Inc.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

Co-expression of bcl-2 and bag-1, apoptosis suppressing genes, prolonged viable culture period of hybridoma and enhanced antibody production

Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

High-Density culture of mouse-human hybridoma in serum-free defined medium

Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.     

Variable functions of bcl-2 in mediating bioreactor stress- induced apoptosis in hybridoma cells

It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures

The influence of Bcl-2 expression on the suppression of apoptosis during the cultivation of an NS0 cell line expressing a chimeric antibody was investigated. Following selection of transfectants in medium containing G418, Western analysis revealed evidence of some up-regulation of endogenous Bcl-2 expression even in the control vector transfectants. Cultivation of the two cell lines in suspension batch cultures clearly demonstrated the enhanced robustness of the bcl-2 vector transfected cells. Suppression of apoptosis resulted in an approximately 20% increase in maximum viable cell number, and a doubling in culture duration compared to the control transfected cells. However, despite the significant affect on viability, Bcl-2 expression did not result in an increase in final antibody titre in comparison with the control cell line. Exposure of cells to various nutrient limited conditions further emphasised the influence of Bcl-2 on cell survival. After 3 days of exposure to serum, glucose, glutamate and asparagine deprivation, the viable cell number and viability were significantly higher in the bcl-2 transfected cell line. When control cells were deprived of all amino acids, there was a complete loss of viability and viable cell number within 3 days. By contrast, the bcl-2 transfected cell line retained greater than 75% of the initial viable cell number and about 70% viability. In response to exposure to 8 mM thymidine (a cytostatic agent) the control cell line underwent complete loss of viability and viable cell number after 6 days. This compared with 18 days for complete loss of viability in the bcl-2 transfected cell line. As under batch culture conditions, there was no difference between the two cell lines in final antibody titre, which indicated that MAb synthesis is limited by nutrient availability during the latter stages of culture in both cases. When fed batch cultures were carried out using a concentrated essential amino acid feed, the bcl-2 cell line exhibited a 60% increase in maximum viable cell number and a 50% increase in culture duration, when compared to the control cell line. Moreover, the bcl-2 cell line exhibited a greater than 40% increase in maximum antibody titre.     

Production of human-mouse chimeric antibody by high cell density perfusion culture

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10⁷ cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10⁷ cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.     

Protective effect of viral homologues of bcl-2 on hybridoma cells under apoptosis-inducing conditions

Targets for metabolic engineering have been identified in a hybridoma cell line to make it more robust in culture toward potential limitations inducing apoptosis. The cells were genetically modified with plasmids harboring endogenous bcl-2 gene and also with viral Bcl-2 homologues, particularly ksbcl-2 and bhrf-1 genes. When cells were exposed to apoptosis-inducing conditions (i.e., glutamine-free medium), the control cells exhibited a decrease in viable cell number within the first 12 h, whereas, for the bcl-2 and ksbcl-2 transfected cell cultures, the viable cell number did not exhibit any clear decrease until after 60 h. Furthermore, hybridoma cells expressing the viral homologue bhrf-1 were even more resistant to cell death, showing a decrease in viability of only 50% at 72 h of culture in glutamine-deprived medium, substantially lower than the 90% viability decrease observed for the control culture. In addition, and most relevant for further bioprocess applications, the cells genetically modified could be brought back to growth conditions even after being exposed to glutamine-deprived conditions during a significant time window, up to 72 h.     

Serum free fed batch production of IgM

Summary Fed batch cultivation of a murine hybridoma secreting IgM in a serum free medium was successfully attempted. Cell growth and IgM productivity remained high for over a month, and compared well to a similar fed batch culture in medium supplemented with 10% fetal calf serum. The average specific secretion rates of antibody in both media were the same. Sufficient inoculum cell density was crucial to the establishment of a viable culture in serum free medium for the cell line, NS6.3, used.     

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.     

Phytohemagglutinin isolectin stimulation of human lympocytes cultured in serum free medium.

The ability of the red kidney bean (Phaseolus vulgaris) Phytohemagglutinin (PHA) isolectins L4 and E4 to transform human lymphocytes cultured in serum free or serum supplemented medium was studied. Previous similar studies done in fetal bovine serum (FBS) supplemented medium have shown L4 to be 30–60 times more potent a mitogen than E4. In serum free conditions, this difference was much less, L4 being only 3–9 times more potent than E4. In serum free medium, optimal mitogenic concentrations of L4 and E4 were 1.1.–3.3 ug/culture and 3.3–10 ug/culture respectively as compared to 3.3–10 ug/culture and 90–270 ug/culture for L4 and E4, respectively, in FBS suplemented medium. L4 stimulated lymphocytes in serum containing medium transform more rapidly than do L4 treated cells cultured in serum free conditions. Fetuin added to serum free cultures of lymphocytes more effectively inhibited transformation induced by E4 than by L4. Although the binding of 1251 E4 and L4 to lymphocytes was greatly reduced by the addition of FBS to the medium, the reduction in E4 binding was much greater than that in L4 binding. Neither L4 or E4 caused the death of lymphocytes cultured in serum free or FBS supplemented medium. These results confirm the previously described difference in mitogenic potential between L4 and E4. However, the results of earlier studies done in FBS supplemented medium include several artifacts related to the differential interaction of PHA isolections with fetuin and other serum glycoproteins.     

Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2

Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. (c) 1996 John Wiley & Sons, Inc.     

Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity

A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors. One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector. The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7. The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium. At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values. Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture. The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture. Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time. The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate. Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time. The volumetric production rate was, however, similar in both cultures. Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients. Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line.     

Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation

Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence oftetracycline) compared to MG12 populations culturedunder tetracycline-containing conditions (bcl-2repressed). However, bcl-2-expressing MG12 cellsshowed no significant retardation of the decline phasecompared to batch cultures in which the dicistronicexpression unit was repressed.Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hencederepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiology.     

Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma

Sericin, a constituent of the silkworm cocoon, was added to the culture of four mammalian cell lines: murine hybridoma 2E3-O,human hepatoblastoma HepG2, human epithelial HeLa and human embryonal kidney 293 cells. The proliferation of all cell lineswas accelerated in the presence of sericin. The hybridoma cellline was further studied. The 2E3-O cell line was so well adapted to serum-free medium that both the proliferation rate and maximum cell density in serum-free ASF103 medium were higher than in RPMI medium supplemented with all lots of FBS tested, and this proliferation was stimulated by the addition of sericin in a dose-dependent manner. Stimulation was observed at sericin concentrations from 0.01 to 0.1 %, although 1% sericin was severely harmful to the culture. In comparison with bovine serum albumin (BSA), a widely used supplement in serum-free medium, sericin had an equivalent effect on the proliferation of the hybridomas and sericin additively stimulated the proliferation with BSA. Although heat easily denatures and inactivates most proteins, the activity of sericin was not affected by autoclaving. In a similar manner to the silkworm-derived sericin, recombinant sericin synthesized in E. coli also stimulated the hybridoma proliferation, irrespective of whether it was autoclaved or filtered. Since BSA is obtained from bovine serum and the risk of infections such as bovine spongiform encephalopathy cannot be eradicated, sericin derived from insects could be a preferable culture medium supplement for stimulating the proliferation of mammalian cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions

TB/C3 hybridoma cells were transected with either pEF-MClneopA or pEF bcl2-MClneopA vectors to produce a control cell line (TB/C3 pEF) and a cell line that overexpresses the "antiapoptotic" human bcl-2 protein (TB/C3 bcl2). Flow cytometry analysis of intracellular bcl-2 protein levels enabled near on-line monitoring of the stability of bcl-2 expression in the absence of drug selection. It was possible to maintain spontaneous selection of cells with the overexpression of bcl-2 protein during semicontinuous cultures at very low dilution rates, where cells were subjected to the selective conditions of nutrient limitation and high toxic metabolite concentrations. Interestingly, cells that overexpressed bcl-2 were adapted to suspension culture conditions significantly faster than control cells. Dual fluorescence staining with acridine orange and propidium iodide allowed for discrimination between viable, apoptotic, secondary necrotic, and necrotic cells, respectively. Compared with the usual trypan blue method of establishing culture viability, dual staining demonstrated that under stressful conditions a significant proportion of cells that excluded trypan blue were also undergoing cell death through apoptosis. In batch cultures the overexpression of bcl-2 more than doubled the membrane intact (MI) cell productive period (the integral of Ml cell density with respect to culture time) and increased the monoclonal antibody (mAb) production by approximately 40% when compared with the control cell line. The overexpression of bcl-2 protein also significantly extended the cell integrity and viability by the suppression of apoptosis in conditions of hypoxia, hyperoxia, glutamine deprivation, glucose deprivation, and serum limitation. The suppression of apoptosis in anaerobic conditions suggests that bcl-2 exerts its antiapoptotic activity by a mechanism that does not involve an oxidative reactive pathway. In conditions of excess thymidine, which suppressed cell proliferation, Ml cell density and specific mAb productivity were further enhanced by the overexpression of bcl-2, which suggests the possibility of accomplishing a controlled proliferation in immortalized cell lines without invoking cell death. Cell size and intracellular mAb were increased for TB/C3 bcl2 cells compared with TB/C3 pEF control cells when analyzed by flow cytometry. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 1-16, 1997.     

Effects of three-dimensional culturing in a fibrous matrix on cell cycle, apoptosis, and MAb production by hybridoma cells

The effects of culturing hybridoma cells in a three-dimensional (3-D) poly(ethylene terephthalate) (PET) fibrous matrix on cell cycle, apoptosis, metabolism, and monoclonal antibody (MAb) production were evaluated by comparing with two-dimensional (2-D) culturing on microcarrier and multiwell plate surfaces. The percentage of cells in the G1/G0 phase increased during the long-term culturing period of approximately 4 weeks. Compared to the 2-D culture systems, cells grown in 3-D matrices had higher MAb productivity for long-term culture. Decreasing serum content in the culture medium increased both MAb productivity and apoptosis. However, the 3-D culture had a greater increase in MAb productivity and a much lower apoptotic rate than the 2-D culture, especially at 0% serum. Most cells in the 3-D fibrous matrix formed large aggregates and were smaller than cells grown on a 2-D surface or in suspension. The smaller cell size allowed cells to survive better in the high-cell-density environment. The fibrous matrix also selectively retained healthy, nonapoptotic cells. These results suggested that the 3-D fibrous matrix contributed to growth arrest, protected cells to better resist low-serum environments, and reduced apoptosis, all of which contributed to the high viable cell density and volumetric MAb productivity in the long-term 3-D culture.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant

Summary Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mm lactate exhibited intracellular lactate and glutamine levels of 40 mm and 4 mm, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mm lactate concentrations. For cells cultivated in medium supplemented with 6 mm pyruvate, intracellular lactate levels were estimated at approx. 40 mm, but these cells also showed an increased intracellular alanine concentration of 22 mm. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.