Repurposing fed-batch media and feeds for highly productive CHO perfusion processes

Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL⁻¹ cell⁻¹ day⁻¹. High productivities and favorable product quality are also achievable.     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

The isolation and characterization of a rumen chitinolytic bacterium

J.S. DICKSON, T.R. MANKE, i.v. WESLEY AND A.L. BAETZ. 1996. Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm² tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log₁₀ cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus , which was non-culturable in broth culture, attained population densities of 10⁹ cells ml^-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm² resulted in increased cell densities.     

Growth of human embryonic fibroblasts at clonal density: concordance with results from mass cultures.

In the past, it has been difficult to grow human diploid fibroblast cells at clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1) population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures at clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture.     

Density-dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells

In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development. In the current study, the effects of cell number and population density on drug-induced conversion of BeWo cells from the cytotrophoblastlike to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of "giant" cells, a process shown to require exogenous purines, and expression of placental (heat-stable) alkaline phosphatase. Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate-induced cell enlargement. Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two-threefold greater in sparsely populated than in densely populated cultures. Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response. When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate-induced cell enlargement was inversely related to population density. Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities. These results indicate that expression of markers of methotrexate-induced differentiation of BeWo cells was inhibited in a density-dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.     

Modulation of competence for genetic transformation in Streptococcus pneumoniae

The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6.8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.     

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells--which have no intrinsic resistance--owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.     

Passaging protocols for mammalian neural stem cells in suspension bioreactors

Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.     

Engineering challenges in high density cell culture systems

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.     

A self-feeding roller bottle for continuous cell culture

The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding.     

The effects of division rate-limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells

Chick embryo fibroblasts serially propagated in media containing division ratelimiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating ³H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated “senescent” cultures returned to 10% serum containing medium had proliferative rates, incorporated ³H-thymidine, and attained saturation densities at confluency similar to younger cells. “Senescent” cells serially propagated in low serum and returned to 10% serum achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis becuase of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative lifespan per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.     

Modeling and optimization of the baculovirus expression vector system in batch suspension culture

A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum-supplemented IPL-41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2-L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque-forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large-scale systems for the production of recombinant products using the baculovirus expression vector system. (c) 1994 John Wiley & Sons, Inc.     

A Single Dynamic Metabolic Model Can Describe mAb Producing CHO Cell Batch and Fed-Batch Cultures on Different Culture Media

CHO cell culture high productivity relies on optimized culture medium management under fed-batch or perfused chemostat strategies enabling high cell densities. In this work, a dynamic metabolic model for CHO cells was further developed, calibrated and challenged using datasets obtained under four different culture conditions, including two batch and two fed-batch cultures comparing two different culture media. The recombinant CHO-DXB11 cell line producing the EG2-hFc monoclonal antibody was studied. Quantification of extracellular substrates and metabolites concentration, viable cell density, monoclonal antibody concentration and intracellular concentration of metabolite intermediates of glycolysis, pentose-phosphate and TCA cycle, as well as of energetic nucleotides, were obtained for model calibration. Results suggest that a single model structure with a single set of kinetic parameter values is efficient at simulating viable cell behavior in all cases under study, estimating the time course of measured and non-measured intracellular and extracellular metabolites. Model simulations also allowed performing dynamic metabolic flux analysis, showing that the culture media and the fed-batch strategies tested had little impact on flux distribution. This work thus paves the way to an in silico platform allowing to assess the performance of different culture media and fed-batch strategies.     

Effects of glucose on the production of recombinant protein C in mammalian cell culture

Effects of glucose on a cultured Chinese hamster ovary cell line producing recombinant human protein C were investigated. After the recombinant cells reached confluency, they were maintained in the medium containing 10% serum and different levels of glucose in either batch or daily-exchange mode. High concentrations of glucose to the cultures yielded higher cell densities. Daily exchanges of media produced higher cell densities than the corresponding batch culture. Total protein C production per cell decreased with time in batch culture, in accordance with the declined glucose metabolism. Supplementation of the media with high levels of glucose diminished both the expression and gamma-carboxylation activities of the recombinant cells. Production of protein C persisted in daily-exchange culture, resulting in a constant production rate of protein C. In this case again, glucose reduced the specific productivity of recombinant protein C. An apparent glucose inhibition constant was determined to be 0.11 mg/mL by Dixon plots. The ability to gamma-carboxylate recombinant protein C was also impaired at the highest level of glucose. From these results, a strategy to maximize recombinant protein C productivity is discussed.     

The influence of culture conditions on mitotic activity in protoplasts derived fromPisum root cortical explants

Summary Protoplast cultures were prepared from explants of the roots of seedling peas. In defined, synthetic media these cultures were mitotically active. A variety of culture conditions were investigated and the influences of these conditions on the mitotic activity of the protoplasts were observed. Marked inhibition of mitoses were observed after exposure to high light intensities, and in the absence of a proper exogenous supply of hormones. The protoplasts showed extreme sensitivity to the nature and concentration of the exogenous hormone supply. The protoplasts were mitotically active at low population densities (6,000–8,000 protoplasts/ ml of medium). They did not divide in cultures in which glucose was supplied as a carbohydrate source, but divided actively in cultures in which sucrose (2%) was supplied. The responses to temperature and pH were similar to most protoplast systems which have been reported. The definition of a wide variety of optimal culture conditions resulted in high mitotic activity in protoplast cultures with low population densities.     

PUROMYCIN RESISTANCE IN HAPLOID AND HETEROPLOID FROG CELLS: GENE OR MEMBRANE DETERMINED?

The frequency of colony formation in monolayers of cultured frog cell lines treated with puromycin was compared in (a) haploid and heteroploid lines and (b) mutagen-treated and nontreated haploid lines. Evidence that resistant colonies result from gene mutation was negative, since the colony frequency is independent of both ploidy and mutagen treatment. A study of five frog cell lines showed that colony formation in puromycin depends on (a) the concentration of puromycin, (b) preselection of the population with puromycin, and, particularly, (c) the capacity of the treated population to survive some exposure to puromycin. One haploid and one heteroploid strain showing stable resistance to puromycin have been isolated; comparison of those variants with sensitive populations has shown that resistance to puromycin is correlated with the cells' capacity to exclude the drug. The evidence for different levels of membrane permeability, combined with evidence for many degrees of resistance among and within cell populations, suggests a model of self-determining membrane units. The evolution of a resistant phenotype may result from changes in the proportion of specific units in the membrane population.     

Use of a Tetrazolium-based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation

ABSTRACT. Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus . at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.     

Cell interactions within nascent neural crest cell populations transiently promote death of neurogenic precursors

We have previously shown that cultured trunk neural crest cell populations irreversibly lose neurogenic ability when dispersal is prevented or delayed, while the ability to produce other crest derivatives is retained (Vogel, K. S. and Weston, J. A. (1988) Neuron 1, 569-577). Here, we show that when crest cells are prevented from dispersing, cell death is increased and neurogenesis is decreased in the population, as a result of high cell density. Control experiments to characterize the effects of high cell density on environmental conditions in culture suggest that reduced neurogenesis is the result of cell-cell interactions and not changes (conditioning or depletion) of the culture medium. Additionally, we show that the caspase inhibitor zVAD-fmk, which blocks developmentally regulated cell death, rescues the neurogenic ability of high density cultures, without any apparent effect on normal, low-density cultures. We conclude, therefore, that increased cell interaction at high cell densities results in the selective death of neurogenic precursors in the nascent crest population. Furthermore, we show that neurogenic cells in cultured crest cell populations that have dispersed immediately are not susceptible to contact-mediated death, even if they are subsequently cultured at high cell density. Since most early migrating avian crest cells express Notch1, and a subset expresses Delta1 (Wakamatsu, Y., Maynard, T. M. and Weston, J. A. (2000) Development 127, 2811-2821), we tested the possibility that the effects of cell contact were mediated by components of a Notch signaling pathway. We found that neurogenic precursors are eliminated when crest cells are co-cultured with exogenous Delta1-expressing cells immediately after they segregate from the neural tube, although not after they have previously dispersed. We conclude that early and prolonged cell interactions, mediated at least in part by Notch signaling, can regulate the survival of neurogenic cells within the nascent crest population. We suggest that a transient episode of cell contact-mediated death of neurogenic cells may serve to eliminate fate-restricted neurogenic cells that fail to disperse promptly in vivo.     

CELL DEATH,SURVIVAL AND PROLIFERATION IN TETRAHYMENA THERMOPHILA. EFFECTS OF INSULIN,SODIUM NITROPRUSSIDE, 8-BROMO CYCLIC GMP,NG-METHYL-L-ARGININE AND METHYLENE BLUE

Cells of the ciliate Tetrahymena thermophila produce compounds that act as autocrine (paracrine) survival and/or growth factors. 8-Bromo cyclic GMP, sodium nitroprusside, hemin, protoporphyrin IX, human recombinant and bovine insulin were tested for their ability to substitute for the cell-produced factors and stimulate cell survival and proliferation. The cells were inoculated into conical flasks in a nutritionally complete, chemically defined medium at known cell densities from 5 to 5000cells/ml. In unsupplemented medium cells at 5 to 500cells/ml (‘low initial cell density cultures’) died within 8h, whereas cells at 1000 and 5000cells/ml (‘high initial cell density cultures’) proliferated with lag phases lasting for up to 4h. In the presence of insulin compounds, hemin, protoporphyrin IX, or 8-bromo cyclic GMP, cells also proliferated at all low initial cell densities. Sodium nitroprusside was effective over two separate concentration ranges: at the nanomolar levels as well at low pico- to femtomolar levels. At initial population densities of up to 50cells/ml the cells at both concentrations of sodium nitroprusside survived about 4-fold longer than the controls. At 500 initial cells/ml, cells at thehigh concentrations of sodium nitroprusside survived about 4-fold longer than those of the control cultures; they proliferated in the low concentrations of sodium nitroprusside. Concentrations of hemin, too low to have any effects on their own, had synergistic effects with sodium nitroprusside. N^G-methyl-L-arginine inhibited proliferation at high initial cell densities. This inhibitory action was reduced by high concentrations of L-arginine, protoporphyrin IX, sodium nitroprusside, or 8-bromo cGMP, but not by insulin. Methylene blue inhibited cell proliferation at high initial cell densities. This inhibition was circumvented by addition of 8-bromo cGMP. The findings that insulin-related material may be released from Tetrahymena and that insulin and sodium nitroprusside increase intracellular cGMP in these cells are discussed in relation to the presented results. Together these observations suggest that cGMP is responsible for supporting cell survival in Tetrahymena and switching the cells into their proliferative mode, and that cell-produced signal molecules and insulin stimulate an NO-dependent guanylate cyclase into producing cGMP.     

Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.