Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

Defined protein and animal component-free NS0 fed-batch culture.

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.

Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.

The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.

Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity.

The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages >100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.

A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells.

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

Yeast extract from Express Five serum-free medium contains factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells.

The yeast extract (of unknown origin) present in the commercially available serum-free medium 'Express Five' contains factors ('yeast extract factors') up to 35 kDa which are essential for growth of Trichoplusia ni insect cells. A yeast extract brand lacking these components could not support growth of T. ni cells. However, cell proliferation was restored by adding chromatographic fractions containing the yeast extract factors. The yeast extract factors were not solely responsible for the growth enhancing effect of yeast extract but some other components, which seem to be generally present in yeast extracts, are also required for T. ni proliferation.

Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures.

Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T. ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25kDa, as well as precursor forms above 48kDa. Metalloproteinase bands below the main band at 48kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor dl-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T. ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.

Defined protein-free NS0 myeloma cell cultures: stimulation of proliferation by conditioned medium factors.

A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, beta-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 x 10(6) cells mL(-1) in this medium. The antibody yield was 195 mg L(-1) in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 x 10(6) cells mL(-1), had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.

Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells.

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

Control of endotoxin release in Escherichia coli fed-batch cultures.

High amounts of outer membrane (OM) components were released in glucose-limited fed-batch (GLFB) cultures at 37 degrees C at specific growth rates approaching 0.05 h(-1). Endotoxin analyses from a 20 degrees C GLFB culture gave similar results. An alternative fermentation technique, the temperature-limited fed-batch (TLFB) technique, reduced the endotoxin concentration in a culture with a biomass concentration of 30 g l(-1) from the 850 mg l(-1) in traditional GLFB cultures to about 20 mg l(-1). The TLFB technique uses the temperature to regulate the dissolved oxygen tension, while all substrate components are unregulated. It appears to be severe glucose limitation that triggers the extensive release of endotoxins rather than a low growth rate. Furthermore, it is not the low temperature that stabilizes the OM when using the TLFB technique. Simulations and experimental data show that this technique results in the same biomass productivity as the GLFB technique.

Protease activity in protein-free NS0 myeloma cell cultures.

Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.

Escherichia coli high-cell-density culture: carbon mass balances and release of outer membrane components.

Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO(2), and acetate was approximately 90% at the end of the culture (25 h, 27 g L(-1) dw). The missing carbon remained as soluble organic compounds in the medium. Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at approximately 1 g L(-1)) contributed to 63% of the extracellular carbon. The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture. Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred. Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the extracellular carbon mass. The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.

Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by (13)C NMR spectroscopy.

Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia coli from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium. Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610).3). When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen. The 'glycine effect' was not due to CO(2) produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production. The metabolism of glycine was further investigated in cultures supplied with [2-(13)C] labelled glycine, and the redistribution of label in the [1-(13)C], [2-(13)C], and [1,2-(13)C] isotopomeres of excreted acetate was analysed by 13C NMR. The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1). Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of [2-(13)C] acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine. At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate. Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate. This switch in metabolism appears to be regulated by quorum sensing.