Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0

The cell cycle is at the center of growth, productivity, and death of mammalian cell cultures. There exists a need to identify and quantify major landmarks in the cell cycle of industrially relevant mammalian cell lines and its association with productivity; central for designing productivity optimization strategies. Herein, we studied the expression of three cyclins, under both perturbed and unperturbed growth, by flow cytometry in batch cultures of GS-NS0. The perturbed systems involved two different DNA synthesis inhibitors, thymidine and dimethyl sulfoxide (DMSO). This approach enables the establishment of characteristic cyclin profiles, timings, and thresholds. In particular, two G₁ class cyclins (D1 and E1), and one G₂ cyclin (B1) were investigated. Cyclin B1 showed a clear cell cycle phase-specific expression increasing during G₂ phase where it was approximately 40% higher when compared to G₁ phase. Similarly, cyclin E1 showed a clear pattern being expressed approximately 10% higher in G₁ compared to G₂ phase and decreased through S phase. Cyclin D1 expression was fairly invariable throughout the cell cycle phases. The observed patterns provide a blueprint of the cell line's cell cycle, which can be used for the development of biologically accurate and experimentally validated distributed cell cycle models.     

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1‐checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. Biotechnol. Bioeng. 2015;112: 141–155. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.     

Fibronectin promotes cell cycle entry in smooth muscle cells in primary culture

Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G(0)/G(1) phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27(KIP1) in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27(KIP1) levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors.     

Rosmarinic acid production in perfused Anchusa officinalis culture: Effect of inoculum size

Summary High cell density and rosmarinic acid (RA) productivity have been achieved by applying periodic culture perfusion to the Anchusa officinalis cell suspension. In this study, the effect of inoculum size on cell growth and RA productivity in the perfused Anchusa culture was investigated. Experimental results showed that RA productivity increased with the inoculum size, up to 4 g dry weight/L. Further increases in the inoculum size did not yield a higher RA productivity regardless of culture perfusion. Moreover, the maximum cell concentration was not affected by the inoculum sizes, from 1 to 11 g dry weight/L. Cell crowding, indicated by high culture packed cell volumes, is believed to be the predominant cause of low productivity in perfused cultures with high inoculum sizes.     

Heterogenous expression of a murine B16 melanoma-associated antigen correlates with cell cycle

Summary A murine monoclonal antibody (MM2-3C6) that reacts with a B16 murine melanoma-associated membrane antigen was used to study the relationship of antigen expression to the cell cycle. Dual-parameter flow cytometric measurements of membrane antigen and DNA revealed that antigen-positive cells were present throughout the cell cycle. Peak antigenic expression was noted during the late log phase of the cell growth curve with negligible antigen-negative population. The emergence of a distinct antigen-negative population (30%–40%) was noted in the late stationary phase. Cell cycle analysis indicated that the negative subpopulation was restricted to the G0/G1 phase, thus, demonstrating antigenic heterogeneity within the tumor cell population. Cell sorting was performed to analyze the origin of such heterogeneity. Following two sequential sortings, the antigen-negative cells became antigenic upon reculture. Again, at the late stationary phase, a distinct antigen-negative population (30%–40%) emerged. The sorted antigen-positive cells showed flow cytometric profiles identical to the sorted antigen-negative population upon reculture. Therefore, in this murine model, it appears that antigen expression is cell cycle dependent and such expression seems to be associated with proliferation.     

Recombinant, Replication-Defective Adenovirus Gene Transfer Vectors Induce Cell Cycle Dysregulation and Inappropriate Expression of Cyclin Proteins

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (ΔE1) or lacking the Ad E3 region in addition to E1 sequences (ΔE1ΔE3) induced G₂ cell cycle arrest and inhibited traverse across G₁/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34^cdc2 protein levels. In some instances, infection with ΔE1 or ΔE1ΔE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation ΔE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in ΔE1 or ΔE1ΔE3 Ad vectors induce G₂ growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.     

NA22598, a Novel Antitumor Compound, Reduces Cyclin D1 Levels, Arrests Cell Cycle at G1 Phase, and Inhibits Anchorage-Independent Growth of Human Tumor Cells

NA22598, a novel antitumor compound isolated from a microbial cultured broth, inhibited the growth of human colon cancer DLD-1 cells in suspension cultures (anchorage-independent growth) severalfold more strongly than in substratum-attached monolayer cultures. It arrested the cell cycle progression at early G1 phase under both these culture conditions. Rb phosphorylation, cyclin D1 expression, and cdk2 activation in G1 progression were all inhibited by NA22598, but the amounts of cdk2 and p27 were not affected. Among these effects the inhibition of cyclin D1 expression was most prominent, and NA22598 was found to inhibit the synthesis of cyclin D1 without affecting mRNA expression or protein degradation. p27 binding to cdk2 was more markedly increased in suspension cultures than in attached cultures by NA22598, but the compound had no effect on total p27. Apparently, the decrease of cyclin D1 induced redistribution of p27 from the cyclin D1/cdk4 to the cyclin E/cdk2 complexes during G1 phase in the suspension cultures. Because p27 is upregulated during suspension culture, a greater amount of it was associated with cyclin E/cdk2, thus producing greater growth inhibition. An agent, like NA22598, which induces the downregulation of cyclin D1 might offer a new anticancer strategy.     

Cell cycle,cell size and mitochondrial activity of hybridoma cells during batch cultivation

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G₂ fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.     

Comparative Metabolic Analysis of CHO Cell Clones Obtained through Cell Engineering,for IgG Productivity,Growth and Cell Longevity

Cell engineering has been used to improve animal cells’ central carbon metabolism. Due to the central carbon metabolism’s inefficiency and limiting input of carbons into the TCA cycle, key reactions belonging to these pathways have been targeted to improve cultures’ performance. Previous works have shown the positive effects of overexpressing PYC2, MDH II and fructose transporter. Since each of these modifications was performed in different cell lines and culture conditions, no comparisons between these modifications can be made. In this work we aim at contrasting the effect of each of the modifications by comparing pools of transfected IgG producing CHO cells cultivated in batch cultures. Results of the culture performance of engineered clones indicate that even though all studied clones had a more efficient metabolism, not all of them showed the expected improvement on cell proliferation and/or specific productivity. CHO cells overexpressing PYC2 were able to improve their exponential growth rate but IgG synthesis was decreased, MDH II overexpression lead to a reduction in cell growth and protein production, and cells transfected with the fructose transporter gene were able to increase cell density and reach the same volumetric protein production as parental CHO cells in glucose. We propose that a redox unbalance caused by the new metabolic flux distribution could affect IgG assembly and protein secretion. In addition to reaction dynamics, thermodynamic aspects of metabolism are also discussed to further understand the effect of these modifications over central carbon metabolism.     

Targeting DYRK1A/B kinases to modulate p21-cyclin D1-p27 signalling and induce anti-tumour activity in a model of human glioblastoma

The dual-specificity tyrosine-regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence-proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi-cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER-239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure-guided drug discovery. Inhibition of DYRK1A/B by VER-239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re-expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth-promoting factors. DYRK inhibition-induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition-induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER-239353 as a treatment for glioblastoma is therefore warranted.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.     

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.     

Application of a serum-free medium for the growth of Vero cells and the production of reovirus

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.     

Expression of BHRF1 improves survival of murine hybridoma cultures in batch and continuous modes

Cell death by apoptosis limits growth and productivity in most animal cell cultures. It is therefore desirable to define genetic interventions to generate robust cell lines with superior performance in bioreactors, either by increasing specific productivity, life-span of the cultures or both. In this context, forced expression of BHRF1, an Epstein–Barr virus-encoded early protein with structural and functional homology with the anti-apoptotic protein Bcl-2, effectively protected hybridomas in culture and delayed cell death under conditions of glutamine starvation. In the present study, we explored the potential application of BHRF1 expression in hybridomas for long-term apoptosis protection under different biotechnological process designs (batch and continuous) and compared it to strategies based on Bcl-2 overexpression. Our results confirmed that long-term maintenance of the anti-apoptotic effect of BHRF1 can be obtained using bicistronic configurations conferring enhanced protection compared to Bcl-2, even in the absence of selective pressure. Such protective effect of BHRF1 is demonstrated both in batch and continuous culture. Moreover, a further analysis at high cell densities in semi-continuous perfusion cultures indicated that the mechanism of action of BHRF1 involves cell cycle arrest in G0–G1 state and this is translated in lower numbers of dead cells.     

Long-term perfusion culture of hybridoma: a "grow or die" cell cycle system

In order to elucidate the hybridoma life cycle and the limiting factors in perfusion systems, we performed cultures in a stirred tank bioreactor, coupled to an external tangential flow filtration unit. Cell density and antibody production in perfusion were consistent with previous studies. The average life span of the cells (2.1-2.2 days), antibody, productivity per cell produced (30-38 mg/10(9) cells) and cell size diameter evolution appeared similar to values observed in batch cultures. These observations highly suggest a similar "grow or die" life cycle. Cell and antibody production, strictly related to the medium perfusion rate, seem to be under the control of the nutrient availability. A hypothesis to explain such a life cycle of hybridoma cells in perfusion systems and a model for viable and dead cell density is proposed.     

Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.