Apoptosis in batch cultures of Chinese Hamster Ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum‐free batch culture of CHO cells in suspension died via apoptosis—as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin‐1‐β‐Converting Enzyme (ICE) family, e.g., CPP32, Mch‐1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoro‐methyl‐ketone (z‐VAD.fmk), was unable to substantially extend the life of a serum‐free batch culture of CHO cells. In addition, z‐VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z‐VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl‐2 expression was able to significantly extend viabilities in CHO batch culture. Bcl‐2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 632–640, 1999.     

Protective effect of Bcl-2 in NSO myeloma cell culture is greater in more stressful environments

In the present study, the protective effects of Bcl-2 over-expression in a suspension culture (without any adaptation) and spent medium (low nutrient and high toxic metabolite conditions) were investigated. In the suspension culture without prior adaptation, the viability of the control cell line fall to 0% by day 7, whereas the Bcl-2 cell line had a viability of 65%. The difference in the viability and viable cell density between the Bcl-2 and control cell lines was more apparent in the suspension culture than the static culture, and became even more apparent on day 6. Fluorescence microscopic counting revealed that the major mechanism of cell death in the control cell line in both the static and suspension cultures was apoptosis. For the Bcl-2 cell lines, necrosis was the major mode of cell death in the static culture, but apoptosis became equally important in the suspension culture. When the NSO 6A1 cell line was cultured in spent medium taken from a 14 day batch culture, the control cell line almost completely lost its viability by day 5, whereas, the Bcl-2 still had a viability of 73%. The viable cell density and viability of the Bcl-2 cell line cultivated in fresh medium were 2.2 and 2.7 fold higher, respectively, than those of the control cultures. However, the viable cell density and viability of the Bcl-2 cultivated in the spent medium were 8.7 and 7.8 fold higher, respectively, than those of the control cultures. Most of the dead cells in the control cell line were apoptotic; whereas, the major cell death mechanisms in the Bcl-2 cell line were necrotic.     

The in-line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality.     

Cell cycle model for growth rate and death rate in continuous suspension hybridoma cultures

As a result of recent advances in flow cytometry, renewed interest is shown in modeling the kinetic behavior of cells in culture on the basis of cell cycle parameters. An important but often overlooked kinetic variable in hybridoma cultures is the cell death rate. Not only the overall cell growth but also the kinetics of nutrient metabolism and monoclonal antibody production have been shown to depend on the cell death rate in continuous suspension hybridoma cultures. The present study shows that the death rate in hybridoma cultures is proportional to the fraction of cells arrested in the G(1) phase of the cell cycle. The steady-state cell age distributions in the various phases of the division cycle have been calculated analytically. A simple mathematical model has been used to produce the profiles of the cycling and arrested cell fractions with respect to the dilution rate. The calculated steady-state growth rate, death rate, and viability profiles are shown to be in agreement with recently published experimental data from continuous suspension hybridoma cultures. (c) 1992 John Wiley & Sons, Inc.     

Apoptosis of Terminally Differentiated Chondrocytes in Culture

During the process of endochondral ossification chondrocytes progress through stages of terminal differentiation culminating in apoptotic death. We have developed a serum-free suspension culture that allows terminal differentiation and facilitates the investigation of factors affecting chondrocyte apoptosis. We have found that chondrocytes not committed to terminal differentiation, i.e., those from the caudal region of chick embryo sterna, a region that remains cartilaginous for some months after the chick hatches, maintained high viability in serum-free suspension culture. A strong dependence of viability on culture density and sensitivity to induction of apoptosis with the protein kinase inhibitor, staurosporine, was consistent with the proposal that these chondrocytes, like nearly all cells, require intercellular communication for survival. Chondrocytes that were committed to terminal differentiation, i.e., those from the cephalic region of chick embryo sterna, a region that is replaced by bone before the chick hatches, expressed the hypertrophic phenotype but maintained their viability in culture for only approximately 6 days. Subsequent cell death was very consistent between cultures and shown to occur by an apoptotic process by analysis of DNA fragmentation and cell morphology. Short-term viability of hypertrophic chondrocytes was independent of culture density and relatively resistant to treatment with staurosporine. Induction of the hypertrophic phenotype in immature chondrocytes committed them to cell death and prevention of expression of the hypertrophic phenotype prevented cell death. We conclude that commitment of chondrocytes to terminal differentiation is associated with a commitment to apoptosis and apoptosis of hypertrophic chondrocytes in growth cartilage does not require initiation by external signals.     

Highly Selective Anti-Cancer Activity of Cholesterol-Interacting Agents Methyl-β-Cyclodextrin and Ostreolysin A/Pleurotolysin B Protein Complex on Urothelial Cancer Cells

Cholesterol content can vary distinctly between normal and cancer cells, with elevated levels in cancer cells. Here, we investigated cholesterol sequestration with methyl-β-cyclodextrin (MCD), and pore-formation with the ostreolysin A/pleurotolysin B (OlyA/PlyB) protein complex that binds to cholesterol/sphingomyelin-rich membrane domains. We evaluated the effects on viability of T24 invasive and RT4 noninvasive human urothelial cancer cells and normal porcine urothelial (NPU) cells. Cholesterol content strongly correlated with cancerous transformation, as highest in the T24 high-grade invasive urothelial cancer cells, and lowest in NPU cells. MCD treatment induced prominent cell death of T24 cells, whereas OlyA/PlyB treatment resulted in greatly decreased viability of the RT4 low-grade noninvasive carcinoma cells. Biochemical and transmission electron microscopy analyses revealed that MCD and OlyA/PlyB induce necrotic cell death in these cancer cells, while viability of NPU cells was not significantly affected by either treatment. We conclude that MCD is more toxic for T24 high-grade invasive urothelial cancer cells, and OlyA/PlyB for RT4 low-grade noninvasive urothelial cancer cells, and neither is toxic for NPU cells. The cholesterol and cholesterol/sphingomyelin-rich membrane domains in urothelial cancer cells thus constitute a selective therapeutic target for elimination of urothelial cancer cells.     

Defining viability in mammalian cell cultures

A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure membrane integrity (a measure of necrotic cell death) and assays that measure apoptotic cells, and show that discrepancies in the measurement of culture viability have a significant impact on the calculation of cell culture parameters and lead to skewed experimental data.     

GFP-FABD2和GFP-MBD标记蛋白对拟南芥悬浮细胞培养及应激响应能力的影响

以转GFP-FABD2和GFP-MBD基因的拟南芥为材料,研究了GFP-FABD2和GFP-MBD这两种细胞骨架标记蛋白对拟南芥愈伤组织诱导、悬浮细胞培养及应激响应能力的影响.结果表明:(1)GFP-MBD标记蛋白延长愈伤的出愈时间,改变愈伤形态,使转基因拟南芥种子的出愈量减少为野生型的59%、悬浮细胞的长短轴比缩小为1.20±0.21、第7天细胞活力下降为0.66±0.09,影响细胞的生长曲线.(2)GFP-FABD标记蛋白虽对愈伤生长影响不大,但却使悬浮细胞的长短轴比显著增加为2.49±1.18、第7天细胞的活力下降为0.87±0.06,造成悬浮细胞生长曲线的改变.(3)通过调整培养条件的激素水平,以上两种细胞骨架标记蛋白对悬浮细胞生长的影响可以得到修复.(4)检测优化条件下培养的GFP-FABD2或GFP-MBD悬浮细胞对温度、渗透压、机械应力等环境改变的应激响应能力,结果未发现与野生型有明显区别.     

Efficient formation of cell spheroids using polymer nanofibers

Spheroid culture has been used for suspension cultures of anchorage-dependent cells. In this study, we developed a new method for the suspension cultures of anchorage-dependent animal cells using polymer nanofibers. Poly(lactic-co-glycolic acid) nanofibers (785?nm in average fiber-diameter, 88?μm in average fiber-length) fabricated by the electrospinning method were added to each suspension culture of human embryonic kidney 293 cells and human dermal fibroblasts. As compared to no addition of nanofibers to the suspension cultures, nanofibers enhanced cell spheroid formation, thereby reducing cell death resulting from a lack of cell adhesion. Efficient formation of spheroids in the presence of polymer nanofibers may be useful for the suspension cultures of anchorage-dependent cells.     

Orbital shaker technology for the cultivation of mammalian cells in suspension

For large-scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior system for suspension cultures of mammalian cells based on orbital shaker technology. We found that "square-shaped" bottles (square bottles) provide an inexpensive but efficient means to grow HEK-293 EBNA and CHO-DG44 cells to high density. Cultures in agitated 1-L square bottles exceeded the performance of cultures in spinner flasks, reaching densities up to 7 x 10(6) cells/mL for HEK-293 EBNA cells and 5 x 10(6) cells/mL for CHO-DG44 cells in comparison to (2.5-4) x 10(6) cells/mL for cultures of the same cells grown in spinner flasks. For 1-L square bottles, optimal cell growth and viability were observed with a filling volume of 30-40% of the nominal volume and an agitation speed of 130 rpm at a rotational diameter of 2.5 cm. Transient reporter gene expression following gene delivery by calcium phosphate-DNA co-precipitation was the same or slightly better for HEK-293 EBNA cells grown in square bottles as compared to spinner flasks. Reductions in cost, simplified handling, and better performance in cell growth and viability make the agitated square bottle a new and very promising tool for the cultivation of mammalian cells in suspension.     

Mathematical characterization of insect cell (Sf-9) death in suspended culture

The effect of baculovirus infection on cell death in suspended cultures was characterized based on work by Wu et al. (1993) Biotech. Bioeng. 41: 104–110 and Wu et al. (1994) Biotechnol. Prog. 10: 55–59. The post infection time can be separated into a constant viability phase characterized by a time delay, td, and a rapid death phase, which is characterized by a specific death rate constant, k. Results indicated that the characteristic time delay decreased with increased multiplicity on infection (MOI). Further, there was only a weak correlation between specific death rate and MOI, for the range of MOI tested. Cell infection and death rates were consistent with a more evenly distributed infection process likely found in suspension cultures.     

The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.     

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.     

Zajdela hepatoma cells cultured in vitro

The goal of this work was to study cellular mechanisms of tumor progression and metastasizing. As a result of explantation of cells of rat Zajdela ascitic hepatoma, we obtained two transplantable cell cultures—monolayer (ZH-ad) and suspension (ZH-fl)—that differ in levels of cell differentiation and tumorigenicity. By using tumor-specific immune serum, we revealed tumor-associated antigens, synthesis of which is reduced or inhibited in ZH-ad cells, in outer membranes of the ZH-fl cells. Intraperitoneal injection into rat of 0.5–12 × 10⁶ ZH-fl cells leads to development of an ascitic tumor and death of 100% of animals, whereas, in the case of administration of ZH-ad cells, to achieve a tumorigenic effect, the minimal dose needs to be elevated to 20 × 10⁶ cells. Clonogenic analysis of the ZH-fl cells revealed three types of the formed clones—nonadhesive sphere colonies and two types of monolayer clones differing in proliferative potential, shape of colonies, and cell composition. Upon reaching a critical size, the spheres disintegrated, with separation of single cells and islands of different sizes, some of them being attached with monolayer formation. Three clonal cell lines were obtained: 1C as a result of expansion of a spherical clone and 4G and 10E from monolayer clones. We established that there is tumorigenicity of the 1C cell line, which, at a dose of 107 cells, led to the development of ascites and to the death of 50% of animals. The presented results indicate the existence in the ZH-fl cell population of tumor-initiating cells generating spherical clones—floating multicellular islets that, in culturing in the complete growth medium, are partly differentiated and are attached with monolayer formation.     

Fluid shear effects on suspension cultures of Morinda citrifolia

The shear susceptibility of cell suspension cultures of the plant cell Morinda citrifolia was investigated by subjecting the cells to the well-defined shear field generated in turbulent flow through a capillary. Suspensions were circulated using a peristaltic pump and average shear stresses between 25 and 350 N m(-2) were generated in the capillary test section. Control experiments were performed to assess the possible contribution of the peristaltic pump to the observed cell damage. There was clear evidence of pump-induced damage at the more severe test conditions and all viability measurements were corrected accordingly. Both shake flask suspension cultures (aged between 9 and 15 days) and repeated batch fermentation cultures, grown in a stirred tank reactor (STR) under a variety of controlled agitation conditions, were tested in the capillary shear loop. The cell damage incurred was evaluated in terms of suspension viability, as determined by a dye exclusion technique. Viability loss was found to conform closely to a first-order model in which the rate constant was observed to increase with the imposed shear stress. Furthermore, a linear relationship was identified between the specific death constant and the cumulative energy dissipated. Post-shear morphological measurements showed that the chain length distribution is shifted toward markedly lower values. In comparison with shake flask cultures, repeated batch fermentation cultures exhibited a marked increase in sensitivity to capillary shear. Based upon the determined morphological characteristics, this result is primarily attributable to the increased chain lengths characteristic of the repeated batch cultures. (c) 1995 John Wiley & Sons, Inc.     

The role of lipid peroxidation in aluminium toxicity in soybean cell suspension cultures

The primary reactions leading to Al toxicity in plant cells have not yet been elucidated. We used soybean (Glycine max [L.] Merr.) cell suspension cultures to address the question whether lipid peroxidation plays an important role in Al toxicity. Upon transfer to an Al-containing culture medium with a calculated Al3+ activity of 15 microM soybean cells showed a distinct and longtime increase in lipid peroxidation within 4 h. At the same time a drastic loss of cell viability was observed. Butylated hydroxyanisole (BHA) and N,N'-diphenyl-p-phenylenediamine (DPPD), two lipophilic antioxidants, were able to almost completely suppress lipid peroxidation in Al-treated cells at a concentration of 20 microM. This effect was dose-dependent for DPPD and was observed at minimum concentrations of 1-2 microM. When lipid peroxidation was suppressed by DPPD or BHA cell viability remained high even in the presence of toxic Al concentrations. These results suggest that Al-induced enhancement of lipid peroxidation is a decisive factor for Al toxicity in suspension cultured soybean cells.     

Enhanced plumbagin accumulation in embryogenic cell suspension cultures of Plumbago rosea L. following elicitation

This study focused on enhancing the production of plumbagin, an anticancer compound, in embryogenic cell suspension cultures of Plumbago rosea. Elicitation techniques have been reported to enhance plumbagin production. Cell suspension cultures raised from embryogenic calli induced from in vitro leaf explants were exposed to different concentrations of jasmonic acid, yeast extract and different auxin combinations. Influence of these on cell growth, biomass and plumbagin production was studied. To our knowledge this is the first report on elicitation of embryogenic cell suspension cultures of P. rosea for enhanced plumbagin production. Elicitor treated suspension cultures exhibited decreased culture viability and increased plumbagin synthesis. A maximum of 5.59-fold enhancement of plumbagin production was observed in cultures added with 1 mg L^?1 naphthalene acetic acid after 6 days of incubation. Viability of cultures decreased with increased concentration of elicitors and prolonged incubation period. Application of elicitors in cell suspension cultures induces defense related responses which lead to increased secondary metabolite production for making the cells adapt to the situation. If the stressed condition persists or is in intolerable level this will eventually lead to programmed cell death and loss of culture viability.     

Relationship of viability and apoptosis to taxol production in Taxus sp. suspension cultures elicited with methyl jasmonate

Taxus cuspidata P991 in plant cell suspension culture is capable of producing the important anticancer agent Taxol (paclitaxel) and related taxanes. High-level production is obtained by elicitation with methyl jasmonate, but successful elicitation leads to loss of cell viability that cannot be recovered by subculture. Here, we test whether the loss of viability is due to a direct effect of methyl jasmonate. Upon subculture, the reduced viability continued in methyl jasmonate elicited cultures, but not in nonelicited control cultures. The growth reduction in elicited T. cuspidata P991 suspension cultures was evaluated by viability reduction measurements using phenosafranin and fluorescein diacetate. The viability reduction does not appear to be related to apoptosis based on DNA laddering analysis because it occurred very late (at day 35) in the culture period. DNA laddering was also found only after day 28 in T. canadensis C93AD (a Taxol-producing cell line) elicited with methyl jasmonate, implying that apoptosis is not the major death mechanism after elicitation. As compared to Taxol-producing cell lines, the viability of a nonproducing cell line, T. canadensis CO93D, was not severely affected by methyl jasmonate, indicating that methyl jasmonate itself is not the primary factor for viability reduction. Based on Northern analysis of taxadiene synthase mRNA from both elicited and nonelicited T. cuspidata P991, methyl jasmonate directly induces the production of this enzyme, which is the first committed step in the biosynthetic pathway for Taxol. As a result, both viability reduction and growth reduction appear related to a high production level of Taxol (and related taxanes) upon methyl jasmonate elicitation, rather than to the direct effect of methyl jasmonate.     

Changes in protein tyrosine phosphorylation during mannose and senescence induced cell death in rice

In mammals protein tyrosine phosphorylation plays an important role in the activation of apoptosis. However, tyrosine phosphorylation associated with cell death has not been examined in plants. We monitored changes in tyrosine phosphorylation during cell death in rice (Oryza sativa L.) suspension cultures. Cell death was induced in the cell cultures by mannose treatment or by allowing the cultures to senescence. We have demonstrated that both mannose and senescence induced DNA fragmentation in rice suspension cells. In the presence of mannose, the tyrosine phosphorylation patterns of mannose treated and non-treated cell proteins are basically the same, except the tyrosine phosphorylation intensity is considerably different. In aged suspension-cultured cells, the occurrence of DNA fragmentation was detected. In addition, the tyrosine phosphorylation pattern was changed. These results suggest that protein tyrosine phosphorylation may have a role in distinct signal transduction pathways responding to mannose and senescence. The expression of a gene that encodes mitogen-activated protein kinase (MAPK), OsMAPK2, is up-regulated during mannose treatment, suggesting the possible involvement of rice MAPK in pathways associated with rice cell death induced by >d-mannose.     

Matrix-assisted cell transfer for intervertebral disc cell therapy

Cell therapy seems to be a promising way to reconstitute degenerated discs. We elucidate the basic aspects of intervertebral disc (IVD) cell therapy to estimate its potential in disc regeneration. Cell transfer efficiency and survival was quantified by luciferase expression after injection of recombinant cells into healthy, nucleotomized or mechanically degenerated rabbit IVDs in vitro, in situ or in vivo. A two-component fibrin matrix was adapted to allow injection of a fluid cell suspension that quickly polymerizes in IVDs. Thirty-five to fifty percent of matrix injected cells remained in the nucleus and transition zone in contrast to a rapid loss of medium-injected cells. Nucleotomy, which reduces intradiscal pressure, was crucial to the survival of the transferred cells over 3 days and nutritional enrichment of the fibrin matrix with potent biomolecules from serum significantly enhanced cell viability. In conclusion, advanced matrix substitutes are needed for efficient transfer and improved cell survival in the low-nutrient intradiscal environment to further improve disc cell therapy.