Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.     

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment

Summary Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 × 10⁸ cells/ cm³ PUF and 3.5 × 10⁷ cells/ cm³ PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 × 10⁷ cells/cm³ culture vessel volume was obtained in a packed-bed culture of Vero cells. Offprint requests to: K. Funatsu     

A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells

PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis. Maintenance of cleavage after lysis is dependent on the composition of the lysis medium; the pH must be around neutrality, MgATP must be present, and the free Ca++ concentration should be 1 microM for optimal constriction to occur. Cleavage can be stopped and reinitiated by raising and lowering the Ca++ levels in the lysis medium. Cleavage in the permeabilized cell is blocked by addition of phalloidin, cytochalasin B, and N-ethylmaleimide-modified myosin subfragment-1 to the lysis medium. This represents the first cell model system for studying cleavage since the pioneering studies of Hoffman- Berling in 1954.     

Constraints-based genome-scale metabolic simulation for systems metabolic engineering

Random mutagenesis and selection approaches used traditionally for the development of industrial strains have largely been complemented by metabolic engineering, which allows purposeful modification of metabolic and cellular characteristics by using recombinant DNA and other molecular biological techniques. As systems biology advances as a new paradigm of research thanks to the development of genome-scale computational tools and high-throughput experimental technologies including omics, systems metabolic engineering allowing modification of metabolic, regulatory and signaling networks of the cell at the systems-level is becoming possible. In silico genome-scale metabolic model and its simulation play increasingly important role in providing systematic strategies for metabolic engineering. The in silico genome-scale metabolic model is developed using genomic annotation, metabolic reactions, literature information, and experimental data. The advent of in silico genome-scale metabolic model brought about the development of various algorithms to simulate the metabolic status of the cell as a whole. In this paper, we review the algorithms developed for the system-wide simulation and perturbation of cellular metabolism, discuss the characteristics of these algorithms, and suggest future research direction.     

Use of genome-scale microbial models for metabolic engineering

Metabolic engineering serves as an integrated approach to design new cell factories by providing rational design procedures and valuable mathematical and experimental tools. Mathematical models have an important role for phenotypic analysis, but can also be used for the design of optimal metabolic network structures. The major challenge for metabolic engineering in the post-genomic era is to broaden its design methodologies to incorporate genome-scale biological data. Genome-scale stoichiometric models of microorganisms represent a first step in this direction.     

Historical reflections on cell culture engineering

Cell culture engineering has enabled the commercial marketing of about a dozen human therapeutic products derived from rDNA technology and numerous monoclonal antibody products as well. A variety of technologies have proven useful in bringing products to the marketplace. Comparisons of the technologies available 15 years ago are contrasted with those available today. A number of improvements in unit operations have greatly improved the robustness of the processes during the past 15 years. Further evolution of the technology is expected in several directions driven by commercial and regulatory pressures. Some problems remain for the next generation of cell culture engineers to solve. This revised version was published online in August 2006 with corrections to the Cover Date.     

Metabolic flux analysis in mammalian cell culture

Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while ¹³C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale ¹³C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.     

A pilot plant for mammalian cell culture

Studies of the possible viral etiology of human leukemia have required large quantities of cultured cells derived from human hematopoietic tissues. Since cultures sufficiently large and free from contamination could not readily be produced according to existing methods, a pilot, cell culture plant has been constructed for the production of mammalian cells in mass quantity. 500-ml to 20-liter trophocell units have already proved to be scientifically and economically practical, as they provide good reliability, excellent growth rates, and sustained yield of human cells. 200-liter stainless steel culture units have now been added to the trophocell system. Five complete 200 liter units are now in operation. The design of the original stainless steel unit was based on that of a stainless steel, jacketed soup kettle. There are no openings in the vessel other than those in the lid, which provide convenient access points for sampling, sensor probes, etc. Environmental parameters, e.g., liquid level, temperature, and pH, are monitored and controlled with commercially available apparatus. Many initial problems connected with the new 200 liter units have been resolved, but operational and design problems remain in the areas of stable instrumentation, cell harvesting, salvaging and reuse of unspent media components, establishment of physiologic steady stale, recovery of virus-containing cells with reculture of the remaining unaffected cells, and the recovery and separation of cell components and special products such as immunoglobulins, interferons, and hormones. A definitive cell plant with culture units of 20, 50, 250, and 1250 liters is now being constructed.     

A mammalian cell culture collection for biotechnology

Conclusion Culture collections are key to the success of biotechnology companies. Protection of patent strains and manufacturing inoculum; standardization of biological materials for research, development and manufacturing; and documentation of organism transfers are essential functions provided by a culture collection in a biotechnology company. Certified, stable mammalian cell cultures will continue to be key in research advances and in manufacturing of biotechnology products in the future.     

Descriptive parameter evaluation in mammalian cell culture

Several methods exist for assessing population growth and protein productivity in mammalian cell culture. These methods were critically examined here, based on experiments with two hybridoma cell lines. It is shown that mammalian cell culture parameters must be evaluated on the same basis. In batch culture mode most data is obtained on a cumulative basis (protein product titre, substrate concentration, metabolic byproduct concentration). A simple numerical integration technique can be employed to convert cell concentration data to a cumulative basis (cell-hours). The hybridoma lines used in this study included a nutritionally non-fastidious line producing low levels of MAb and a nutritionally fastidious hybridoma with high productivity. In both cases the cell-hour approach was the most appropriate means of expressing the relationship between protein productivity and cell population dynamics. The cell-hour approach could be used as the basis for all metabolic population parameter evaluations. This method has the potential to be used successfully for both prediction and optimization purposes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Step-fortifications of nutrients in mammalian cell culture

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated. (c) 1993 John Wiley & Sons, Inc.     

Advancing vascular tissue engineering: the role of stem cell technology

Atherosclerosis and heart disease are still the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts but the patency of such grafts is limited compared to natural materials. Tissue engineering, whereby living tissue replacements can be constructed, has emerged as a solution to some of these difficulties. This, in turn, is limited by the availability of suitable cells from which to construct the vessels. The development of prosthesis using progenitor cells and switching these into endothelial cells is an important and exciting advance in the field of tissue engineering. Here, we describe recent developments in the use of stem cells for the development of replacement vessels. These paradigm shifts in vascular engineering now offer a new route for effective clinical therapy.     

Microfluidic cell culture models for tissue engineering

Microfluidic systems have emerged as revolutionary new platform technologies for a range of applications, from consumer products such as inkjet printer cartridges to lab-on-a-chip diagnostic systems. Recent developments have opened the door to a new set of opportunities for microfluidic systems, in the field of tissue and organ engineering. Advances in the design of physiologically relevant structures and networks, fabrication processes for biomaterials suitable for in vivo use, and techniques for scaling towards large, three-dimensional constructs, are converging towards therapeutic applications of microfluidic technologies in engineering complex tissues and organs. These advances herald a new generation of microfluidics-based approaches designed for specific tissue and organ applications, incorporating microvascular networks, structures for transport and filtration, and a three-dimensional microenvironment suitable for supporting phenotypic cell behavior, tissue function, and implantation and host integration.     

Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%.     

Reverse engineering and analysis of large genome-scale gene networks

Reverse engineering the whole-genome networks of complex multicellular organisms continues to remain a challenge. While simpler models easily scale to large number of genes and gene expression datasets, more accurate models are compute intensive limiting their scale of applicability. To enable fast and accurate reconstruction of large networks, we developed Tool for Inferring Network of Genes (TINGe), a parallel mutual information (MI)-based program. The novel features of our approach include: (i) B-spline-based formulation for linear-time computation of MI, (ii) a novel algorithm for direct permutation testing and (iii) development of parallel algorithms to reduce run-time and facilitate construction of large networks. We assess the quality of our method by comparison with ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and GeneNet and demonstrate its unique capability by reverse engineering the whole-genome network of Arabidopsis thaliana from 3137 Affymetrix ATH1 GeneChips in just 9 min on a 1024-core cluster. We further report on the development of a new software Gene Network Analyzer (GeNA) for extracting context-specific subnetworks from a given set of seed genes. Using TINGe and GeNA, we performed analysis of 241 Arabidopsis AraCyc 8.0 pathways, and the results are made available through the web.