Anchorage-dependent Mammalian Cells Research Articles

Growth of anchorage-dependent mammalian cells on microstructures and microperforated silicon membranes

Anchorage-dependent cells (mouse fibroblasts L929 and 3T3) were cultivated on microstructures made by semiconductor technology. Both cell lines showed normal growth on silicon surfaces covered with microelectrode arrays as well as on microperforated silicon membranes with square pores made by anisotropic etching (5, 10 or 20 μm edge length at the top and 1.2, 6.2 or 16.2 μm at the bottom). The cells spread over the 5 and 10 μm pores, but mostly failed to cover the 20 μm ones. They were able to cross the silicon membrane through the pores and to grow and spread on the under side of the membrane. Small pores (about 1 μm2) impeded but did not prevent cells crossing the membrane. Medium and large pores were freely crossed. Negative dielectrophoresis was used to achieve accurate positioning of cells above pores or to repel them from the chip surface (a.c., square wave, 2.5 V peak-to-peak, 5 MHz). The results are discussed with respect to their microtool applications for single-cell technologies.

Cultivation of anchorage-dependent mammalian cells and production of various metabolites

Cell culture is an important tool for the production of various biologically active products such as pharmaceuticals, viral vaccines, viral insecticides, monoclonal antibodies, polypeptides and growth factors. These products are produced by normal, transformed and genetically engineered cells. The majority of these cells are anchorage dependent and require a solid surface to attach to in order to proliferate, metabolize nutrients and produce biomolecules. The need for cell culture was intensified with the enhanced development of mammalian genetic engineering, which raised a new demand for advanced cell culture technologies. Intensive studies have been undertaken by scientists to develop new technologies for large-scale cell culture. A major effort was invested to develop cell culture systems with high ratios of surface area to volume. At present there is a wide variety of systems, each with its own advantages and limitations. In this short review, the various large-scale cell culture systems are described. Cell culture technology in general, and in particular the substrates to which the cells adhere, play important roles in the product formation of a given macromolecule; therefore the system should be chosen very carefully and adapted to the biological process. The production process of tissue plasminogen activator from normal human fibroblasts and a new cell carrier, “Fibra-cel”, which is made of non-woven polyester fabric, are presented as examples in order to demonstrate the state of the art in this field.

Surface coating ? No improvement of shear stress resistance of anchorage dependent mammalian cells

The growth of adherent Baby Hamster Kidney cells (BHK 21) on surfaces coated with different substances (FCS, Con. A, PHA, cholesterol, collagen) was investigated. A positive influence on cell viability could only be observed when using a fetal calf serum (FCS) coat. To evaluate the effects of coating on shear stress sensitivity, experiments in a home made flow chamber were carried out. For comparison further investigations were conducted for several serum concentrations in the medium. The results demonstrate that the latter had a more beneficial effect on shear stress resistance than coating.

The application of a new type of sintered glass carriers for the cultivation of anchorage‐dependent mammalian cells

AbstractCylinder‐shaped sintered glass carriers were successfully used for immobilizing and cultivating cells of murine line L929. The process has been performed in a fixed‐bed bioreactor (working volume of 150 ml) with perfusion and aeration of the medium (total volume of 3.5 l). The maximum cell density of 1.23 × 107cells/ml was achieved when the basal medium (MEM + 10% bovine serum) was enriched with glucose (up to 2 g/l) and L‐glutamine (up to 4 mM). The carriers have proved to be nontoxic, mechanically stable and reusable.

Real-time measurement of anchorage-dependent cell adhesion using a quartz crystal microbalance.

An in situ technique based on the mass-sensitive piezoelectric quartz crystal microbalance (QCM) was applied to continuously monitor the attachment and detachment of anchorage-dependent mammalian cells on a metal surface. Specifically, we have demonstrated that the attachment of Vero cells to metal surfaces on the piezoelectrically active area of the QCM results in decreases in the QCM resonant frequency that can be monitored conveniently in real time. Lysis and detachment of Vero cells caused by vesicular stomatitis virus (VSV) infection can also be monitored readily. Our results demonstrate that the QCM is a viable technique for monitoring anchorage-dependent cell attachment and detachment on surfaces (caused by stimulatory, inhibitory, or cytopathic effects).

A cellular automation model for the growth of anchorage-dependent mammalian cells used in vaccine production

In the production of human vaccines, the kinetics of anchorage-dependent cell growth on microcarriers is complicated by their spatial environment. As the cells begin to cover the surface area available for growth, the growth rate gradually decreases until the entire surface is covered, and growth ceases. This contact inhibition of cell growth makes it difficult to accurately determine key kinetic parameters such as the specific growth rate of a culture. In this paper, a simple, but useful cellular automation model is proposed which is capable of decoupling the effect of contact inhibited cell growth from the overall kinetics of the culture. The automaton model is a good qualitative representation of cellular growth on microcarrier cultures. Moreover, quantitative comparison with experimental data is very good. The model is capable of fitting growth curves initiated at different inoculation levels by taking into account the initial cell attachment distribution for microcarrier cultures. Information generated by the automation on the dynamic nature of the specific growth rate of a culture has been coupled to a kinetic model describing the dynamics of key nutrients and metabolites in both the batch and perfusion modes of bioreactor operation, and these results are compared to experimental data.

Surface immobilization of anchorage‐dependent mammalian cells

Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (< or = 2-3 h) attachment of inoculated cells (> or = 95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.

Growth Monitoring of Mammalian Cells on Microcarriers by Image Analysis

The growth of anchorage-dependent mammalian cells on microcarriers is monitored via analysis of light microscopy images during the first hours of culture. The method, taking into account the spherical shape of the microcarriers, provides data on the number of cell clusters and the extent of coverage of the microcarriers by the cells in function of the culture age.

Development of the optimal inoculation conditions for microcarrier cultures

The environmental conditions under which anchorage-dependent mammalian cells are grown are not necessarily those under which a culture should be initiated. Cell attachment is a physical process, and those factors which affect forces involved in cell attachment differ from the biological factors which affect cell growth. We have conducted an extensive experimental study to define clearly the optimal environmental conditions for MRC-5 cell attachment onto microcarriers. These inoculation conditions are particularly important when the serial propagation of mammalian cells on microcarriers is considered as in a human vaccine production process. The conditions which were investigated are: initial serum content (% v/v), initial pH, inoculation level (cells/bead), agitation rate (rpm), and the concentration of microcarriers (g/L). The initial distribution of attached cells was found to have a significant affect on the overall efficiency of anchorage-dependent cell cultures, and was used to evaluate attachment efficiency. Based on the experimental results, we propose an optimized protocol for the inoculation of microcarrier cultures.

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10(7) cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.

A viscous pump bioreactor

The fluid dynamic design and characterization of a low mechanical stress agitator and bioreactor vessel for large-scale bioprocessing using anchorage dependent mammalian cells is considered. The complex and fragile nature of mammalian cells puts stringent constraints on the design of agitators for stirred tank bioreactors. Traditional agitators have difficulty meeting the competing requirements of fluidization and mixing while maintaining low enough mechanical stress levels to avoid cell damage. A rotating disc agitator design is proposed. Flow visualization and laser Doppler velocimeter measurements reveal fluidization of microcarriers with a gentle (low turbulence level), highly three-dimensional flow characterized by good mixing at low hydrodynamic shear stress levels.

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

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 X 10(8) cells/cm3 PUF and 3.5 X 10(7) 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 X 10(7) cells/cm3 culture vessel volume was obtained in a packed-bed culture of Vero cells.

Hydrogel microspheres from crosslinked poly(methyl methacrylate): synthesis and biocompatibility studies

Smooth, perfectly spherical, highly hydrophilic microspheres have been prepared from crosslinked poly(methyl methacrylate) microspheres by alkaline hydrolysis in ethylene glycol at elevated temperatures. These microspheres absorb varying quantities of water depending upon the extent of hydrolysis. Subcutaneous implantation studies on rabbits demonstrated that the microspheres are biocompatible. Implantation studies in the renal arteries of dogs demonstrated the occlusion effect produced by the microspheres. Microspheres could be made radiopaque by the incorporation of barium sulphate. Potential uses envisaged for these microspheres in the biomedical area are that of artificial emboli for endovascular embolization and as microcarriers for the growth and propagation of anchorage dependent mammalian cells.

Growth of anchorage dependent mammalian cells on glycine-derivatized polystyrene in suspension culture.

Glycine-derivatized polystyrene beads were prepared and used as microcarriers to grow normal cells of human embryonic kidney, rhesus monkey kidney, an human foreskin fibroblasts in suspension cultures. Growth of the cells on polystyrene beads derivatized with other amino acids, peptides, and carboxylic acids also was investigated.

Instrumentation for Process Control in Cell Culture

This chapter discusses the instruments and measurement techniques used by the fermentation industry. It assesses the potential of these and other emerging techniques for use in cell culture. An important step in the development of an instrument system is the selection of the quantities to be measured. The choice generally depends upon the overall aim of the project and the nature of the biological system that is utilized. Instrumental methods can be classified into two general categories: offline and on-line. The cultivation of anchorage-dependent mammalian cells is one of the main problems in the large-scale production of cellular products and virus vaccines. A primary difficulty is of providing large, accessible surfaces for cell growth. A number of techniques proposed as means of solving this problem include the roller bottle, the multiplate propagator, the spiral plastic film bottles, the artificial capillary propagators, and the membrane tubing reels.