Suspension Cultures Of Mammalian Cells Research Articles

Advances in the research and applications of orange fluorescent protein

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

Isolation of High-Molecular-Weight DNA from Suspension Cultures of Mammalian Cells Using Proteinase K and Phenol.

This procedure is the method of choice for purification of mammalian genomic DNA from suspension cultures when large amounts of DNA are required, for example, for Southern blotting. Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 × 107 cultured aneuploid cells (e.g., HeLa cells).

Thermal stability of purified and reconstituted CFTR in a locked open channel conformation

CFTR is unique among ABC transporters as the only one functioning as an ion channel and from a human health perspective because mutations in its gene cause cystic fibrosis. Although considerable advances have been made towards understanding CFTR’s mechanism of action and the impact of mutations, the lack of a high-resolution 3D structure has hindered progress. The large multi-domain membrane glycoprotein is normally present at low copy number and when over expressed at high levels it aggregates strongly, limiting the production of stable mono-disperse preparations. While the reasons for the strong self-association are not fully understood, its relatively low thermal stability seems likely to be one. The major CF causing mutation, ΔF508, renders the protein very thermally unstable and therefore a great deal of attention has been paid to this property of CFTR. Multiple second site mutations of CFTR in NBD1 where F508 normally resides and small molecule binders of the domain increase the thermal stability of the mutant. These manipulations also stabilize the wild-type protein. Here we have applied ΔF508-stabilizing changes and other modifications to generate wild-type constructs that express at much higher levels in scaled-up suspension cultures of mammalian cells. After purification and reconstitution into liposomes these proteins are active in a locked-open conformation at temperatures as high as 50°C and remain monodisperse at 4°C in detergent or lipid for at least a week. The availability of adequate amounts of these and related stable active preparations of homogeneous CFTR will enable stalled structural and ligand binding studies to proceed.

Semliki forest virus-based expression of recombinant GPCRs.

Due to their importance as targets for drug development, rapid and consistent high-level production of G protein-coupled receptors (GPCRs) has become an essential part of drug discovery. Alphaviruses, particularly recombinant Semliki Forest virus (SFV) particles, have provided the means for expression of a number of GPCRs in a broad range of mammalian host cell lines for pharmacological characterization by determination of receptor binding activity and functional coupling to G proteins. The rapid high-titer virus particle production has made it possible to study a large number of GPCRs in parallel. Moreover, large-scale production in adherent and suspension cultures of mammalian cells has provided sufficient amounts of GPCRs for purification and subsequent structural studies. Furthermore, the high preference for neuronal delivery of SFV particles has allowed functional and localization studies of recombinant proteins in hippocampal slice cultures, in primary neurons, and in vivo.

Scale-up and predictability in process development with suspension cultures of mammalian cells for recombinant protein manufacture: comments on a trend reversal

Scale-up and predictability in process development with suspension cultures of mammalian cells for recombinant protein manufacture: comments on a trend reversal

Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure

Transient transfection of mammalian cells in suspension culture has recently emerged as a very useful method for production of research-scale quantities of recombinant proteins. The most commonly used cell lines for this purpose are suspension-adapted HEK and CHO cells. We report here that the plasma exposure in mice of an IL-23R extracellular domain Fc fusion protein (IL23R–Fc) differed dramatically depending on whether the protein was prepared by transient transfection of HEK or CHO cells. Specifically, IL23R–Fc expressed using CHO cells had about 30-fold higher in vivo plasma exposure compared to the HEK-expressed protein. In contrast to their differing plasma exposures, the HEK- and CHO-expressed proteins had equivalent in vitro biological activity. Characterization of the CHO- and HEK-expressed IL23R–Fc proteins indicated that the differences in in vivo plasma exposure between them are due to differential glycosylation.

The Kinetics of Polyethylenimine-Mediated Transfection in Suspension Cultures of Chinese Hamster Ovary Cells

The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI-DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2 x 10(6) cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the rate and the level of PEI-DNA uptake in serum-free minimal medium were found to be dependent on the PEI-DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein production.

Inoculation and Passaging of Mammalian Cell Suspension Cultures

INTRODUCTIONThis protocol describes the growth and passaging of mammalian cells in suspension culture using a simple dilution procedure. In situations in which total replacement of medium is desired, the cells can be gently pelleted by centrifugation and resuspended in the appropriate medium.

Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli & mammalian cells in suspension culture

Preparation of isolated cells and microorganisms for ultrastructural examination always provides a challenge in terms of adequate immobilization of the cells and prevention of subsequent sample loss and damage during various steps of sample processing. Using a positively charged nylon membrane substrate we demonstrate that it is possible to easily immobilize and retain a sample of isolated cells in culture for a wide variety of microscopy-based techniques. Radiolabelled E. coli cells when immobilized on the charged membrane were seen to be highly resistant to detachment when subjected to the normal sample processing procedures associated with microscopy. In contrast cells on regular millipore membranes were rapidly lost during sample preparation. We demonstrate the utility of charged nylon membranes for a wide variety of microscopy based analysis including scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), TEM based immunogold labelling, laser confocal microscopy and SEM based elemental analysis.

New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells

We present a new approach for biomass assessment in cell culture using a disposable microcentrifuge tube. The specially designed tube is fitted with an upper chamber for sample loading and a lower 5 microL capillary for cell collection during centrifugation. The resulting packed cell volume (PCV) can be quantitatively expressed as the percentage of the total volume of the sample. The present study focused on the validation of the method with mammalian cell lines that are widely used in bioprocessing. Using several examples, the PCV method was shown to be more precise, rapid, and reproducible than manual cell counting.

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.

A technique for determining the shear sensitivity of mammalian cells in suspension culture

A murine hybridoma was subjected to constant shear rates within a couette viscometer for periods of 15 hours. Shear effects on cells were determined by live cell counts, cell viability and by the release of the cytoplasmic enzyme lactate dehydrogenase into the culture medium. Cell damage was observed at a shear rate of 870s-1 but not at 420s-1.

Animal cell cultivation for production of biological substances with a novel perfusion culture apparatus

Specific standard methods of a new perfusion culture are described for growth and maintenance of mammalian cells in suspension culture at high density. The new system of perfusion consists of a suspension vessel containing a cone-type cell-sedimentation column as cell separator and serving as well for axis of impeller. In this report, Namalva cells (human lymphoblastoid cells), adapted to a serum-free medium, have been grown in this perfusion system. The cultured cells and the conditioned medium were separated in the cell-sedimentation column; cell-free expended medium was drained from culture vessel to effluent vessel. The cell-sedimentation column device has been used for the propagationin vitro of Namalva cells to densities 7 to 10 × 106 cells/ml in serum-free medium supplemented with insulin, transferrin, sodium pyruvate, selenious acid, and galactose.

Five‐year perspective of the large‐scale growth of mammalian cells in suspension culture

AbstractThe Cell Culture Center at the University of Alabama in Birmingham was set up to produce large quantities of cells and their products from suspension cultured cell lines. This system has now been in operation for over five years and has been effective in producing large quantities of mammalian cells of murine and human origin. This article describes the system and some growth parameters which have been of importance for large‐scale mammalian cell growth.

Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures.

A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10(9) cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O2 and CO2 were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10- to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4- ad 40-liter reactor also has been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10 x 10(6) cells/ml with a total yield of 8.7 x 10(11) cells from 360 liters of medium.

Cyclic nucleotides and growth regulation in suspension cultures of mammalian cells

Cyclic nucleotides and growth regulation in suspension cultures of mammalian cells

Kinetics of Drug Transport and Concurrent Metabolism in Human Cell Cultures I: Theory

A method for evaluating the passive permeability and single-step metabolism of drugs in suspension cultures of mammalian cells was formulated assuming linear kinetics. It was assumed that the metabolizing enzymes are driven by endogenous substrates present in steady-state quantities. The presence of the drug in radiolabeled tracer quantities was assumed to cause only a small perturbation from the endogenous steady-state operating point. The time course solutions for the drug and its metabolite are given in terms of macroscopic constants, and their physical interpretations are given in terms of metabolic and transport parameters.

The application of rapid kinetic techniques to the transport of thymidine and 3‐O‐methylglucose into mammalian cells in suspension culture

Techniques are described by which the transport of nutrients into mammalian cells in suspension can be measured at intervals of 1.5 seconds. By application of these techniques, the existence of a saturable (Km = 85 muM), non-concentrative, transport system for thymidine was demonstrated in Novikoff rat hepatoma cells depleted of ATP. At concentrations of thymidine less than the Km, this system operated at velocities sufficient to nearly completely equilibrate intra- and extra-cellular thymidine pools within 8 seconds. In phosphorylating cells, the transport system operated with similar rapidity, so that intracellular phosphorylation was rate-limiting for the incorporation of thymidine into nucleotides. Uptake of 3-O-methylglucose occurred at comparable velocities, attaining 90% of equilibrium between internal and external pools within 25 seconds. Uptake of cytosine by simple diffusion was 100 times slower.

Systems approach to the study of drug transport across membranes using suspension cultures of mammalian cells V. Simultaneous passive transport and biosynthesis

A physical model is described for the simultaneous enzymatic bioconversion of a nonelectrolyte solute and the passive transport of both the solute and product of the enzymatic reaction out of cells in culture suspension. The plasma membrane is assumed to be the rate-determining transport barrier. This model provides the basis for the experimental design and analysis of the Michaelis-Menten kinetic parameters of simple enzymatic reactions in situ, the phenomenological transport parameters and other factors. The primary set of differential equations describing the quasisteady state rate of change in the concentration of the solute and product within the cell due to enzyme reaction and transport are given. These are nonlinear and must be solved by numerical methods. However, analytical mathematical expressions have been derived for various cases in the limit when the rate of enzymatic reaction is first or zero order.

Systems Approach to Study of Solute Transport across Membranes Using Suspension Cultures of Mammalian Cells IV: Uptake and Release Kinetics of Sterols

The kinetics of uptake and release of desmosterol, cholesterol, and β‐sitosterol by Burkitt lymphoma cells at 26°, pH 7.3, and isoosmotic conditions were quantitatively and mechanistically interpreted. The data are in agreement with the model involving the passive transport of the unbound sterol across the ratedetermining plasma membrane, with rapid distribution within the heterogeneous cell interiro. Effective permeability (Pe) and partition (Ke) coefficients of the sterols were inversely proportional to the serum concentration in the external media due to sterol‐serum binding. These results are consistent with the mechanism in which only the unbound solute in the external solution participates in the membrance transport process. At al serum levels, Pe and Ke increased with increasing sterol polarity: desmosterol > cholesterol > β‐sitosterol.