Resolution of cells by centrifugal elutriation

Cell separation using the Beckman elutriator depends upon the flow rate of the medium and the centrifugal field employed. Changes in either the centrifugal field or the flow rate can be used to elute fractions of cells based on size. Even when these variables are held constant in the Beckman J21C centrifuge, a periodic pulse of cells is eluted. We have found that this anomolous elution is related to the temperature control system which gave a periodically pulsed temperature drop in the centrifuge well. The elution resulting from this change in temperature caused a shift in the modal cell size of the fraction eluted at a particular flow rate and centrifugal field. Because of this, the fractions have a larger size dispersion than fractions collected under conditions where refrigeration-related temperature fluctuations do not occur. We conclude that the temperature control system of the Beckman J21C centrifuge used with the Beckman elutriation rotor produces temperature fluctuations which prevent maximum resolution of cells.     

Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes

A method to obtain several highly enriched populations of testis cell types from rats of a single age is described. Single cell suspensions from immature rat testes were prepared after enzymatic removal of interstitial cells. Cells were separated on the basis of size into four fractions (bulk preparations) or eight fractions (analytical preparations) by centrifugal elutriation. These elutriator fractions were further separated by equilibrium density centrifugation in Percoll gradients. In this manner, populations of 2 X 10(7) type A spermatogonia (51% purity), 3 X 10(7) type B spermatogonia (76% purity), 5 X 10(7) zygotene/early pachytene spermatocytes (56% purity), 3 X 10(7) midpachytene spermatocytes (70% purity), and 4 X 10(7) Sertoli cells (89% purity) could be obtained from 50 immature rats within 6 h after killing. Purities, determined by examination of cytologic smears, were verified by Coulter volume and flow cytometric DNA determinations. These separation methods were used to obtain cell populations for characterization of levels and synthesis of high mobility group proteins in the early stages of spermatogenesis.     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min^–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 10⁶ cells ml^–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process.     

Centrifugal elutriation (counterstreaming centrifugation) of cells

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

Centrifugal elutriation (counterstreaming centrifugation) of cells.

Lindahl first described the separation of cells by velocity sedimentation utilizing a special technique (counterstreaming centrifugation) that was later modified slightly and renamed centrifugal elutriation. Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as cells in particular phases of the cell cycle. The capacity of the elutriator to separate large numbers of cells is its chief advantage. The purities of the separated cells have not been compared with the purities of cells separated by other methods in most cases; such comparisons would permit more sophisticated comparison of elutriation with other techniques for velocity sedimentation.     

A genomically/chemically complete module for synthesis of lipid membrane in a minimal cell

A minimal cell is a hypothetical cell defined by the essential functions required for life. We have developed a module for the synthesis of membrane precursors for a mathematical minimal cell model. This module describes, with chemical and genomic detail the production of the constituents required to build a cell membrane and identifies the corresponding essential genes. Membranes allow selective nutrient passage, harmful substance exclusion, and energy generation. Bacterial membrane components range from lipids to fatty acids with embedded proteins and are structurally similar to eukaryotic cell membranes. Membranes are dynamic structures and experimental analyses show great variations in bacterial membrane composition. The flexibility of the model is such that different membrane compositions could be obtained in response to simulated changes in culture conditions. The model's predictions are in close agreement with the observed biological trends. The model's predictions correspond well with the experimental values of total lipid content in cells grown in chemostat culture, but less well with data from batch growth. Cell shape and size results agree especially well for data for growth rate relative to maximum growth rate larger than 0.5; and DNA, RNA, and protein predictions are consistent with experimental observations. A better understanding of the simplest bacterial membrane should lead to insights on the more complex behavior of membranes of higher species as well as identification of potential targets for antimicrobials.     

Efficient recovery of gamma-poly (glutamic acid) from highly viscous culture broth.

An efficient strategy for the separation and recovery of gamma-polyglutamic acid (gamma-PGA) from highly viscous broth was developed. This strategy was divided into two processes: The first was to separate gamma-PGA from highly viscous culture broth; the second was to concentrate gamma-PGA solution by ultrafiltration for the reduction of the amount of alcohol required during recovery process with precipitation. By lowering the pH value of culture broth to 3, the viscosity of culture broth and the zeta potential of cell could be reduced to a sixth of the original value at 35 degrees C and a third, respectively. After the acidification of culture broth the energy demand for the separation of gamma-PGA from culture broth by centrifugation could be reduced to 17% of that without it when the centrifugal force was 22,000g. The amount of alcohol required for precipitation could be reduced to a fourth of that generally used without concentration by concentrating 20 g gamma-PGA/L solution to 60 g gamma-PGA/L at pH 5 by ultrafiltration with hollow-fiber membrane cartridge (MWCO 500,000).     

Cell separation by countercurrent centrifugal elutriation: recent developments

Countercurrent centrifugal elutriation (CCE) is a cell separation technique that separates particles predominantly according to their size, and to some degree according to their specific density, without a need for antibodies or ligands tagging cell surfaces. The principles of this technique have been known for half a century. Still, numerous recent publications confirmed that CCE is a valuable supplement to current cell separation technology. It is mainly applied when homogeneous populations of cells, which mirror an in vivo situation, are required for answering scientific questions or for clinical transplantation, while antibodies or ligands suitable for cell isolation are not available. Currently, new technical developments are expanding its application toward fractionation of healthy and malignant tissue cells and the preparation of dendritic cells for immunotherapy.     

Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation

Synchronization of cells and nuclei is a powerful technique for the exact study of regulatory mechanisms and for understanding cell cycle events. Counterflow centrifugal elutriation is a biophysical cell separation technique in which cell size and sedimentation density differences of living cells are exploited to isolate subpopulations in various stages of cell cycle. Here, a protocol is described for the separation of phase-enriched subpopulations from exponentially growing Chinese hamster ovary cells at high-resolution power of elutriation. The efficiency of elutriation is confirmed by measuring the DNA content fluorimetrically and by flow cytometry. The resolution power of elutriation is demonstrated by the ability to fractionate nuclei of murine pre-B cells. The installation and elutriation by collecting 16-30 synchronized fractions, including particle size analysis, can be achieved in 4-5 h.     

Novel method for continuous cell separation by density gradient centrifugation: evaluation of a miniature separation column

A compact bench-top model of the centrifuge enables continuous cell separation based on density differences. The apparatus holds a small separation disk equipped with a circular channel (8 mL capacity) separated by a septum. A set of isotonic Percoll media with different densities is continuously introduced at one terminal and collected from the other. Under a centrifugal force field, cell suspension introduced into the proximal portion of the channel results in continuous separation of cells according to their densities. The performance of the apparatus was demonstrated with the separation of human buffy coat containing nucleated cells (>10(8)) among a large population (10(10)) of RBC. The results indicated that the method is capable of separating a large number of nucleated cells, with minimum damage, for a few hours of operation wherein neutrophils are well resolved from lymphocytes. The method may be applied to other types of samples including cord blood, blood from small animals, cultured cells, pancreatic beta cell islets, malaria parasites, sperm cells, etc.     

Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation

Cell separation by counterflow centrifugal elutriation (CCE) or free flow electrophoresis (FFE) is performed at lower frequency than cell cloning and antibody-dependent, magnetic or fluorescence-activated cell sorting. Nevertheless, numerous recent publications confirmed that these physical cell separation methods that do not include cell labeling or cell transformation steps, may be most useful for some applications. CCE and FFE have proved to be valuable tools, if homogeneous populations of normal healthy untransformed cells are required for answering scientific questions or for clinical transplantation and cells cannot be labeled by antibodies, because suitable antibodies are not available or because antibody binding to a cell surface would induce the cell reaction which should be investigated on purified cells or because antibodies bound to the surface hamper the use of the isolated cells. In addition, the methods are helpful for studying the biological reasons for, or effects of, changes in cell size and cellular negative surface charge density. Although the value of the methods was confirmed in recent years by a considerable number of important scientific results, activities to further develop and improve the instruments have, unfortunately, declined.     

Characterization of bombesin receptors on canine antral gastrin cells

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mathematical model for the growth of a single cell of E. coli on a glucose/glutamine/ammonium medium

Extension of a single cell model of E. coli B/r to make predictions of culture response to variations in glutamine/glucose/ammonium ion concentrations is described. A biphasic glutamine transport system, a nitrogen metabolism scheme that includes glutamate dehydrogenase (GDH), glutamine synthetase/glutamate synthase (GS/GOGAT), the glutaminase routes, and a transaminase mechanism for glutamine carbon usage are added to the prototype model. The predictions of the extended model with regard to nutrient concentrations and cell size compare well with the experimental data and the prototype model predictions, demonstrating the capability of the integrated kinetic model to illustrate important enzymological interactions in a biological system. The discrepancies between the experimental data and the model predictions on growth yield suggest that a more detailed regulatory system of the TCA cycle is required for a more accurate energy budget.     

A semi-empirical glycosylation model of a camelid monoclonal antibody under hypothermia cell culture conditions

The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars’ levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.     

A simple model to describe transient differences between cell number and biomass growth rates of Escherichia coli

Information on the response of a microbial culture to dynamic environmental conditions is necessary for the design of transient operation processes. However, most attempts at modelling culture response have been directed at describing the steady-state behavior. Thus, there is a need for adequate dynamic models for process design. Simulations of nutrient shifts were completed using a "single-cell" model for Escherichia coli. It was discovered that the specific mass growth rate and the specific number of cells growth rate were different under transient conditions, whereas at steady state (balanced growth) these rates are equivalent. Using these observations, a simple delay model to describe the transient behavior of the two growth rates is formulated and tested. The model contains as state variables only the readily measurable macroscopic quantities (biomass, cell number, and limiting nutrient). This model agreed well with the predictions of the single-cell model.     

Characterization of the separation properties of the Beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12--35 micron diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at ech collection rotor speed) and a long collection method (10--15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The long collection method significantly improved the homogeneity of the collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.     

Testing a Mathematical Model of the Yeast Cell Cycle

We derived novel, testable predictions from a mathematical model of the budding yeast cell cycle. A key qualitative prediction of bistability was confirmed in a strain simultaneously lacking cdc14 and G1 cyclins. The model correctly predicted quantitative dependence of cell size on gene dosage of the G1 cyclin CLN3, but it incorrectly predicted strong genetic interactions between G1 cyclins and the anaphase-promoting complex specificity factor Cdh1. To provide constraints on model generation, we determined accurate concentrations for the abundance of all nine cyclins as well as the inhibitor Sic1 and the catalytic subunit Cdc28. For many of these we determined abundance throughout the cell cycle by centrifugal elutriation, in the presence or absence of Cdh1. In addition, perturbations to the Clb-kinase oscillator were introduced, and the effects on cyclin and Sic1 levels were compared between model and experiment. Reasonable agreement was obtained in many of these experiments, but significant experimental discrepancies from the model predictions were also observed. Thus, the model is a strong but incomplete attempt at a realistic representation of cell cycle control. Constraints of the sort developed here will be important in development of a truly predictive model.     

Nonlinear dynamics of eucaryotic pyruvate dehydrogenase multienzyme complex: decarboxylation rate,oscillations, and multiplicity

Pyruvate conversion to acetyl-CoA by the pyruvate dehydrogenase (PDH) multienzyme complex is known as a key node in affecting the metabolic fluxes of animal cell culture. However, its possible role in causing possible nonlinear dynamic behavior such as oscillations and multiplicity of animal cells has received little attention. In this work, the kinetic and dynamic behavior of PDH of eucaryotic cells has been analyzed by using both in vitro and simplified in vivo models. With the in vitro model the overall reaction rate (nu(1)) of PDH is shown to be a nonlinear function of pyruvate concentration, leading to oscillations under certain conditions. All enzyme components affect nu(1) and the nonlinearity of PDH significantly, the protein X and the core enzyme dihydrolipoamide acyltransferase (E2) being mostly predominant. By considering the synthesis rates of pyruvate and PDH components the in vitro model is expanded to emulate in vivo conditions. Analysis using the in vivo model reveals another interesting kinetic feature of the PDH system, namely, multiple steady states. Depending on the pyruvate and enzyme levels or the operation mode, either a steady state with high pyruvate decarboxylation rate or a steady state with significantly lower decarboxylation rate can be achieved under otherwise identical conditions. In general, the more efficient steady state is associated with a lower pyruvate concentration. A possible time delay in the substrate supply and enzyme synthesis can also affect the steady state to be achieved and leads to oscillations under certain conditions. Overall, the predictions of multiplicity for the PDH system agree qualitatively well with recent experimental observations in animal cell cultures. The model analysis gives some hints for improving pyruvate metabolism in animal cell culture.     

Characterization of the separation properties of the beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12–35 μm diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at each collection rotor speed) and a long collection method (10–15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.