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.     

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.     

Theory and practice of centrifugal elutriation (CE). Factors influencing the separation of human blood cells

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that selective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5 X 10(8) rbc) resulted in better separations than low cell loads (5 X 10(7) rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

Theory and practice of centrifugal elutriation (CE)

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×10⁸ rbc) resulted in better separations than low cell loads (5×10⁷ rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

DIFFERENTIAL ENRICHMENT OF CELLS FROM EMBRYONIC RAT CEREBRA BY CENTRIFUGAL ELUTRIATION

—Centrifugal elutriation was used to obtain different populations of cells dissociated from 16-day-old rat embryo cerebra. The cell populations recovered were viable and could be maintained for several weeks in vitro. Sterile conditions were maintained throughout a preparation. Rat pups were removed by Caesarean section, the cerebra dissected and the cells dissociated by brief exposure to trypsin (0.125%, 6 min). An equivalent volume of elutriation medium (Dulbecco's medium containing 1% fetal calf serum, sodium bicarbonate, penicillin and streptomycin, EDTA, and deoxyribonuclease) was added to the trypsin-cell suspension, the dissociated cells pelleted, resuspended in elutriation medium and counted. Up to 4 x 10⁸ cells were injected into the previously sterilized elutriator. Seven fractions were usually recovered from a preparation. The first fraction contained primarily red blood cells and cell debris, which could not be maintained in vitro. Upon culture, fraction 2 consisted of predominantly non-neuronal cells, while fractions 3–6 contained neuronal and non-neuronal cells. The morphological characteristics of the neurons differed in these fractions. Fraction 7 contained cells that had reaggregated during the elutriation procedure and exhibited a variety of cell types in vitro.     

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Temperature regulation during centrifugal elutriation and its effect on cell separation

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulations have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6 degrees C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3 degrees C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.     

Protein factors in extracts of ribosomes isolated from L-929 cells during various phases of the cell cycle

Centrifugal elutriation has been utilized in order to separate cultures of L-929 fibroblasts into subpopulations containing cells at different stages of the cell cycle. The subpopulations were characterized by Coulter counter volume determination, [3H]thymidine label into DNA and flow cytometry. When a population of early G1 cells was returned to roller culture it was shown to progress through the cell cycle in a synchronous manner. Ribosomal factor extracts were prepared from cells at various phases during the cell cycle. The amounts of protein in the extracts varied greatly, being lowest in early G1 phase and showing a peak during S phase. Polyacrylamide gel electrophoresis demonstrated that there were differences in the protein species present in the extracts. Some proteins were present in the same amounts throughout the cell cycle, whereas others appeared to show a form of cyclical behaviour.     

Differentiation of primary embryonic neuroblasts in purified neural cell cultures from Drosophila

Embryonic neuroblasts of Drosophila are undifferentiated precursor cells that give rise to the central nervous system. Centrifugal elutriation has been employed to fractionate embryonic cells on the basis of size. A fraction of large cells was found to be greatly enriched for neuroblasts, whereas mesodermal precursor cells were completely excluded. This allowed a second step of purification, based upon adhesion to glass, to provide virtually pure cultures of neural cells. The cells in these cultures had the properties of neurons of the Drosophila CNS: They gave rise to ganglion-like clusters from which neurites extended on the culture substrate, and they expressed the enzyme, acetylcholinesterase, and the cell surface antigens recognized by antisera raised against horseradish peroxidase. The production of large-scale neuronal cell cultures will be useful for immunological and molecular studies of neural cell differentiation.     

Temperature regulation during centrifugal elutriation and its effect on cell separation

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulation have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6°C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3°C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.     

Relationship between cell size, cell cycle and specific recombinant protein productivity

Centrifugal elutriation was used to produce cell cycle enrichedfractions of four commercially relevant recombinant cell lines,chosen to allow for variation in properties due to construct,expression system and parent cell type, from normally growingheterogeneous batch cultures. As these fractions had identicalculture histories and had not been subjected to any insult orstress which was likely to have adversely affected cellularmetabolism, they were ideal for further study of cellularproperties. Specific productivity, cell size and cell cyclestate of replicate elutriated fractions were measured for eachcell line. Results showed that cell size was the major cellulardeterminant of productivity for all cell lines examined. Productformation was not restricted to any particular cell cycle phaseand in all cases, production occurred irrespective of cell cyclephase. Specific productivity was lowest when the majority ofcells in the fraction were G₁, intermediate when themajority of cells in the fraction were S phase and greater whenthe majority of cells in the fraction were in G₂/M. However, the evidence suggests that size is the major cellulardeterminant of productivity; the apparent relationship betweencell cycle and productivity is secondary and can simply beascribed to the increasing size of cells as they progress thoughthe cell cycle. Thus, in addition to cell density and viabilitycell size is the cellular parameter which should be incorporatednot only into mathematical models of recombinant mammalian cellproduction processes but also into process monitoring andcontrol strategies.     

Characterization of the efficiency of a cell separation process by the extent of elimination of a contaminating cell type

A stepwise approach to the selection of an appropriate technique for a cell separation problem is presented in which the preparative purification of cells is linked to their analytical separation. We have introduced the extent of elimination of a contaminating cell type from the cell type which one chooses to purify, as a separation parameter that characterizes the efficiency of a separation process independently of the relative cell composition of the starting material. In order to compare different separation techniques, a preparative fraction boundary needs to be chosen between the cell types. We defined this boundary in terms of the physical property on which the separation is based such that yield and purity of the isolated cell suspension are optimized simultaneously. With this analytical approach, it was found that a similar elutriation technique separated human and equine mononuclear cells equally well and that the separability of human monocytes and lymphocytes improved when the cells were separated by increasing the limiting sedimentation coefficient value of the elutriation chamber in small increments.     

Synthesis of ribosomal proteins during the cell cycle of the yeast Saccharomyces cerevisiae.

Centrifugal elutriation was used to separate yeast cells by their cell cycle position. The rate of synthesis of ribosomal proteins showed a constant exponential increase through the cell cycle.     

A BROAD SPECTRUM ARTIFICIAL SEA WATER MEDIUM FOR COASTAL AND OPEN OCEAN PHYTOPLANKTON1

A new artificial seawater medium has been tested with 83 strains of coastal and open ocean phytoplankton from 11 different algal classes. The cultures were carried through four transfers, representing a period of eight weeks for most species. Only three species could not be maintained in the enriched artificial seawater, and 16 species, mainly from the Prymnesiophyceae and Dinophyceae, had reduced final cell yields compared to those grown in enriched natural seawater. Since 77% of the species tested grew equally well in enriched artificial or natural seawater and more than 95% could be maintained in the artificial medium, this recipe is useful over a broad spectrum of species. The artificial seawater base was enriched with a modified ES enrichment solution; the primary modifications were the omission of Tris and the addition of Si. Enriched medium was autoclaved without precipitation by lowering the pH before autoclaving. This was accomplished by adding equimolar amounts of Na-HCO₃ and HCl which produced NaCl and CO₂ during the heating process. When no pH buffer was used, precipitation could only be avoided by autoclaving the artificial seawater base as two separate salt solutions (with Ca and Sr separated from CO₃^?2 and SO₄^?2), cooling, mixing and aseptically adding the sterilized enrichment solution.     

Monitoring of HAB species in the Mediterranean Sea through molecular methods

A qualitative and semi-quantitative polymerase chain reaction(PCR)-based assay was developed for the detection of severalpotentially Harmful Algal Bloom (HAB) species and genera belongingto Dinophyceae, Bacillariophyceae and Raphydophyceae. Oligonucleotideprimers were designed based on Internal Transcribed Spacer (ITS)–5.8Sribosomal DNA (rDNA) sequences available in public databaseor identified in this study. The specificity and sensitivityof the PCR assay were validated using clonal cultures and thennatural seawater samples, as well as the known copy number ofplasmids containing the target ITS–5.8S rDNA regions.A filter system for collecting mixed phytoplankton cells coupledto a target species-specific PCR assay was performed on spatialand temporal series of net and surface seawater samples duringcoastal water monitoring carried out in several localities ofthe Mediterranean Sea. The application of PCR allowed a rapiddetection of various genera and species-specific potential HABtaxa in all examined natural samples. Qualitative and semi-quantitativePCR results obtained from field samples were compared with microscopic[light microscope (LM)] examinations. The two methods gave comparableresults, and the molecular assay was able to detect HAB targetcells at concentrations not detectable by microscopy or thoseof uncertain identity. The highest values of positive detectionof potential HAB taxon presence obtained by PCR assay comparedwith the microscopic examination ranged from 67 to 8.0%.     

Characterization of natural cytotoxic effector cells isolated from rat liver

Nonparenchymal liver cells (NPC) from normal untreated female Wistar/Furth rats were tested for natural cytotoxicity in a 4-hour 51Cr release assay against the murine lymphoma YAC-1, the murine mastocytoma P815, and the syngeneic rat mammary carcinoma TMT-081 tumor cell lines. NPC exerted strong cytotoxicity against all three target cells. In contrast, fresh spleen cells displayed cytotoxicity only against YAC-1, although after culture for 24 h at 37 degrees C cytotoxicity was displayed against all three target cells. Fresh spleen cells contained 2-15% large granular lymphocytes (LGL) as assessed by Giemsa staining whereas NPC contained 10-23% LGL and 10-25% Kupffer cells. Centrifugal elutriation produced fractions that were increased in one or the other of the cell types. More cytotoxic activity was observed in the fraction containing more LGL. The cytolytic activity of fresh spleen cells could be eliminated by either in vivo or in vitro treatment with anti-asialo-GM1 antiserum. On the other hand, the cytolytic activity of NPC was resistant to in vivo treatment, but was partially sensitive to in vitro treatment. Furthermore, the activity of cultured spleen cells was also partially sensitive to in vitro treatment. NPC and cultured spleen cells also were more resistant to suppression by prostaglandin E2 and nordihydroguaiaretic acid than fresh spleen cells. We conclude that LGL is mainly responsible for natural cytotoxicity of NPC and that some effector cells in NPC may be highly activated.     

Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a Dinoflagellate)

The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.     

Regulation of RNA synthesis in yeast III

Summary Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse-long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.