[Physical methods of separating cells of the immune system].
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Animals↗
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Another way to separate cells.
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Cell Separation↗
Antigenic characteristics of normal and malignant human hematopoietic cells. Potential use of antigenic differences in cell separation and marrow transplantation.
Recent studies using cytotoxic and cell separation techniques have identified differentiation-related antigens on human hematopoietic cells. These results, combined with bone marrow transplantation studies, have yielded a picture of antigenic modulation from the pluripotent hematopoietic stem cell through the differentiated end cells.
Antigens, Surface↗
First results obtained in France with the latest model of the Fresenius cell separator: AS 104.
In Besançon, we carried out 40 plateletphereses with the latest model of the Fresenius cell separator AS 104 to check this new system against the new generation of cell separators, according to the following criteria: less than 2x10 6 leukocytes (before filtration) and more than 5x10 11 platelets. The results show that platelet concentrates contained 5.04+/-0.88x10 11 platelets in a total volume of 435+/-113 mL. The mean platelet recovery was 40.95+/-4.86% (from 31.7 to 51.6). The leukocyte content was 2.28+/-5.48x10 6 and the red blood cell contamination was 3.48+/-2.38x10 8. The quality of the platelets was very satisfactory. There was no problem with donor biocompatibility or procedure safety, few adverse donor reactions (0.6%) and good therapeutic efficiency of platelet concentrates.
Cell Separation↗
Cell expansion and single-cell separation induced by colchicine in suspension-cultured soybean cells.
Single plant cells have been obtained without the preparation of protoplasts by culturing pieces of soybean callus tissue with colchicine. Cell expansion and separation were evoked by colchicine (1 mM) within a week of culture. Microscopic observation showed that cells took on a spherical shape in the presence of colchicine and then separated into single cells. Addition of colchicine to the culture medium did not affect the composition of cell wall polysaccharides, but a uronic acid-rich extracellular polysaccharide appeared during cell expansion and separation. Addition of microtubule stabilizers, glycerol (300 mM) or dimethyl sulfoxide [3% (vol/vol)], inhibited the secretion of the polysaccharide as well as cell expansion and separation. The extracellular polysaccharide elicited by colchicine was isolated by ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-6B from the conditioned medium of colchicine-treated soybean cells. The purified 18-kDa polysaccharide immediately enhanced cell expansion and separation when added to soybean callus tissue cultured in medium containing colchicine, even at low concentrations (0.1 mM). The polysaccharide was composed of galacturonic acid and, after digestion with a pectinase preparation, had no effect on the cells. Methylation analysis suggests that the polysaccharide consists of approximately 100 sequential alpha-1,4-galacturonic acids. The galacturonan increased the viability of separated cells cultured in medium containing colchicine, and the single cells obtained did not produce a wound-response callose. (Aminoethoxyvinyl)glycine, a specific inhibitor of ethylene production, extensively decreased the cell expansion and separation but did not inhibit the formation of the extracellular polysaccharide, suggesting that the polysaccharide may exert its effect by stimulating ethylene production.
Journal Article↗
Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm texture and cell separation during prolonged storage of apple.
Growth and maturation of the edible cortical cells of apples (Malus domestica Borkh) are accompanied by a selective loss of pectin-associated (1-->4)-beta-D-galactan from the cell walls, whereas a selective loss of highly branched (1-->5)-alpha-L-arabinans occurs after ripening and in advance of the loss of firm texture. The selective loss of highly branched arabinans occurs during the overripening of apples of four cultivars (Gala, Red Delicious, Firm Gold, and Gold Rush) that varied markedly in storage life, but, in all instances, the loss prestages the loss of firm texture, measured by both breaking strength and compression resistance. The unbranched (1-->5)-linked arabinans remain associated with the major pectic polymer, rhamnogalacturonan I, and their content remains essentially unchanged during overripening. However, the degree of rhamnogalacturonan I branching at the rhamnosyl residues also decreases, but only after extensive loss of the highly branched arabinans. In contrast to the decrease in arabinan content, the loss of the rhamnogalacturonan I branching is tightly correlated with loss of firm texture in all cultivars, regardless of storage time. In vitro cell separation assays show that structural proteins, perhaps via their phenolic residues, and homogalacturonans also contribute to cell adhesion. Implications of these cell wall modifications in the mechanisms of apple cortex textural changes and cell separation are discussed.
Carbohydrates↗
A novel method of cell separation based on dual parameter immunomagnetic cell selection.
We describe a novel method of cell purification involving two stage immunomagnetic selection which permits isolation of cells based on a second cell surface marker without the need for removal of beads used in the first selection step. This approach takes advantage of the size differences between commercially available immunomagnetic beads and/or particles and their differing properties in terms of attraction to magnetic fields of various strengths. The first stage of separation involves positive selection of cells using the Miltenyi MiniMacs system, utilising 50 nm MicroBeads and a MiniMacs magnet. Cells obtained from this procedure--still rosetted with 50 nm MicroBeads--can then be subjected to further positive or negative selection using either streptavidin M280 or anti-rat M450 Dynabeads, without the need for prior bead removal, since the strength of the magnetic field of the Dynal separator is sufficient to attract the larger Dynabeads but not the MicroBeads. Here, we show that this system can be used to isolate a number of cell types including very rare target cell populations such as haemopoietic stem cells, using two different surface markers without perturbing subsequent functional capacity.
Animals↗
[A new separation protocol (DRBCP-F) for automated blood component donation with the MCS 3p cell separator for collection of leukocyte depleted erythrocyte concentrates and plasma].
Previously published studies on automated blood component donation with the MCS 3p cell separator proved fairly good quality of the collected red blood cells (RBC) and fresh frozen plasma (FFP), with the disadvantage of a low hematocrit of the filtered RBC and a high platelet contamination of the FFP (RBCP-F protocol.) The DRBCP-F protocol was designed to eliminate the above-mentioned disadvantages and to provide 1 unit of leuko-depleted (filtered) RBC, 2 units of FFP, and additionally 1 platelet concentrate (PC) from the buffy coat. Twenty automated blood component collections (2 cycles, Latham bowl at 5,500 rpm, 230 ml isotonic saline for volume balance, PAGGS-M as additive solution) were performed. The RBC were filtered in a closed system after storage at 4 degrees C for 24 h. Blood cell counts and biochemical parameters of the RBC were determined initially and after 49 days. PC were separated from buffy coat after a soft spin. The volume of the RBC amounted to 293 +/- 12 ml (mean +/- SD) with a hematocrit of 0.61 +/- 0.05 l/l. Residual leukocytes after filtration were found to be 0.04 x 10(6) +/- 0.06 per unit. After storage, the following data were obtained: hemolysis 0.38%, ATP 2.1 +/- 0.4 mumol/g Hb, 2,3-diphosphoglycerate (2,3-DPG) 1.4 +/- 0.3 mumol/g Hb, ph 6.3 +/- 0.1, potassium 6.4 mmol per unit, and LDH in the supernatant was 219 U/l. None of the RBC showed bacterial growth after 49 days. The volume of the collected FFP was 398 +/- 32 ml, with 3.4 +/- 3.5 x 10(3) residual platelets and 5 +/- 12 leukocytes per microliter. Platelet concentrates contained 90.2 +/- 32 x 10(9) platelets in 88 +/- 14 ml plasma. Automated blood donation with the DRBCP-F protocol provided RBC with very low residual leukocyte counts, adequate hematocrit and good metabolic status up to 49 days, and FFP with low platelet contamination. The platelet concentrates were even superior to those prepared from whole blood using the buffy coat method. The storable leuko-depleted RBC are suitable for transfusion of chronically transfused patients in whom primary HLA sensitization should be prevented.
Adult↗
[Thrombocytapheresis with the (Baxter) A 201 cell separator--initial data of biocompatibility].
The cell separator A 201 was a new platelet pheresis prototype designed to carry out a discontinuous apheresis while platelet-rich plasma (PRP) is sampled continuously. After donation, a platelet concentrate (PC) and a fresh frozen plasma was collected from the PRP by the plasma cell-C device. The PC contained 2.6 +/- 0.7 x 10(11) platelets with a leucocyte contamination of 3.9 +/- 3.9 x 10(8) and an erythrocyte contamination of 3.0 +/- 3.0 x 10(8). The fresh frozen plasma was nearly cell-free. Prior, during and after apheresis we analysed hemolysis and coagulation parameters. There was no evidence for hemolysis. The analysis of the coagulation factors and of the Thrombin-Antithrombin III-complex, however, gave a hint of an activation of coagulation.
Biocompatible Materials↗
A simple technique for evaluation of methods of cell separation.
A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.
Antibodies, Monoclonal↗
Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast.
Here we report functional characterization of the essential fission yeast Skp1 homologue. We have created a conditional allele of skp1 (skp1-3f) mimicking the mutation in the budding yeast skp1-3 allele. Although budding yeast skp1-3 arrests at the G(1)/S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes. A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity. Other skp1-3f cells as well as the skp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation. Subsequent identification of 16 fission yeast F-box proteins led to identification of the product of pof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 mutants, and Pof6 localizes to septa and cell tips. Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex. These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.
Alleles↗
Differentiation of antibody-forming cells in toad spleen. A study using density and sedimentation velocity cell separation.
Antibody-forming cells (AFC), developing in toad spleen after stimulation with polymerized flagellin, were studied with an immune adherence assay. Differentiation was followed by several parameters: thymidine uptake to monitor dividing cells; equilibrium density centrifugation in albumin gradients to monitor cell density; microscopic measurements and sedimentation velocity separation to monitor cell size; stained preparations to follow cell morphology. Almost all AFC observed early in the response were dividing cells; the proportion of dividing AFC dropped to 4% 2 wk after stimulation. The earliest AFC detected (3 days) formed a relatively homogeneous light density population, and were purified 17-fold by equilibrium density centrifugation. As the response developed, additional denser peaks were found, so that late in the response dense AFC predominated. Dividing AFC were confined to the light density region throughout the response. Cell diameter measurements revealed that the earliest AFC were all very large cells. In a manner analogous to the density changes, smaller AFC appeared as the response developed until they finally comprised the majority of the AFC population. Dividing AFC were always relatively large, but encompassed a wide range of sizes. Sedimentation velocity separation was employed in a closer study of the immature AFC; they were purified 140-fold by this procedure. The earliest AFC consisted of several readily separable size populations in the range 9-18 micro diameter. The presence of separate peaks related by factors of two in volume suggested that the largest cells undergo a series of halving divisions before entering a division growth cycle. The results suggest an AFC differentiation sequence from a very large, light density, dividing "blast" cell to a nondividing cell with the size, density, and morphological appearance of a small lymphocyte. Stages of this sequence can be defined and selected out for investigation, using sedimentation velocity and equilibrium density centrifugation as complementary techniques.
Animals↗
Cell separation with staphylococcal protein A immunoadsorbent surfaces.
An 'indirect" cell separation technique was developed using protein A, in which each specific antiserum was first reacted with the cells to be processed. Separation was then achieved on antibody/protein A immunoadsorbent surfaces, where the final antibody coupled to the protein A was one which was directed against the specific antisera previously reacted with the cells. Only small volumes of specific antisera were required to effect separation either on a small or large scale with excellent specificity, cell viability and total cell recoveries. Theoretically, the technique would function with any Ig class of specific antisera from any species and would not be restricted to mammalian protein A binding IgG specific antisera, allowing considerable expansion in the number of antisera able to be utilized.
Antibodies↗
Blood conservation in cardiac operations. Cell separation versus hemofiltration.
The effects of hemoconcentration performed during and after extracorporeal circulation by either centrifugation (cell separation group, n = 20) or hemofiltration (n = 20) were investigated in 40 patients undergoing elective aorta-coronary bypass grafting. Interest was focused on the quality of the blood concentrated from the blood remaining in the extracorporeal circuit and on the reaction of the patients after retransfusion of the concentrated products. Hemofiltration was easy to perform and produced whole blood quicker than the cell separation technique. Coagulation studies revealed no significant differences in heparin concentration, levels of fibrinogen and antithrombin III, or platelet counts. Various coagulation parameters tended to normalize completely and more quickly after hemofiltration than after centrifugation. None of the patients had severe bleeding postoperatively. Free hemoglobin levels were not affected by hemofiltration; elastase concentration was higher only immediately after retransfusion of the concentrated blood, with no effect on organ function. We conclude that both methods were effective means of hemoconcentration during extracorporeal circulation and in salvaging the diluted pump blood after extracorporeal circulation. Loss of plasma fraction is an important disadvantage in the centrifugation technique, which can be avoided by hemofiltration; derangement in colloid osmotic pressure and coagulation parameters was less pronounced after hemofiltration. Costs were lower, as well. Therefore, when a high volume of cardioplegic solution and two-stage cannulation are used, hemofiltration seems to be the method of choice for blood conservation during cardiac operations.
Aged↗
Advantages of using a cell separator and metrizamide gradients for human islet purification.
Human islet transplantation has a high rate of failure, often due to primary nonfunction, which suggests that islets are damaged during the processing of the pancreas. The preparation of human islets for transplantation is still a complex process that requires large teams of surgical and laboratory personnel. To overcome this problem, we have adopted the use of the IBM 2991 COBE cell separator and a metrizamide/Ficoll density medium that is easy to prepare. Twenty-seven pancreatic glands have been processed using the COBE cell separator, 23 of which were purified in metrizamide/Ficoll gradients and 4 in bovine serum albumin gradients. The results show an improvement of recovery and viability in these preparations when compared retrospectively with manual gradients. More importantly, the time required for purification was shortened to one fourth the usual time and total processing time is about half as long. Moreover, a team of two laboratory staff was regularly able to prepare islets for transplantation, reducing the separation time from 7 hr to 3.5 hr. We conclude that the automatic cell separator and metrizamide-based separation medium are useful modifications of current islet purification methods.
Adolescent↗
Assessment of efficacy of cell separation techniques used in the enrichment of foetal erythroblasts from maternal blood: triple density gradient vs. single density gradient.
The aim of this study was to determine the efficacy of cell separation with single density and triple density-gradient techniques in the yield of foetal erythroblasts isolated from maternal blood. Maternal blood was obtained from 20 singleton pregnancies at 11-14 weeks of gestation immediately before foetal karyotyping by chorionic villus sampling. In each woman, the blood sample was divided into two portions; one portion was used for single density-gradient separation and the other, for triple density-gradient separation. Magnetic cell sorting (MACS) was subsequently performed with anti-CD71/antiglycophorin-A. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the gamma, epsilon and zeta globin chains. The percentage of foetal cells positive for each stain was calculated. Fluorescence in situ hybridization (FISH) for X- and Y-chromosomes was also performed. Comparison was made in the proportion of enriched foetal cells between the two separation methods for each CD71 and glycophorin-A (GPA) antibody. The percentage of erythroblasts enriched from maternal blood that stained positive for gamma, epsilon and zeta globin chains and with Kleihauer-Giemsa was significantly higher in the triple density-gradient separation fractions compared with the single density-gradient fractions with both anti-CD71 and GPA MACS. FISH analysis for the Y-chromosome confirmed the increase in foetal cell proportion in the triple density-gradient samples. Isolation of foetal erythroblasts from maternal blood using triple density-gradient separation and MACS is more effective with regard to foetal cell yield and purity than single density-gradient separation and MACS.
Cell Separation↗
Development of a model system to compare cell separation methods for the isolation of fetal cells from maternal blood.
Three major methods have been described for the isolation of fetal cells from maternal blood: fluorescence-activated cell sorting (FACS), immunomagnetic beads, and magnetic-activated cell sorting (MACS). To date, no study has directly compared fetal cell recovery using each of these methods. Here we describe our system using a "model' male fetal cell mixed into female peripheral blood mononuclear cells. Fetal cell yields and purities were assayed by a quantitative polymerase chain reaction (qPCR) using chromosomes Y- and 7-specific sequences. Fetal cell recovery was investigated by selection of CD71+ cells or depletion of CD45+ cells. Our data demonstrated variation in fetal cell recovery for all methods tested, although CD71+ selection by FACS gave the best and most consistent results.
Antigens, CD↗
Design of hollow fiber modules for uniform shear elution affinity cell separation.
Large-scale monoclonal antibody based systems for the selection of cell subsets will play a prominent role in the development of hematotherapy and graft engineering. Hollow fiber systems for affinity cell separation rely on the generation of uniform fluid shear stress at the lumenal attachment interface. Potential mechanisms for nonuniformity of lumenal wall shear stress are fiber wall permeation fluxes driven by the pressure gradient along individual fibers and the influence of inlet header dynamic pressure on the radial distribution of axial flow within the fiber module. Dimensional analysis and numerical solution of the flow field within the lumen of a hollow fiber module illustrate the main physical criteria for design of hollow fiber modules. There will be a nearly uniform distribution of flow within the fiber bundle provided that the dynamic inlet pressure is small in comparison with the pressure drop along fibers. Fiber wall permeation fluxes will have a negligible effect on axial flow rate for nonporous membranes such as Cuprophan.
Antibodies, Monoclonal↗