Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

Separation of megakaryocytes from rat bone marrow cells using velocity sedimentation in an isokinetic gradient of ficoll in tissue culture medium

A technique for the purification of rat megakaryocytes is described. Velocity sedimentation in a previously described isokinetic gradient of Ficoll (polysucrose) in tissue culture medium was more effective than isopycnic sedimentation for the purification of megakaryocytes and resulted in preparations of megakaryocytes which contained 2.4 ± 0.8% (range 1.85–3.60%) megakaryocytes. Megakaryocytes exhibited a broad range of density between 1.06 and 1.15 gm/ml. The inaccuracy which is inherent in the use of velocity sedimentation without isopycnic sedimentation as a means of particle size analysis is discussed.     

Release of chemical mediators from partially purified human lung mast cells.

Human lung mast cells dispersed by enzymatic digestion of human lung fragments were concentrated to greater than 50% purity by sedimentation in isopycnic and velocity gradients. The dispersed lung mast cells had a characteristic ultrasturctural appearance including granules with a scroll or reticular structural appearance including granules with a scroll or reticular structure surrounded by perigranular membranes. Histamine and preformed eosinophilotactic activity sedimented with mast cells on isopycnic gradients, and mast cells and these mediators were separated from the bulk of the other lung cells after velocity gradient sedimentation. The histamine content of isolated lung mast cells was calculated to range from 1.0 to 5.5 pg/cell. The quantity of SRS-A generated with anti-IgE or specific antigen was relatively limited but confined to the mast cell-rich fractions and associated with release of histamine and eosinophilotactic activity.     

Mass isolation of pole cells from Drosophila melanogaster.

Crude cell suspensions were obtained from 3 × 10⁶ pregastrula staged embryos. These cells fractionated into three bands on Renografin density gradients. Using ultrastructural characteristics for identification, pole cells were localized in the two denser bands. EM analyses also revealed a striking difference in the frequency of lipid vacuoles found in the pole-cell cytoplasm versus the cytoplasm of other embryonic cells. This difference has enabled us to identify pole cells by light microscopy using a neutral lipid stain. Through detailed analyses of the Renografin fractions with this stain, we have shown that pole cells resolve into two to three density classes. Evidence is presented which suggests that these density classes reflect different developmental age classes. The size differences between the pole cells and other embryonic cells contained in the enriched pole-cell fractions from the density gradients has enabled us to use sedimentation velocity centrifugation for additional enrichment. A distinct lower band of cells was obtained which consisted of 80% pole cells. Using these procedures, 1–2 × 10⁷ pole cells can be obtained daily.     

Cellular composition of fractions of mouse testis cells following velocity sedimentation separation

Identification of cells has been made in stained smears of cell suspensions prepared from mouse testes and separated by velocity sedimentation at unit gravity. Comparison of various methods of producing suspensions demonstrated that the best cell separations were achieved using suspensions prepared with trypsin. Various fractions obtained following separation contained 29% Sertoli cells sedimenting at about 14 mm/h, 17% Leydig cells at 11 mm/h, 73% pachytene spermatocytes at 9.5 mm/h, 54% binucleate spermatids and 14% secondary spermatocytes at 6.7 mm/h, 77% round spermatids at 4.5 mm/h, 21% elongating spermatids and 74% cytoplasmic fragments detached from these spermatids at 2.1 mm/h and 37% late spermatids at 0.75 mm/h. The resolution of different size classes of cells was essentially complete, but separation of different types of cells was limited by the occurrence of multinucleate forms of the cells and by fragments of damaged elongated spermatids. Most cells, however, appeared to be intact on light microscopical examination.     

Isolation of basolateral membranes from columnar cells of the proximal colon of the guinea pig

A method for an analytical isolation of plasma membranes from columnar cells (colonocytes) of the proximal colon of the guinea pig is described. Isolation of the colonocytes was performed by a mild EDTA-chelation method. After homogenization, two subsequent sucrose gradient centrifugations (isokinetic and isopycnic) yielded a plasma-membrane fraction which was enriched 12-fold in (Na+ + K+)-ATPase activity and 8-fold in adenylate cyclase activity. It is suggested that the purified membrane fraction consists mainly of basolateral membranes of the colonocytes. Due to the lack of suitable marker enzymes, no evidence for enrichment of the brush-border membranes was obtained. Histochemical studies demonstrated that alkaline phosphatase is absent from the luminal membrane of the surface epithelial cells of the proximal colon of the guinea pig.     

Separation of proximal tubule cells from suspensions of rat kidney cells by free-flow electrophoresis

Suspensions of rat kidney cells obtained by disaggregation of the kidney with 0.25% trypsin were separated by electrophoresis. Previously, we found a correlation between cells with histochemically demonstrable alkaline phosphatase (HDAP) and cells with brush borders which established that HDAP is a useful marker for rat proximal tubule cells (Kreisberg et al., '77). The starting suspension of cells for electrophoresis consisted of 38.4 +/- 5.7% nucleated cells with HDAP, 39.8 +/- 5.7% nucleated cells without HDAP, and 20.8 +/- 9.2% red blood cells. After electrophoresis, the purest fraction contained 85.8 +/- 3.5% nucleated cells with HDAP, 8.4 +/- 2.2% nucleated cells lacking HDAP, and 5.8 +/- 2.8% red blood cells; 91.9 +/- 2.4% of the nucleated cells in the purest fractions had HDAP.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

The evaluation of AKR leukemia virus purity: the requirement for both velocity and isopycnic centrifugation methods.

The purity of AKR murine leukemia virus obtained by isopycnic centrifugation was compared with the purity obtained by combining velocity sedimentation and isopycnic centrifugation methods. Evaluation of AKR and Rauscher viral purity by electron microscopy and by analysis of [³H]uridine-labeled viral RNA demonstrated that the velocity centrifugation step is essential for the removal of contaminants banding in the viral density region (1.19 – 1.15 g/ml). For studies requiring relatively pure oncornavirus preparations, a combination of both velocity sedimentation and isopycnic centrifugation steps are suggested. Viral recovery of about 50% was obtained by the method described.     

SHORT-TERM CULTURES OF MOUSE MARROW CELLS SEPARATED BY VELOCITY SEDIMENTATION

Our experiments were designed to identify the source of the increased number of cells forming colonies in culture (CFU-C) detected after short-term culture of mouse marrow cells over 'feeders' of renal tubules. Accordingly, marrow cell suspensions were fractionated by velocity sedimentation and aliquots of each fraction cultured for 2 days over tubule 'feeders'. We found a greater increase of CFU-C in suspensions of slowly sedimenting cells as compared to rapidly sedimenting cells. Colcemid and vinblastine were used to alter the peak sedimentation velocity of marrow suspensions by changing the distribution of cells in the cell cycle. After such manipulations, the peak increase in CFU-C continued to be associated with fractions containing slowly sedimenting cells. Such fractions also contained most of the pluripotent stem cells identified by the spleen colony technique.     

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.     

Identification and characterization of monolayer cultures of sheep trophoblast cells maintained in bicameral culture chambers

Enriched epithelial cell and fibroblast fractions were isolated from ovine placentomes by isopycnic centrifugation of collagenase/DNAse-dispersed cells through a density gradient of 45% Percoll. The epithelial cells formed confluent monolayers when plated onto filters impregnated with a 50-microns layer of Matrigel in medium containing 10% fetal bovine serum. These cells were maintained in dual environment culture chambers in serum-free medium for at least 12 days. The epithelium had a polarized appearance similar to that found in vivo only when cells were plated at high density (10(7)/cells/cm2). The epithelial monolayer consisted predominantly of a single population of uninucleate cells with intracellular features similar to those previously described for ovine trophoblast both in vivo and in vitro. These cells stained positively with an antiserum to alpha-keratin, a marker specific to epithelial cells, and no staining was observed with antisera raised against binucleate cells or leucocyte-common antigen. Binucleate cells were detected by microscopy and immunostaining in the pellet of cells obtained from the Percoll gradient but were rarely seen in the epithelium. The epithelial monolayer excluded 3H-inulin, added to the basal chamber, from the apical chamber, thus demonstrating the formation of a permeability barrier similar to that found in vivo. The maintenance of a monolayer of pure ovine trophoblast cells in vitro, which retain the characteristics of the epithelium in vivo, will enable the study of many cellular functions of the trophoblast.     

A novel lectin-gold density perturbation eliminates plasma membrane contaminants from Golgi-enriched subcellular fractions

The availability of pure Golgi fractions is a prerequisite for documenting the composition of the membranes of the Golgi Complex and comparing and contrasting this organelle with the rough endoplasmic reticulum. In a companion article, we have described a subcellular fractionation protocol for rat myeloma cells which effectively eliminates rough microsomes from Golgi-enriched fractions. Nevertheless, a major overlap with plasma membrane remains. We have therefore developed a novel density perturbation procedure to eliminate plasma membrane contaminants. By binding a conjugate of wheat germ agglutinin and colloidal gold to cells at 4 degrees C before homogenization we cause extensive sedimentation of plasma membrane markers to the "mitochondrial pellet" as well as a major shift in the isopycnic density of these markers. The differential and isopycnic sedimentation of several Golgi markers is unaffected in lectin-gold treated cells. The Golgi-enriched fractions obtained by isopycnic sedimentation are therefore of high purity. This procedure may be of general use for either purifying or eliminating plasma membrane-derived vesicles. Adaptations of the method might be equally useful for density perturbation of intracellular organelles.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

New revival of an old biomarker: characterisation of buccal cells and determination of genetic damage in the isolated fraction of viable leucocytes

Buccal cells serve as targets to assess oral exposures. We have refined isolation methods to characterise yield, viabilities, types of cells and baseline levels of genetic damage. Buccal cells were isolated from mouthwashes of 27 volunteers. They were characterised microscopically and different methods (using antibody-labelled magnetic beads, filtration and gradient centrifugation) were compared to separate epithelial cells from leucocytes. Viability of cells, DNA damage, and activity of glutathione S-transferase (GST) were measured with dye exclusion, microgelelectrophoresis, and biochemically. Mouthwashes contained approximately equal amounts of epithelial cells and leucocytes with detectable GST-activities. Repetitive determinations with mouthwashes from four individuals yielded per sample (3.5+/-1.4)x10(6) epithelial cells and (4.7+/-3.9)x10(6) leucocytes with viabilities of 8 and 94%, respectively. Epithelial cells could not be isolated using antibody-labelled beads, but cell separation with the leukocyte-specific antibody CD45 succeeded, yielding 37% leucocytes with a purity of 95% and viability of 65%. Filtering the mouthwash through a 10 microm filter yielded 57% leucocytes, with 86% purity and 94% viability. When using density gradient centrifugation as the separation method, the recovery of leucocytes was low (22%), but good results were scored for purity (95%) and cell viability (88%). This method was used to isolate leucocytes, which were then subjected to a micro-scale comet assay-modification. It was found that buccal leucocytes obtained from smokers had more DNA damage than cells from non-smokers. In conclusion, suspensions of buccal cells consist in approximately equal parts of epithelial cells and leucocytes. Only leucocytes are sufficiently viable for measuring parameters of cytotoxicity and genotoxicity or for studying modulation of gene expression. The cells are useful targets of non-invasive biomarkers, which could be incorporated as tools in many types of intervention studies.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.     

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.     

Separation of isolated hamster intestinal epithelial cells by velocity sedimentation on Ficoll gradients

Isolated hamster intestinal epithelial cells can be separated by velocity sedimentationion on 2–10% Ficoll gradients into three subpopulations of cells which differ in morphology, biochemistry, physiology, and membrane components. These subpopulations are not pure but are enriched in a single cell type to the extent that differences in cell function can be observed. The proliferative crypt cells are separated from the digestive-absorptive villus cells. A third subpopulation with a distinctive morphology is also obtained. Quantitation of DNA recoveries from the gradients indicates that this population constitutes approximately one-third of the epithelial cell population. These carrot-shaped cells are found adjacent to the digestive-absorptive columnar epithelial cells on the villus. The two types of villus cells differ in glycolipid or glycoprotein components of the brush border as shown by lectin binding experiments with the isolated cells. The gradient data also suggest that only one-third of the intestinal epithelial cell population is responsible for most monosaccharide absorption in hamster small intestine.