Average Cell Density Research Articles

Lipid composition of Balb/c3T3, SV3T3, and Concanavalin A-selected revertant cells grown in media containing lipid-depleted serum.

The effects of growth in media supplemented with lipid-depleted fetal calf serum (LDS-media) on morphology, saturation density, and lipid composition were studied in Balb/c3T3, SV3T3, and Concanavalin A selected SV3T3 revertant cells (SV3T3 Rev cells). Cells grown in media containing complete fetal calf serum (FCS-medium) or reconstituted FCS (RS-medium) were used as controls. Growth in LDS-media reduced saturation densities of both SV3T3 and SV3T3 Rev cells while it affected only slightly the saturation density of normal parental cells. Similar inhibitory effects on growth were also induced by exposure of RS-medium. Growth in LDS-medium did not change the typical morphology of the three cell lines. 3T3, SV3T3, and SV3T3 Rev cells grown in LDS-medium showed an accumulation of triacylglycerols and free fatty acids together with a reduction of free cholesterol. All these changes were also present, however, in cells grown in these changes were also present, however, in cells grown in RS-Medium. Growth in LDS-medium induced an increase of 16:1 and 18:1, a decrease of 20:4, and an accumulation of 20:3 (n-9) in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol + phosphatidylserine of 3T3 cells. By contrast, only a slight accumulation of 20:3 (n-9) accompanied by a moderate increase of monoenoic acids was found in the phospholipids of SV3T3 cells grown in LDS-medium. SV3T3 Rev cells grown in LDS-medium showed changes in phospholipid fatty acids composition similar to those found in SV3T3 cells grown under the same conditions.

Asymmetrical Division of Saccharomyces cerevisiae in Glucose-limited Chemostat Culture

Saccharomyces cerevisiae S288C/1 was grown in a glucose-limited chemostat at population doubling times (τ) of 80 to 736 min. Estimates of the daughter cycle time (D), the parent cycle time (P) and the budded period (B) were obtained from bud scar analyses and equations derived from the Hartwell & Unger model of asymmetric cell division. D, P and B all showed biphasic linear relationships to τ, quantitatively different from estimates for the same strain in batch culture. Median cell volume and dry weight per cell increased at the faster growth rates, but the average cell density reached a minimum at τ = 150 min. The contiguous array of bud scars on parent cells became increasingly irregular as the doubling time increased from 140 min. At the fastest growth rates a small percentage of filamentous forms were observed due to failure of cells to undergo cell separation.

HISTOCOMPATIBILITY AND CORNEAL GRAFT ENDOTHELIUM

Endothelia of 102 clear human corneal grafts in 95 patients were photographed in vivo with a specular microscope. The relation of the endothelial cell density to the HLA and ABO compatibility of the donor‐recipient pairs were studied. In addition, the effects of vascularization and reversible rejection on the endothelial cell density were analysed.A negative correlation was found between the HLA incompatibility and the endothelial cell density of the graft. The group with 0–1 (2) mismatches had the highest endothelial cell density (1356 ± 510 cells/mm2). This result was better than in the group with 2 (3) mismatches, but the cell difference was not statistically significant. A statistically significant difference in average endothelial cell densities was found between the groups with 0–1 (2) mismatches and with 3–4 mismatches, the latter having 880 ± 475 cells/mm2 (P < 0.01). There was also a statistically significant difference in average endothelial cell densities between the group with 0–1 mismatch and the untyped group (P < 0.01). The difference in cell densities between the group with 2 (3) mismatches and the more incompatible groups together (3–4 mismatches and the untyped group) was statistically almost significant (P < 0.05).The ABO‐compatible pairs had 18% more endothelial cells than the ABO‐incompatible pairs. The difference was not statistically significant, but when the ABO‐compatible pairs were compared with all the others (incompatible and untyped), the difference in mean cell density between the two groups was statistically almost significant (P < 0.05).Comparison of the effect of tissue incompatibility on graft endothelial cell density separately in the patients with vascularized and non‐vascularized corneas showed that in both categories the endothelial cell density was highest in the group with 0–1 (2) mismatches.In 8 patients with a reversible rejection episode the average cell density was 20% less than in those with an uneventful postoperative period. The importance of rejection on the endothelium in some special cases is discussed.

EFFECTS OF CORNEAL PRESERVATION, DONOR AGE, CADAVER TIME AND POSTOPERATIVE PERIOD ON THE GRAFT ENDOTHELIUM

The endothelia of 102 clear human corneal grafts in 95 patients were photographed in vivo with a specular microscope. The endothelial cell densities of the grafts were counted from a area of 0.01 mm2. The follow-up period averaged three years. The patients' ages ranged from 16 to 77 years and that of the donors from 8 to 89 years. The average endothelial cell density for all 102 transplants was 1169 +/- 478 cells/mm2. Corneal transplants stored in MK medium (N = 19) have more cells than cryopreserved grafts (N = 42) or grafts stored in the moist chamber (N = 41); this last group was used as control material. The differences in cell densities were statistically significant. The cell density of cryopreserved grafts was significantly higher than in grafts stored in moist chamber. Only in those grafts in which moist chamber storage had been used was endothelial cell density dependent on time of storage. With an increase in cadaver time (time from death to enucleation), the endothelial cell density of the graft decreased. An inverse correlation was found between the cell density of the graft and donor age. Good results were still found with healthy donors over 60 years. The endothelial cell density of the grafts showed a steady decrease during the long-term post-operative period, the rate of cell loss being much higher in graft endothelium than in normal endothelium during ageing.

Marrow adherent cell colony-stimulating activity production in acute myeloid leukemia

Provision of colony-stimulating activity (CSA) by human marrow cells was determined utilizing culture techniques in vitro to assess intramedullary cellular interactions on human granulopoiesis. CSA production from marrow cells of 21 patients with acute myeloid leukemia (AML) was evaluated and compared to normal marrow by testing the capacity of conditioned media from adherent marrow cells to promote granulocytic colony formation in agar of relatively-light-density nonadherent human marrow target cells. Morphologic, cytochemical, density, and phagocytic characteristics of normal marrow cells suggested that marrow CSA production was provided by middensity adherent cells including those of the monocyte-macrophage series. Decreased marrow cell CSA provision was found in 13 of 21 studies of patients with AML at diagnosis or relapse (p

Quantitative Corneal Endothelial Evaluation in Intraocular Lens Implantation and Cataract Surgery

We analyzed pre- and postoperative corneal endothelial photographs of 33 intraocular lens patients and found an average endothelial cell density loss of 14%. Twenty-six cataract patients, similarly examined, showed a 13% mean endothelial cell loss. In 14 of the intraocular lens patients, a second postoperative evaluation of the central endothelium, at a later date, revealed a trend toward continuing cell loss. Our results indicate fewer endothelial cells were lost after intraocular lens implantation than has been previously reported. This may be accounted for by our considering only patients with normal corneas for lens implantation, and by our constant maintenance of the anterior chamber with an air bubble during lens implant surgery.

The in vitro cell fusion of embryonic chick muscle without DNA synthesis

A system has been developed for the in vitro development of chick skeletal muscle monolayers, in which a burst of synchronous fusion occurs, such that some 40% of the spindle-shaped cells fuse in a 10-hr period. Cells inhibited from synthesizing DNA by ara-C do fuse, but at a later time than the normal burst. If ara-C is added to cultures 6 hr or more before the normal fusion time, fusion is delayed, but no delay results when the drug is added after this time. A medium change will delay the fusion if done 4 hr or more before fusion, but gives no delay if done later. Cells grown in conditioned medium fuse some 10 hr earlier than controls, even in the presence of ara-C, as do cultures prepared at higher than normal cell densities. The data suggest that muscle cell fusion is independent of DNA synthesis in vitro, but depends upon a modification of the culture medium to a sufficient degree required for initiating the synthetic program for fusion.

Somatic cell mutation detection and quantification of X-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10 4 normal cells per 60 mm diameter culture dish and an AG concentration of 8·10 −6 M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments. Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10 −6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10 −6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10 −9 per R per cell at 75 R, 7.49·10 −8 per R per cell at 125 R, 6.87·10 −8 per R per cell at 150 R and 2.16·10 −7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants. Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.

Method for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors.

A procedure has been developed for isolating mutants which are defective with respect to nicotinamide adenine dinucleotide (NAD) metabolism. It is based on the well known V-factor requirement of Haemophilus parainfluenzae. This procedure was used to isolate a series of mutants from Escherichia coli. The pyridine metabolism of wild-type and mutant E. coli cells falls in one of four distinct classes. Class A includes wild-type E. coli and represents strains that are normal with respect to pyridine metabolism. Class B mutants have altered internal pools of NAD. The intracellular NAD concentration of different class B mutants varies over a 10-fold range. Class C mutants excrete pyridine mononucleotides, and class D mutants excrete NAD. The production of pyridine nucleotides by class C and D mutants exceeds that of wild-type E. coli by a factor of at least ten. The mutant strains generally have normal generation times and achieve normal cell densities in minimal medium.

PH as a determinant of cellular growth and contact inhibition.

1) Both the growth rate and the maximum population density of several normal, virus-transformed, and cancer cells were markedly pH-dependent; the optimum varied from pH 6.9 to 7.8. At the optimum pH, some diploid human cells attained population densities comparable to those of cancer or virus-transformed cells. Contact inhibition of growth is facilitated by repeated fluctuations of pH in nonphysiological ranges, and may not be an intrinsic and necessary attribute of diploid cells in culture. 2) At pH 8.3, at which there was little or no cellular multiplication, the protein content per cell increased 2- to 5-fold over a period of 10-16 days, and was slowly reversed to normal concentrations on restoration of pH to the optimal range. 3) Uridine uptake by contact-inhibited human cell cultures was stimulated by refeeding with salt solution, and to the same extent as by complete (serum-supplemented) growth medium; that immediate increase did not involve the reinitiation of cellular growth and multiplication. Contact inhibition was, however, reversed in 2-4 days by an appropriate increase in the serum concentration of the medium.

The influence of normal cells on the proliferation of tumour cells in culture

The effect of population density of normal fibroblastic cells on the proliferation of Rous, polyoma and sarcoma 180 cells was studied. The growth of Rous-transformed chick, rat and hamster cells was not inhibited by dense sheets of normal mouse or chick cells. Polyoma and S180 cell growth was inhibited by crowded mouse or rat cells, but not by crowded chick, quail or goose cells. Contact or close apposition of cells was essential for the transmission of growth-inhibitory influences. The transformation of chick cells freshly infected with Rous sarcoma virus was inhibited by dense mouse cells but not chick cells. Both mouse and chick cells exerted a feeder effect on tumour cell growth in monolayer and suspension. The proliferation of mouse and chick cells seeded at different densities was analysed.

Stimulation of DNA synthesis in cultures of mouse embryo fibroblast-like cells.

AbstractStimulation of the DNA synthesis and mitoses in stationary cultures of mouse embryo fibroblast‐like cells was induced by various agents such as ribonuclease, digitonin, fresh medium and commercial preparations of hyaluronidases. Time sequence of stimulation was similar in experiments with all these agents. Cells were activated to enter S phase from GI phase. The rise of the number of DNA‐synthesizing cells was preceded by a latent period of about 8–12 hours with the maximal number of DNA‐synthesizing cells being observed at 16–24 hours. Mitotic wave was observed after the wave of DNA synthesis. Stimulation of DNA synthesis and mitosis was not preceded by any significant decrease of an average cell density in the culture. The progeny of activated cells had no greater chance than other cells to be activated again when stimulation was repeated.It is concluded that similar proliferative reactions can be induced in stationary cultures by a variety of diverse agents. Possible role of cell surface changes in the induction of these reactions is discussed.

The conjunctival impression cytology between the diagnosed cases of dry eye and normal individuals

The dry eye or tear film dysfunction is a common ophthalmic syndrome. To compare the results of conjunctival impression cytology between dry eye patients and normal individuals. A case control study including consecutive cases of dry eye syndrome was carried out. Individuals without dry eye were taken as control. Impression of conjunctiva with cellulose acetate filter paper was taken from inferonasal bulbar conjunctiva and was stained with Periodic Acid- Schiff (PAS) and counter-stained with haematoxylin and eosin. goblet cell density. There was a female preponderance in dry eye disease. Of 114 dry eye cases, 49.2% eyes showed decreased or absent goblet cell density. In 72 normal individuals 73.7% eyes showed normal goblet cell density and 26.3% of eyes showed decreased or absent goblet cells (p less than 0.001). The tear break-up time (TBUT) test was significantly more likely to be less than 10 seconds in cases as compared to the controls ( OR = 19.36, 95% CI = 7.56 - 52.52). Similarly, the goblet cell density was likely to be significantly reduced in cases with dry eye syndrome (OR= 2.25, 95% CI = 1.26 - 4.02, p = 0.003). Goblet cell density significantly reduces in dry eye syndrome. The impression cytology is a useful test for the diagnosis of dry eye syndrome.

Automatic identification and measurement of cells by computer.

A visual-input computer system was used to automatically locate and perform measurements with high accuracy on single cells from microscopic slides. The nuclear radius, irregularity, area, and density of normal and malignant cells were automatically measured. These studies agreed with hand measurements showed differences normal and malignant cervical cells on Papanicolaou smears.

Quantitative studies of superior cervical sympathetic ganglia in a variety of primates including man. II. Neuronal packing density

AbstractThe volumes of a sample of primate superior cervical sympathetic ganglia were measured and related to body weight and to the number of ganglionic neurons. Estimates of volumes of the ganglia varied between 1.956 mm3 in squirrel monkey and 173.530 mm3 in a human specimen. Average cell densities for the ganglia ranged from 4,455 cells/mm3 in a human ganglion to 32,528 cells/mm3 in a squirrel monkey ganglion. Mean cell territories varied from 0.0000307 mm3 in a squirrel monkey ganglion to 0.0002245 mm3 in a human ganglion.Analysis of the data reveals striking trends of correlation between body size, volume of ganglia, and average cell territories. Since similar correlations have been described for other types of neuronal cell aggregates, it is suggested that for any given nucleus, ganglion or cortical area, the neuronal packing density varies as a function of body size.

The production of bacteriophage μ2

AbstractIn three series of experiments, 3‐l., 20‐l., and 150‐l. bacterial cultures were grown in stirred, deep culture vessels to average bacterial cell densities of 71 × 108, 63 × 108, and 43 × 108 viable organisms per milliliter, respectively, and then infected with phage. The average yield of progeny phage in each case was ca. 3000 mpfu (minimum plaque‐forming units) per cell. Thus, the average mass of phage obtained in the 3‐l. experiments was not less than 124 mg./l., calculated from the plaque counts, assuming a particle size of 3.6 × 106 Daltons for the μ2 phage. This is about twentyfold higher than is obtainable by conventional methods in aerated, shaken culture flasks. The actual phage yields are probably much higher than the minimum values calculated from plaque counts. For example, in the case of one of our culture lysates which was purified at King's College, the efficiency of plating was shown to be only 19%. The carbon dioxide evolution rate of cultures was measured and used as a guide to the time at which phage should be added. In this way, greater control of cultural conditions was obtained than is possible in shaken flasks. For the best yield of phage per milliliter of culture, the optimum time for phage infection was such that bacterial lysis just prevented the carbon dioxide evolution rate from reaching its potential maximum. The major factor influencing the phage yield per milliliter of culture was the aeration capacity of the culture vessel used. All had maximum aeration capacities much higher than those obtainable in shaken culture flasks. Cultures grown and infected in 3‐l. Vessel operated under conditions of low aeration gave poor yields of phage. The reason for this are discussed.

Nucleic Acid Content of Various Areas of the Rat Brain.

Summary and ConclusionsNucleic acid analyses were made on the various areas of the rat brain using the ultraviolet absorption method. It was found necessary to use fresh tissues to obtain correct ribonucleic acid concentrations since analyses in some areas (cerebellum and hypothalamus) were lower when the tissues had been kept frozen. Enzymatic analysis indicated that the higher concentration of 5'nucleotidase in cerebellum than in cortex might be indirectly responsible for the loss of the ribonucleic acid during the freezing and thawing processes. Deoxyribonucleic acid concentration in the cerebellum is approximately 4-5 times the amount found in the other areas. Ribonucleic acid content of the brain decreased in the order: cerebellum, hypothalamus, cerebral cortex, thalamus, white matter and medulla. Average cell densities, weight/cell, and total cells/area were calculated from the deoxyribonucleic acid concentrations. Close agreement between the calculated cell densities and direct measurements were found.