Growth and metastasis of Lewis lung carcinoma in the footpad of mice

When Lewis lung carcinoma cells were transplanted into the footpad of mice, exponential growth of tumour was seen from the 4th day. Intravasation of tumour cells was first seen at this time, then increased steadily up to the 10th day. Tumour cells entered the circulation by destructive degeneration of the vascular endothelium and also by transcellular passage, mainly through capillaries, venules and thin-walled tumour vascular channels. Multiple sites of entry of tumour cells into the circulation were frequently found. The rate of intravasation of tumour cells into the circulation was the same at the proximal, middle and distal portions of the tumour.     

Red blood cell life span in the ovine fetus

Red cell life span within the fetal circulation has not been reported, although erythrocyte life span has been studied in the adult and newborn. The present study quantified red cell life span in 12 chronically catheterized fetal sheep at 97-136 days gestation (term = 150 days) with the use of autologous red cells labeled with [(14)C]cyanate. Cyanate forms a permanent covalent bond with hemoglobin and acts as a permanent red cell label. In the fetuses, blood (14)C activity decreased in a curvilinear fashion with time and reached 50% of the initial activity at 16.4 +/- 1.6 (SE) days. In contrast, (14)C activity of autologous red cells in two adult ewes decreased linearly with time as expected, reached 50% of the initial (14)C activity in 59 days, and yielded life spans of 117 and 121 days. Computer modeling and parameter optimization taking into account growth and skewed life span distribution were used to analyze the (14)C disappearance curve in each fetus. The mean life span of all red cells in the fetal circulation was 63.6 +/- 5.8 days. Mean red cell life span increased linearly from 35 to 107 days as fetal age increased from 97 to 136 days (r = 0.83, P < 0.001). Life span of cells produced at the time of labeling was significantly greater than the mean life span. Fetal growth rate estimated from parameter optimization was 3.28 +/- 0.72%/day; this compared well with the rate of 3.40 +/- 0.14%/day calculated from fetal weights at autopsy. Mean corpuscular volume decreased as a function of gestational age, but the decrease was small compared with the large increase in red cell life span. We conclude the following: 1) red cell life span in the fetal circulation is short compared with the adult; 2) red cells in younger fetuses have shorter life spans than in near-term fetuses; 3) the curvilinear disappearance of labeled red cells in the fetus appears to be due primarily to an expanding blood volume with fetal growth; and 4) red blood cell life span in a growing organism will be significantly underestimated unless the expansion of blood volume with growth is taken into account.     

Variables controlling somatomedin production by cultured human fibroblasts

Somatomedin is secreted by multiple types of cultured cells including human fibroblasts. Since the control of somatomedin (Sm) production by fibroblasts may be an important regulatory step in cell division, we undertook studies to define the variables in tissue culture experimental design that may have significant effects on the secretion of Sm. Cell density was an important parameter in determining basal Sm production rates. Cultures plated at 2.5 X 10(4) cells/well produced 0.38 +/- 0.06 U/ml/10(5) cells whereas an increase in culture density to 6.2 X 10(4) cells/well was associated with a decrease in Sm production per cell to 0.23 +/- 0.04 U/ml/10(5) cells (P less than .01). Cultures stimulated by platelet-derived growth factor (PDGF) showed a similar reduction in Sm production with increasing cell density. Epidermal growth factor was stimulatory (5.4-fold increase) in low-density cultures but had no effect when high-density cultures were used. If the experiments were initiated between 2 and 4 days after the last media change there were no significant differences in basal or PDGF-stimulated Sm concentrations. Between days 5 and 9 however, there was a progressive increase in the basal Sm production rate. The duration of incubation was an important variable since Sm production increased during the first 12 h in noncycling cells and showed an accelerated increase between 4 and 8 h in cycling cells. Cells between the eighth and 12th passage had similar basal Sm production (0.22 +/- 0.04 U/ml/10(5) cells) rates; cells between the 19th and 20th passages had significantly higher basal Sm production rates (0.41 +/- 0.05 U/ml/10(5) cells) (P less than .01). These results suggest that several variables, particularly cell density and passage number, are critical variables when quantitating the effect of hormones and growth factors on Sm production by cultured fibroblasts.     

Lipid metabolism during the initiation of lactation in the rat. The effects of starvation and tumour growth.

1. The effects of starvation post partum (24 h) and tumour growth pre partum on the initiation of lactation in the rat were studied. 2. Tumour growth decreased food intake at 24 h, but not at 2 days post partum. 3. Pup growth rate increased with hyperphagia; starvation and tumour burden decreased pup growth, and starvation decreased maternal body weight. 4. Starvation decreased gastrointestinal-tract mass; tumour growth decreased gastrointestinal-tract and mammary-gland mass. 5. Mammary-gland DNA-synthesis rate was high immediately post partum, but decreased by day 3 of lactation; starvation and tumour burden decreased this rate, and also decreased gastrointestinal-tract DNA-synthesis rate. 6. Arteriovenous differences for glucose and lactate across the mammary gland did not change with time, nor were they affected by the tumour. Starvation decreased arterial glucose and lactate, and the gland extracted less glucose but produced lactate. 7. Mammary-gland lipogenesis was sensitive to starvation and to tumour growth. 8. In contrast with the gradual development of mammary-gland lipogenic enzyme activities, lipoprotein lipase activity was high in the gland by 2 days post partum; starvation or tumour burden decreased the activity. 9. The mammary gland is sensitive post partum to decreased food intake, and to tumour presence. The effects of the latter are apparently independent of hypophagia.     

Effects of thyroxine on mitochondrial morphology and uptake of 35S in embryonic chicken livers

Ultrastructural analyses of reactions of mitochondria in hepatocytes of chicken embryos to low levels of exogenous thyroxine (T4) reveal that such reactions (overall swelling accompanied by disruption of crest geometry) first take place at about 10 days of incubation, T4 having been administered on the 6th day. Physically altered mitochondria may be seen after 11-12 days of incubation but are no longer evident by 13 days. Correlated with the initial evidence of T4 effects on mitochondria at 10 days of incubation is a spurt in hepatocyte proliferation. The time correlation observed between T4 induced mitochondrial changes in morphology and abrupt increases in rates of cell proliferation, suggests that liver nuclear receptors for thyroxine are unavailable prior to 9-10 days of incubation. Golgi complexes within the hepatocytes appear to be especially active in the production of electron-opaque vesicles from at least the 8th day of incubation to 11-12 days. Uptake of 35S (probably into chondroitin sulphates) was found to be fifteen times greater on the 8th day of incubation than at 15 days. This correlates with the period of heightened activity of the Golgi complex. In livers exposed to T4 on the 6th day, uptake of 35S was higher on the 9th and 10th days of incubation as compared to controls.     

Cell proliferation in Walker tumour growing in the stomach wall.

Tumour cells from a Walker carcinosarcoma 256 were implanted in the gastric mucosa in rats. The tumour grew and infiltrated the lamina propria and the submucosal space after 7 days. It appeared to grow faster in the submucosal space than in the lamina propria. The cell proliferation was therefore studied separately in: (1) the tumour in the lamina propria, (2) the main tumour mass and (3) the tumour periphery, defined as the cells located within the outer 100-120 mum of the tumour. Mitoses arrested with vinblastine, cells labelled with tritiated thymidine and the grain count per labelled cell were studied at the three different sites. The rate of cell proliferation in the tumour was highest in the lamina propria, lower in the centre of the main tumour mass, and lowest at the periphery. Cell loss might explain the discrepancy between the rate of cell proliferation and the actual tumour growth. The factors that influence tumour cell proliferation in the different parts of the tumour are discussed.     

Tumor promoters retard the loss of a transient subpopulation of cells in low passage Syrian hamster cell cultures

Early passage normal diploid Syrian hamster (SH) fetal cell cultures contain a transient subpopulation of contact-insensitive (CS-) cells which lack density-dependent inhibition of cell division. The size of this CS- subpopulation decreases during in vitro passage by conversion of the CS- cells to contact-sensitive (CS+) cells. Approximately 10-15 population doublings after the frequency of the CS- cells has declined to below 0.001%, mass cultures cease proliferating and exhibit cellular senescence. Cultures with higher initial numbers of CS- cells exhibit longer in vitro proliferative life spans than cultures with smaller initial numbers of CS- cells. Active tumor promoting phorbol esters (12-O-tetra-decanoyl-phorbol-13-acetate [TPA] and phorbol-12,13-didecanoate [PDD]) retard the decline in the proportion of CS- cells during in vitro passage, while the inactive tumor promoting phorbol ester, 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD) has no effect on the rate of loss of the CS- cells. In addition, continuous treatment from secondary culture with TPA or PDD extends by approximately twofold the in vitro proliferative life span of SH fetal cell cultures. Treatment must, however, begin at passage 1 or 2 when the CS- cells are still present. After the proportion of the CS- cells has decreased to less than 0.001% as in passage 6 cultures, promoters have no effect on the life span of the culture. This finding that exposure to promoters results in both a prolonged maintenance of the CS- cellular subpopulation, as well as an extension of in vitro proliferative life span, suggests that the conversion of CS- cells to CS+ cells is involved in the mechanism of in vitro senescence.     

Long-term in vitro cell culture of Sinclair swine melanoma.

The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in cluture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell cultures of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18.     

Suppressive effect of adrenalectomy on growth of L1210 leukemic cells in ascites

This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF1 mice. Varying doses of 1.5 x 10(4), 5.0 x 10(5), and 1.5 x 10(6) viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x 10(4) or 5.0 x 10(5) tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1.5 x 10(6) cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 micrograms dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using [125I]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x 10(5) labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time.     

Replicative senescence, telomere shortening and cell proliferation rate in Gaddi goat's skin fibroblast cell line

We assessed aging in continuous donor skin fibroblast cell line GGM5 up to the 25th passage by in vitro replicative senescence, telomere dynamics and chromosomal abnormalities. Cell proliferation rate increased from 0.84+/-0.26 (primary cells) to 1.20+/-0.17 (13-15 passage group) per day and reduced to 0.65+/-0.14 in 22-25 passages. Cell proliferation rate was reduced by 45.7% after 87.62 CPDs. Cell viability reduced from 100% to 97.4% up to the 25th passages. Frequency of beta gal(+) cells increased in successive passages and days in culture. The correlation coefficient between frequency of beta gal(+) cells and growth rate was -0.50 to -0.61. Loss of mean TRF length was 13.8 nucleotides (passage 15) to 95.4 nucleotides per cell division in later passages. All cells showed Robertsonian translocation in 22-25 passaged cells. The SCNT pre-implantation embryos production was highest (22.5%) in donor cells used from 10-15 passages as compared to early (相似文献   

A karyotype study of the cells of the African green monkey kidney cell line 4647 cultured long term in media with different sera

The karyological analysis of the cell line 4647 used for production of a killed vaccine to Hepatitis A virus was run in the 98th, 107th, 117th and 127th passages by the routine and C, G, and Ag methods of differential chromosome staining. A considerable balancing of the chromosome composition at 107-127 passage levels is shown. The cells of line 4647 present a significant heterogeneity, as to the number of chromosomes, and do not belong to any distinct modal class. The modal number of chromosomes ranged from 61 to 66 and from 121 to 125 for hyperdiploid and polyploid cells, respectively. The stable modal class of cells was established in the tetrasomic region, when culturing in the medium containing 10% CS from the 107th passage, and in the medium containing 10% FBS from the 117th passage, which conforms to one of the WHO requirements asserted to the substrate cells.     

CELL PROLIFERATION IN WALKER TUMOUR GROWING IN THE STOMACH WALL

Tumour cells from a Walker carcinosarcoma 256 were implanted in the gastric mucosa in rats. The tumour grew and infiltrated the lamina propria and the submucosal space after 7 days. It appeared to grow faster in the submucosal space than in the lamina propria. The cell proliferation was therefore studied separately in: (1) the tumour in the lamina propria, (2) the main tumour mass and (3) the tumour periphery, defined as the cells located within the outer 100–120 μm of the tumour. Mitoses arrested with vinblastine, cells labelled with tritiated thymidine and the grain count per labelled cell were studied at the three different sites. The rate of cell proliferation in the tumour was highest in the lamina propria, lower in the centre of the main tumour mass, and lowest at the periphery. Cell loss might explain the discrepancy between the rate of cell proliferation and the actual tumour growth. The factors that influence tumour cell proliferation in the different parts of the tumour are discussed.     

High-titer adenovirus vector production in 293S cell perfusion culture

Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 x 10(5) cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 x 10(6) cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 x 10(6) cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 x 10(6) cells/mL. One of the cultures was set at 37 degrees C and the two others at 35 degrees C. After a rapid initial cell loss, the average cell density stabilized at 5.75 x 10(6) cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 x 10(9) infectious viral particles/mL (IVP/mL) at 37 degrees C and 7.8 x 10(9) IVP/mL at 35 degrees C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 degrees C can improve viral titer by 2.4-fold compared to 37 degrees C, in accordance with a previous study on adenovirus batch production.     

Long-term in vitro cell culture of sinclair swine melanoma

Summary The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in culture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell culture of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18. This work was supported by a grant from the NIH, NCI (2 P01 CA 08023-11A1).     

Bovine embryonic stem cell-like cell lines cultured over several passages

Summary A total of 14 microsurgically produced zona pellucida-free bovine demi-blastocysts were cultured for 3 days in tissue culture medium (TCM) 199 supplemented with 10% heat-inactivated newborn calf serum (NBCS). Developing embryos were continuously cultured in TCM 199 plus 10% NBCS on a feeder-layer of murine embryonic fibroblasts, that had been incubated with mitomycin C (10 g/ml) for 3 h prior to the onset of embryo cultivation to block mitotic activity of the fibroblasts. After 2 days, 3 expanded blastocysts were attached to the feeder-layer and both trophoblastic cells and inner cell mass (ICM) cells became apparent on the 9th day of culture in 2 out of the 3 expanded blastocysts. Five days later, the ICM cells were disaggregated by a short-term trypsin treatment. The resulting dissociated clumps were seeded on a new murine embryonic fibroblast feeder-layer and covered with modified minimum essential medium (MEM)-Alpha with 10% fetal calf serum (FCS), 0.1 mm mercaptoethanol, 4.5 g/l glucose and 20 mm HEPES-buffer (=passage 0). To prevent differentiation of the cells, approximately 1/3 of the MEM-Alpha was replaced by MEM previously incubated on cell line 5637 containing leucaemia inhibitory factor (LIF) for 3 days. Colonies of embryonic stem cell (ES)-like cells were observed 5 days after the 1st passage. These colonies were repeatedly passaged at approximately 2-week intervals. Two bovine ES-like cell lines were established, which grew considerably slower than murine ES cells, but were lost after the 4th passage, possibly because of toxic effects of a new FCS batch. After cytogenetic analysis, 16 out of 18 metaphase plates contained an euploid number of chromosomes with 2 X-chromosomes and 58 autosomes. Distribution of G-banding on the chromosomes of ES-like cells was in accordance with the diploid set of the bovine genome. ES-like cells were fused to in vitro matured bovine oocytes and, upon successful fusion, cultured in vitro over 5 days. Successful fusion was observed in 79.8% (67/84), 31.3% initiated cleavege and 10.4% reached the 8–16 cell stage at termination of culture. Offprint requests to: H. Niemann     

Allometric study of the lachrymal and Harderian glands of the rat during postnatal life.

The authors performed an allometric study of the growth of the rat's lachrymal and Harderian glands, during postnatal life. From the analysis of the results, they could conclude: (1) the growth of these glands in relation to body weight, during postnatal life, could be considered similar, following the allometric law; (2) the differential growth of the glands occurred in two stages: from birth until the 15th day and from the 15th day until the final period of life studied; (3) the two stages of development were separated by a critical period, during which an abrupt modification of the allometric coefficient occurred; (4) during the first days of postnatal life, the development of the Harderian gland was characterized by a high rate of growth and, just after eyelid disjunction and during rest of postnatal life, by a rate of allometric of growth less than 1. It is interesting to observe that the lachrymal and Harderian glands' critical period of development on the 15th day of postnatal life coincides with the time at which the eyelids of the animal open.     

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones

The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours. A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase). Labelled mitoses curves and metaphase accumulation after colchicine injection show that the duration of the S, G2 and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days. Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G2 duration. The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G1-S transition when it is higher in the evening.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

提高人胚胎干细胞建系率的优选培养体系与方法

目的:建立一种从废弃胚胎中提高囊胚形成率和质量的培养体系,寻找多种促进内细胞团(ICM)数目增多、贴壁、增值的方法,提高人胚胎干细胞(human embryonic stem cell,hESC)建系效率,建立人胚胎干细胞库。方法:将179枚IVFDay3废弃的胚胎放入优选培养体系中培养(G2.5培养液中添加10%人血清蛋白,人白细胞抑制生长因子(hLIF),碱性成纤维细胞生长因子(bFGF))。到Day7将形成的囊胚全部用机械法分离ICM,接种于丝裂霉素C灭活处理的原代小鼠胚胎成纤维细胞(MEF)上,培养8-9天,每4-5天传代1次。结果:优选培养体系的囊胚形成率为29.1%(52/179),其中A级囊胚形成率为11.2%(20/179),50个ICM贴壁生长,20个出现克隆形态,成功建立11株hESC(FY-hES-11至FY-hES-21)。11株hESC均具有共同的多能性生物学特性。结论:优选培养体系可以明显提高囊胚形成的质量,促进ICM的增值,纯熟的机械切割法可以避免损伤ICM并提高其贴壁率,原代灭活的MEF饲养层可以明显促进细胞增殖。