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1.
Cell population kinetics were followed in 9L tumour spheroids as they grew from aggregates of about 80 micron in diameter to over 800 micron. The kinetic parameters measured were cell cycle time, spheroid-doubling time, and growth fraction; from these the cell loss factor phi was calculated. The rate of cell shedding from the surface was also measured, so that the contribution of shedding to the overall cell loss could be evaluated. The major findings include significant elongation of the cell cycle, a low rate of cell death in spheroids below 500 micron in diameter, and a relatively high GF in large spheroids. The results also indicated that 9L spheroid kinetic parameters may be strongly influenced by the culture methodology. 相似文献
2.
Synthetic human gonadotropin releasing hormone (GnRH) injections were used for induction of spermatozoon release followed by cloacal lavage or mechanical stimulation of sperm release in Lepidobatrachus laevis . Light microscopic observations of Lepidobatrachus laevis spermatozoa indicated an acrosomal segment with a length of 4.1 μm delineated by an indentation, a nuclear region of 12.6 μm in length and a midpiece of 0.87 μm in length. The tail was 54.9 μm long by 1.35 μm wide with two lateral axial fibers and a central undulating membrane. At the electron microscopic level, the unusual tail had two complete axonemes that emanated from the distal centriole. The tail also contained two axial fibers 77 nm in diameter medial to the axonemes and was connected by an undulating membrane. An unusual accessory cell adherent to the head of the spermatozoon was noted in freshly obtained suspensions of spermatozoa. Spermatozoa with the accessory cell were motile and a subsequent loss of motility was correlated with the shedding of the accessory cell. 相似文献
3.
Abstract. Based on biological observations and the basic physical properties of tri-dimensional structures, a mathematical expression is derived to relate the growth rate of multicellular spheroids to some easily measurable parameters. This model involves properties both of the individual cells and of the spheroid structure, such as the cell doubling time in monolayer, the rate of cell shedding from the spheroid and the depth of the external rim of cycling cells. The derived growth equation predicts a linear expansion of the spheroid diameter with time. The calculated growth rate for a number of spheroid cell types is in good agreement with experimental data. The model provides a simple and practical view of growth control in spheroids, and is further adapted to include parameters presumably responsible for the growth saturation in large spheroids. 相似文献
4.
CELL CYCLE KINETICS IN AN IN VITRO TUMOR MODEL 总被引:1,自引:0,他引:1
Cell cycle kinetic parameters of multicell spheroids in vitro have been estimated using thymidine labeling techniques and autoradiography. Both the mitotic index and thymidine labeling index decreased in larger spheroids, whereas the duration of the cell cycle, as determined by two independent methods utilizing labeled mitoses or labeled cells, was essentially independent of spheroid size or age. These results suggest that the tumor-like growth exhibited by spheroids is the result of a decreasing growth fraction and a large apparent cell loss, rather than a general elongation of the cell cycle. 相似文献
5.
Adam P. Rago Dylan M. Dean Jeffrey R. Morgan 《Biotechnology and bioengineering》2009,102(4):1231-1241
Tissue fusion and cell sorting are processes fundamental to developmental biology with applications in tissue engineering. We have designed a fusion assay to investigate the factors governing the fusion of microtissues and the cell sorting that occurs after fusion. Normal human fibroblast (NHF) spheroids were self‐assembled and cultured for 1, 4, or 7 days, then combined in trough shaped recesses. Over a 24‐h period the spheroids fused to become a rod shaped microtissue and the kinetics and extent of fusion could be quantified by assessing rod contraction. By varying the amount of spheroid culture time prior to fusion (1–7 days), the rate of fusion, the coherence of the building units (as measured by fusion angle) and the steady state length of the structure could be easily controlled. Longer pre‐culture times for the spheroids resulted in slower fusion, less coherence and increased length of rod microtissues. The fusion kinetics and steady length of rods formed by smaller versus larger spheroids (~100 vs. 300 µm diameter) were indistinguishable, even though smaller spheroids had twice the surface area and greater numbers of contacts between units. Both small and large spheroids were strongly influenced by spheroid pre‐culture time. Pre‐culture time could also be used to control cell sorting and cell position when combinations of NHFs and H35s, a rat hepatocyte cell line, were fused to form heterotypic microtissues. Control of fusion and cell position are important parameters for creating functional heterotypic microtissues as well as the use of microtissues as building units to create larger tissue structures. Biotechnol. Bioeng. 2009;102: 1231–1241. © 2008 Wiley Periodicals, Inc. 相似文献
6.
Optical coherence tomography (OCT) was used to monitor the dynamics of tumour spheroid formation by the hanging drop method.
In contrast to microscopy, the estimates obtained using OCT for the volume of the spheroid, were consistent with the measured
changes in cell number as a function of time. The OCT images also revealed heterogeneous structures in the spheroids of ∼200 μm
diameter. These corresponded to the necrotic regions identified by fluorescence of propidium iodide stained cells. 相似文献
7.
Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle. 相似文献
8.
Jacques Landry James P. Freyer Robert M. Sutherland 《Journal of cellular physiology》1981,106(1):23-32
During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exonential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytoflurometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell-to-cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis. 相似文献
9.
A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of 3 H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to 3 H-thymidine in vivo and 14 C-thymidine 40-104 min later in vitro , the proportion of cells labelled with 3 H alone to those labelled with 14 C(±3 H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm). 相似文献
10.
Summary We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability
to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic
center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGU-U1 cells
placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and consistently than cells
derived from monolayer culture. Spheroid diameter increased at a rapid rate of ∼100 μm/d in multiwell dishes, and necrosis
occurred only in spheroids of diameter >1 mm. Spheroids placed in spinner culture at a higher concentration (∼1.5 spheroids/ml)
grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture
was maintained at ∼190 μm over a wide range of spheroid diameters (400 to 1000 μm). Sequential trypsinization of spheroids,
which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from
varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids.
This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and
grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models
to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human
bladder biopsies to form spheroids.
Supported in part by a grant from the National Cancer Institute of Canada and by grant CA29526 NCI through the National Bladder
Cancer Project, U.S.A. 相似文献
11.
Multicellular spheroids provide a physiologically relevant platform to study the microenvironment of tumors and therapeutic applications, such as microparticle-based drug delivery. The goal of this study was to investigate the incorporation/penetration of compliant polyacrylamide microparticles (MPs), into either cancer or normal human cell spheroids. Incorporation of collagen-1-coated MPs (stiffness: 0.1 and 9 kPa; diameter: 15–30 µm) into spheroids (diameter ∼100 µm) was tracked for up to 22 h. Results indicated that cells within melanoma spheroids were more influenced by MP mechanical properties than cells within normal cell spheroids. Melanoma spheroids had a greater propensity to incorporate and displace the more compliant MPs over time. Mature spheroids composed of either cell type were able to recognize and integrate MPs. While many tumor models exist to study drug delivery and efficacy, the study of uptake and incorporation of cell-sized MPs into established spheroids/tissues or tumors has been limited. The ability of hyper-compliant MPs to successfully penetrate 3D tumor models with natural extracellular matrix deposition provides a novel platform for potential delivery of drugs and other therapeutics into the core of tumors and micrometastases. 相似文献
12.
In situ oxygen consumption rates of cells in V-79 multicellular spheroids during growth 总被引:2,自引:0,他引:2
The rate of consumption of oxygen by V-79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 micron in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 micron diameter to a value one-fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid-derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell-cell contact, cell packing, and cell volume were not critical parameters. Plateau-phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V-79 spheroids to account for the remainder of the reduction observed in this system. 相似文献
13.
Cerebral Cortical Glucose Utilization in the Conscious Rat: Evidence for a Circadian Rhythm 总被引:10,自引:8,他引:2
P. D. Crane L. D. Braun E. M. Cornford A. M. Nyerges W. H. Oldendorf 《Journal of neurochemistry》1980,34(6):1700-1706
Abstract: The presence of a circadian rhythm of glucose utilization was demonstrated in vivo in rat cerebral cortex. The activity pattern of the rats, living in a controlled lighting regimen with lights on from 7 a.m. to 7 p. m., appeared to coincide with the rate of glucose consumption in the brain. The rate of utilization was measured at 3-h intervals throughout the day and was found to fall from a maximum at 3 a.m. of 0.98 ± 0.13 μmol min−1 g−1 to a minimum of 0.70 ± 0.08 μmol min−1 g−1 at 3 p. m. Brain glucose also varied with time and its fluctuating level weakly correlated with its rate of utilization. Animals entrained on a 5-h (4: 30-9: 30 p. m.) feeding schedule had a similar circadian rhythm, with only a slight increase in amplitude. Reversal of the light cycle caused a disruption in the normal rhythm, but utilization still varied significantly with time of day. The results both indicate the potential error that can be encountered in experiments done at different times of the day and stress the need for awareness of time of day as a factor in measurements of alterations of metabolic rate in the brain. 相似文献
14.
Adult female specimens of Liza ramada were pinealectomized and sham-pinealectomized, and the development of their ovaries was followed over a period of 14 weeks and compared with those of untreated controls. Pinealectomized specimens exposed to long photoperiod (16L/8D) for 14 weeks, showed undeveloped ovaries, with a maximum oocyte diameter of less than 100 μm, and a gonadosomatic index of 0·6 similar to that of sham-pinealectomized and control specimens. In pinealectomized females exposed to short photoperiod (8L/16D) for 6 weeks, the mean diameter of oocytes was 270 μm v . 155 μm in control and sham-pinealectomized specimens. After 9 weeks, the oocytes in pinealectomized specimens reached 430 μm as against 265 μm in the controls. Within 14 weeks of pinealectomy, mean oocyte diameter was 480 um while it was 400 μm in controls and sham-pinealectomized specimens. It is tentatively concluded that the pineal complex has an inhibitory effect on ovarian function in Liza ramada exposed to short photoperiod. 相似文献
15.
Characteristics of the reproductive cycle of Basilichthys microlepidotus (Jenyns), an atherinid fish living in a South American Mediterranean arid zone, are analysed. Specimens 9-170 mm in total length were captured from August 1982 to April 1984 in the Rio Choapa, in northern Chile (31%%45' S, 71°15' W). Analysis of gonadosomatic index fluctuations, and determination of the proportion of mature males and females captured during the study period, allowed the estimation of the duration of the reproductive cycle.
Females 70–170 mm in total length contained 2000–9000 intraovarian oocytes, of which approximately 80% were vitellogenic (100–1300 μm in diameter) and exhibited a polymodal distribution. A further 4 to 15% of the total oocytes, measured 1700–2000 μm in diameter and appeared ready to be spawned. In 30% of the examined ovaries some of the largest oocytes exhibited signs of degeneration, or atresia. Juveniles approximately 30 mm in total length were captured over most of the study period. Results strongly suggest that B. microlepidotus has multiple spawnings. 相似文献
Females 70–170 mm in total length contained 2000–9000 intraovarian oocytes, of which approximately 80% were vitellogenic (100–1300 μm in diameter) and exhibited a polymodal distribution. A further 4 to 15% of the total oocytes, measured 1700–2000 μm in diameter and appeared ready to be spawned. In 30% of the examined ovaries some of the largest oocytes exhibited signs of degeneration, or atresia. Juveniles approximately 30 mm in total length were captured over most of the study period. Results strongly suggest that B. microlepidotus has multiple spawnings. 相似文献
16.
Song H Jain SK Enmon RM O'Connor KC 《In vitro cellular & developmental biology. Animal》2004,40(8-9):262-267
Summary Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo
simulation provided an insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture
of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental
cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145
aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation
versus 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55±0.05 for
DU 145 aggregates with projected areas exceeding 2000 μm2 to a value approaching 0.75±0.02 (P<0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same
time period (P<0.05). Combined, these data suggest that motility events govern the overall rate of spheroid restructuring. This information
has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production. 相似文献
17.
RALPH INABNIT BILL CHOBOTAR JOHN V. ERNST 《The Journal of eukaryotic microbiology》1972,19(2):244-247
SYNOPSIS. Thirty-two of 48 raccoons examined were infected with a previously undescribed species of Eimeria which is herein named E. procyonis. Of the 32 infected animals, 10 also harbored E. nuttalli and 1 had Isospora sp. oocysts.
The ellipsoid to ovoid oocysts of E. procyonis measured 23.4 × 18.0 (16–29 × 13–24) μm; its sporocysts measured 12.1 × 9.3 (11.5–15 × 7–10) μm, each containing a slightly flattened substiedal body. The sporocyst residuum consisted of numerous scattered granules each ∼1 μm in diameter. The oocyst wall was double-layered. The outer layer appeared rough and pitted, measuring 1.5 μm, except at the micropyle where it was 1 μm thick.
The oocysts of the Isospora sp. measured 16.8 × 13.7 (16–18.5 × 12.5–15.5) μm. The wall consisted of a single layer ∼0.5 μm thick. The sporocysts measured 11.2 × 9.1 (9.5–11.5 × 8–10) μm, and each contained 4 elongate sporozoites. The oocysts of E. nuttalli measured 17.5 × 13.6 (12-21 × 11-15) μm, with a smooth single-layered wall approximately 0.7 μm thick. The sporocysts measured 12.2 × 7.1 (9-13 × 5.5–11) μm. Each sporocyst had a thin, dark, Stieda body and the sporocyst residuum consisted of many fine granules. 相似文献
The ellipsoid to ovoid oocysts of E. procyonis measured 23.4 × 18.0 (16–29 × 13–24) μm; its sporocysts measured 12.1 × 9.3 (11.5–15 × 7–10) μm, each containing a slightly flattened substiedal body. The sporocyst residuum consisted of numerous scattered granules each ∼1 μm in diameter. The oocyst wall was double-layered. The outer layer appeared rough and pitted, measuring 1.5 μm, except at the micropyle where it was 1 μm thick.
The oocysts of the Isospora sp. measured 16.8 × 13.7 (16–18.5 × 12.5–15.5) μm. The wall consisted of a single layer ∼0.5 μm thick. The sporocysts measured 11.2 × 9.1 (9.5–11.5 × 8–10) μm, and each contained 4 elongate sporozoites. The oocysts of E. nuttalli measured 17.5 × 13.6 (12-21 × 11-15) μm, with a smooth single-layered wall approximately 0.7 μm thick. The sporocysts measured 12.2 × 7.1 (9-13 × 5.5–11) μm. Each sporocyst had a thin, dark, Stieda body and the sporocyst residuum consisted of many fine granules. 相似文献
18.
《Biochemical Engineering Journal》2010,51(3):116-121
A scaffold-free tissue construct was formed by assembling endothelial cell-covered spheroids, and medium perfusion through the tissue construct was investigated using hydrostatic pressure-driven culture circuit. Primary rat hepatocyte spheroids covered by human umbilical vein endothelial cells (HUVECs) were assembled in culture chambers with a cylindrical culture space of 2 mm in diameter, and then medium was perfused through the assembled spheroids for 48 h. The medium flow rate through the culture chamber was measured over the perfusion culture time, which decreased during the first several hours, then increased or remained low depending on the amount of spheroids in the culture chamber. Histochemical analyses showed single tissue construct formation by spheroid fusion when cultured from 2 × 105 nuclei spheroids, with the loss of boundaries between the spheroids. Moreover, a viable cell region was found at the center of the tissue construct in several locations. Poor adhesion was found between spheroids cultured from 4 × 105 nuclei spheroids. The total nuclei density in cultured tissue constructs was estimated to be about half of that in HUVEC-covered hepatocyte spheroids.This study demonstrated the possibility of medium perfusion through scaffold-free tissue constructs by assembling endothelial cell-covered spheroids, promising for a large tissue construct culture in vitro. 相似文献
19.
The in vitro proliferation kinetics of a cell line derived from a patient with American Burkitt's lymphoma were investigated at three different growth phases: lag (day 1), exponential (day 3) and plateau (day 5). The growth curve, labeling and mitotic indices, percentage labeled mitosis (PLM) curves and DNA content distributions were determined. The data obtained have been analysed by the previously developed discrete-time kinetic (DTK) model by which a time course of DNA distributions during a 10-day growth period was characterized in terms of other cell kinetic parameters. The mean cell cycle times, initially estimated from PLM curves on days 1, 3 and 5, were further analysed by the DTK model of DNA distributions and subsequently the mean cell cycle times with respect to DNA distributions during the entire growth period were determined. The doubling times were 39·6, 31·2 and 67·2 hr, respectively, at days 1, 3 and 5. The mean cell cycle time increased from 23·0 to 37·7 hr from day 3 to day 5 mainly due to an elongation of the G1 and G2 phases. A slight increase in the cell loss rate from 0·0077 to 0·0081 fraction/hr was accompanied by a decrease in the cell production rate from 0·0299 to 0·0184 fraction/hr. This calculated cell loss rate correlated significantly with the number of dead cells determined by trypan blue exclusion. Analysis of the number of dead cells in relation to the cell cycle stage revealed that a majority of cell death occurred in G1 (r= 0·908; P < 0·0001). There was a good correlation between the in vitro proliferation kinetics at plateau phase of this Burkitt's lymphoma derived cell line and the in vivo proliferation kinetics of African Burkitt's lymphoma (Iversen et al., 1974), suggesting the potential utility of information obtained by in vitro kinetic studies. 相似文献
20.
Dramatic differences of cells behavior exist between cells cultured under classical 2D monolayers and 3D models, the latter being closer to in vivo responses. Thus, many 3D cell culture models have been developed. Among them, multicellular tumor spheroid appears as a nice and easy-to-handle 3D model based on cell adhesion properties. It is composed of one or several cell types and is widely used to address carcinogenesis, or drugs screening. A few and recent publications report the use of spheroids to investigate electropermeabilization process. We studied the response of spheroids to electrical field pulses (EP) in terms of their age, diameter or formation technique. We found that small human HCT-116 colorectal spheroids are more sensitive to electric field pulses than larger ones. Indeed, the growth of spheroids with a diameter of 300 μm decreased by a factor 2 over 4 days when submitted to EP (8 pulses, lasting 100 μs at a 1,300 V/cm field intensity). Under those electrical conditions, 650 μm spheroids were not affected. These data were the same whatever the formation method (i.e. hanging drop and nonadherent techniques). These observations point out the fact that characteristics of 3D cell models have to be taken into account to avoid biased conclusions of experimental data. 相似文献