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1.
Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age)
have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained
in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics
(penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml;
fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO2: 95% air.
The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning
and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and
interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria,
rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive
staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.
This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute. 相似文献
2.
Linda J. Loretz Catherine A. Reznikoff 《In vitro cellular & developmental biology. Plant》1988,24(4):333-342
Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells
(HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency
(CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded
in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×105 lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum.
Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions
in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining
for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder
cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone
supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented
MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%.
This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral
fellowship. 相似文献
3.
David Kirk Susumu Kagawa Gudrun Vener K. Shankar Narayan Y. Ohnuki Lawrence W. Jones 《In vitro cellular & developmental biology. Plant》1985,21(3):165-171
Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human
ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock
1, low Ca2+ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine,
0.1 mM each; hydrocortisone, 2.8×10−6
M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin
fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype.
Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a
population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed
that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca2+ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free
epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important
in future studies of carcinogenesis.
This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD. 相似文献
4.
Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media 总被引:3,自引:0,他引:3
David Danielpour Terry L. Riss Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(1):42-52
Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated
DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When
ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate
was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect
on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors
in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal
growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium
equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth
rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion
may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.
This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255. 相似文献
5.
Lawrence E. Shapiro Neil Wagner 《In vitro cellular & developmental biology. Plant》1988,24(4):299-303
Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium
is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum
containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these
two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium
was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the
absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium
from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free
culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells.
This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes
of Health grants CA 24604-09 and CA 16463-14. 相似文献
6.
Yasuhiro Tomooka Stephen E. Harris John A. McLachlan 《In vitro cellular & developmental biology. Plant》1985,21(4):237-244
Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained
in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically,
immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number
was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10
ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required
eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin,
and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free
media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides
a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells. 相似文献
7.
Susumu Yoshida Shigeo Kasuga Yuzo Hirao Tohru Fuwa Shizutoshi Nakagawa 《In vitro cellular & developmental biology. Plant》1987,23(7):460-464
Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein
when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations
as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much
lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the
cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic
mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture.
Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis
and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram
DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin. 相似文献
8.
Terramani TT Eton D Bui PA Wang Y Weaver FA Yu H 《In vitro cellular & developmental biology. Animal》2000,36(2):125-132
Summary The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial
cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells
(HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated
in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate-coated culture dishes were
also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly
used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell
proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth
supplement (ECGS) (50μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement
in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were
grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types
I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented
with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate-coated culture dishes offer no advantage. 相似文献
9.
Lisa J. Leiderman J. Allan Tucker Vincent W. Dennis 《In vitro cellular & developmental biology. Plant》1989,25(10):881-886
Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to
that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's
medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml).
Cells in serum-free medium seeded at 1×104 per cm2 grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium.
Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and
tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this
transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney
cells grown in serum-free medium. 相似文献
10.
Floyd M. Price Richard F. Camalier Raymond Gantt William G. Taylor Gilbert H. Smith Katherine K. Sanford 《In vitro cellular & developmental biology. Plant》1980,16(2):147-158
Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed,
by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two
media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer
system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10%
horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC
168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after
only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human
epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes
and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor,
hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth
of the epithelial cells. 相似文献
11.
Calvin D. Roskelley Nelly Auersperg 《In vitro cellular & developmental biology. Plant》1990,26(5):493-501
Summary We have developed a method that separates rat adrenocortical cells by density into populations which retain zone specific
properties in primary culture. Two different parenchymal populations were obtained and designated 2FASC (1.034 g/ml, 18.0
μm cell diameter) and 7GLOM (1.069 g/ml, 11.7 μm cell diameter). In freshly isolated cell suspensions the physical characteristics
and differential steroidogenic responses to adrenocorticotropin and angiotensin II suggested that 2FASC cells originated predominantly
from the zona fasciculata and 7GLOM cells from the zona glomerulosa. In primary culture (Dulbecco's Modified Eagle's Medium-F12
medium with 15% horse serum and 2.5% fetal bovine serum) the two populations exhibited different morphologies. 2FASC cells
retained lipid and formed cohesive epithelial monolayers that remained stationary for 3 wk. 7GLOM cells were initially epithelial
but rapidly lost lipid, spread, and assumed fibroblastic shapes. Both cell types were ositive for the cholesterol side-chain
cleavage cytochrome P-450 by immunofluorescence. Therefore, the morphologic changes seen in 7GLOM cultures were due to modulation,
not fibroblastic overgrowth. This phenotypic plasticity may reflect the mesodermal origin of the adrenal cortex, and the subcapsular
location of 7GLOM cells in vivo. In contrast, cells such as 2FASC which are located deeper in the cortex seem to have a more
restricted, fully committed parenchymal phenotype.
This work was supported by a studentship to C. D. R., and by a grant and research associateship to N. A., from the National
Cancer Institute of Canada. 相似文献
12.
Gary D. Stoner Curtis C. Harris David G. Bostwick Raymond T. Jones Benjamin F. Trump Elizabeth W. Kingsbury Elliott Fineman Carnell Newkirk 《In vitro cellular & developmental biology. Plant》1978,14(7):581-590
Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90
doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated
fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are
multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar
nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10
μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration
of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria,
Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding
junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred
as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after
treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones
are different as well as the period of time in which the clones can be propagated in vitro.
This work was supported in part by Y01 CP 60204 and N01 CP 43237. 相似文献
13.
E. I. Isaeva N. A. Mazurkova M. O. Skarnovich G. P. Troshkova L. N. Shishkina R. Ya. Podchernyayeva 《Applied Biochemistry and Microbiology》2011,47(8):737-743
Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material
obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a
strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon
Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as
a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the
viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml). 相似文献
14.
Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(9):911-920
Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed
of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate.
Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained
MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA,
insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors
in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml
concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic
for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA.
This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells
without interfering activities known to be present in serum.
This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American
Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc. 相似文献
15.
David G. Chilton Betty H. Johnson Laurence Danel-Moore Simon Kawa E. Brad Thompson 《In vitro cellular & developmental biology. Plant》1990,26(6):561-570
Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously
cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium
of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine
serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar
to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for
3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell
morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived
lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited
increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine
synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be
completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially
sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone
in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results
suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid
hormone action may be pursued more precisely in a clearly defined culture medium.
This work was conducted in conjunction with the Walls Medical Research Foundation. 相似文献
16.
Summary L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2
mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO5 medium). Growth in WO5 medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the
WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and
earlier declines in viable cell counts. Cell death occurred rapidly in WO160 medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when
no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have
been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained
in spinner cultures in WO5 medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×107 plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO5 medium.
This investigation was supported by Public Health Service Research Grant HE 08214 from the Program Projects Branch, Extramural
Programs, National Heart and Lung Institute; The World Health Organization; and The Hormel Foundation. 相似文献
17.
M. Emura U. Mohr M. Riebe M. Aufderheide D. L. Dungworth 《In vitro cellular & developmental biology. Plant》1988,24(7):639-648
Summary Proliferative and differentiative responses to various doses of vitamin A (VA) were studied in the predifferentiated cells
of a fetal Syrian hamster pulmonary epithelial line (M3E3/C3), which were cultured on a collagen gel in a hormone-supplemented
medium. These predifferentiated cells possessed well-developed endoplasmic reticulum (ER) and Golgi apparatus. At VA doses
higher than 8 μg/ml, periodic acid Schiff and slightly alcian blue positive mucuslike granules were produced, which were also
detectable electron microscopically. These mucuslike products were rich in sialic acid and resembled quite well those from
primary cultures of tracheal epithelial cells of Syrian hamster sucklings when analyzed by column chromatography on various
types of gel. At all VA doses studied (2.4, 8, 24 μg/ml), cells grew exponentially with an average population doubling time
of around 74 h, whereas in the absence of VA they had a linear growth rate and a population doubling time of 158 h between
Days 4 and 11. The uptake of [3H]glucosamine into the whole cell homogenates showed a peak at Day 8, irrespective of VA doses (0 to 24 μg/ml), and at the
highest VA dose (24 μg/ml) it exceeded by twofold the control (0 μg/ml) level. At the same time, [14C]thymidine demonstrated a high peak of uptake on Day 8 at 8 and 24 μg/ml VA. There was virtually no difference between 0
and 2.4 μg/ml VA, with both doses yielding much lower peaks. Based on the results currently presented and previously reported,
three successive stages were hypothesized for the mucous differentiation processes in M3E3/C3. The process from the first
undifferentiated stage to the second predifferentiated stage with well-developed ER and Golgi apparatus requires both collagen
gels and hormones. Differentiationn from the second stage to the third secretory stage with mucous granules is stimulated
by VA. These observations indicate that the cell line M3E3/C3 could provide a new system for investigating the mechanisms
of mucus differentiation by VA.
This study was partly supported by a grant for Humanisierung des Arbeitslebens from Bundesministerium für Forschung und Technologie 相似文献
18.
Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal
rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of
immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the
Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's
medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10
ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over
a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the
alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like
projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin,
progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing
in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network
with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development
occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane.
In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this
model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells.
This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health,
Bethesda, MD. 相似文献
19.
Dr. Donna M. Peehl Richard G. Ham 《In vitro cellular & developmental biology. Plant》1980,16(6):526-540
Summary A survey of commercially availabla media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal
keratinocytes (HK) when supplemented with 10 μg/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather
than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal
clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 μg/ml
HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly
from MCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth
of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of
calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal
growth of HK occurs at a very low level of Ca2+ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium.
However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the
Ca2+ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDB 151 with
10 μg/ml HC and 1.0 mg/ml FBSP.
This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna
M. Peehl in partial fulfillment of the requirements for the Ph.D. degree.
This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute
on Aging. 相似文献
20.
S. H. Mousavi Z. Tayarani-Najaran M. Asghari H. R. Sadeghnia 《Cellular and molecular neurobiology》2010,30(4):591-598
The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study
of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present
study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD
conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and
100 μg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated
with different concentrations of N. sativa extract (15.62–250 μg/ml) and TQ (1.17–150 μM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production
was measured by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells
toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62–250 μg/ml) and TQ (1.17–37.5 μM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase
in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 μg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 μM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that
N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise
the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders. 相似文献