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1.
Primary and long term epithelial cell cultures from human fetal normal colonic mucosa 总被引:3,自引:0,他引:3
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 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees 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. 相似文献
2.
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
3.
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
4.
Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's
minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated
in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced
by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further
cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations
of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures.
Supported by National Cancer Institute Contract N01-CP-75914. 相似文献
5.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
6.
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
7.
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
8.
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
9.
Dharam P. Chopra Richard L. Shoemaker Gregory W. Taylor Patricia A. Mathieu 《In vitro cellular & developmental biology. Animal》1991,27(1):13-20
Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free
medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary
extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes
between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively
with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl− channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic
acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin
glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will
be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations
with the tracheal mucosal epithelial cells already established.
This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes. 相似文献
10.
Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland 总被引:1,自引:0,他引:1
Nivedita Ganguly Ranjan Ganguly Nozer M. Mehta Linda R. Crump M. R. Banerjee 《In vitro cellular & developmental biology. Plant》1981,17(1):55-60
Summary Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to
pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 μg/ml), aldosterone (1 μg/ml), growth
hormone (5 μg/ml), cortisol (5 μg/ml), and prolactin (80 ng/ml, present as a contaminant in 5 μg/ml growth hormone). The alveolar
growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation
time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA
probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed
that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas
the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation
was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These
results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved
in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis
culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum
mammary gland in vivo.
This work was supported by Department of Health, Education and Welfare Grants CA11058 and CA25304 from the National Cancer
Institute. 相似文献
11.
Ursula J. Behrens Fiorenzo Paronetto 《In vitro cellular & developmental biology. Plant》1984,20(5):391-395
Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two
effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×105 cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated
cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not
detectable by light microscopy, can be removed by the addition of macrophages (2×105/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes
with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects
to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost.
This work was supported by Veterans Administration Research Funds. 相似文献
12.
Terry L. Riss' David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1989,25(2):136-142
Summary The effect of 17β-estradiol (E2) on growth of GH4C1 rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum.
Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T3), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E2 responsive only when growth was retarded by reducing the T3 concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E2 to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with
charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E2 to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing
and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects
in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors.
This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society
grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc. 相似文献
13.
Exogenous fibronectin is not required for organogenesis in vitro 总被引:1,自引:0,他引:1
I. Thesleff P. Ekblom P. Kuusela E. Lehtonen E. Ruoslahti 《In vitro cellular & developmental biology. Plant》1983,19(12):903-910
Summary The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ
cultures. Transferrin (50μg/ml) was a strong mitogen for kidney cells, whereas, the addition of soluble fibronectin (50 to
250 μg/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium
did not support tooth differentiation. however, fibronectin was not a necessary serum component because fibronectin-free serum
supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added
human fibronectin at 50 μg/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney
basement membrane area was observed when fibronectin concentration was 250μg/ml. The mesenchymal stroma and the basement membranes
of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies,
indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin
as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product
of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate
that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ
explants in vitro.
This work was supported by grants from the Sigrid Jusélius Foundation, Finska L?kares?llskapet, Finnish Life Insurance Companies
and Grant CA 28896 and Cancer Center Support Grant CA 30199 from the National Cancer Institute, Department of Health and Human
Services, Washington, D.C. 相似文献
14.
Microvascular endothelium and pericytes: High yield,low passage cultures 总被引:17,自引:0,他引:17
Mary Pat Carson Christian C. Haudenschild 《In vitro cellular & developmental biology. Plant》1986,22(6):344-354
Summary Cultured microvascular endothelial cells (MEC) have become a valuable model for studies of microvascular physiology and pathology.
Most current techniques involve manual removal of undesirable cell types or cloning, require one to several months, and yield
high population doubling level cultures derived from a relatively small sample of the original population. We have devised
a technique to more rapidly produce larger numbers of MEC. This method provided primary cultures consisting predominantly
of MEC within 1 wk. The technique involves selective aspiration of gray matter from the bovine cerebral cortex followed by
homogenization, sieving, enzymatic dissociation, and then dense plating (104 to 105 vessel fragments/cm2) onto gelatin- or fibronectin-coated plastic. Typical yields were 0.1 to 0.5 × 106 fragments/g of aspirated gray matter. The optimal culture medium for these cells was 15% equine plasma derived serum, 20%
conditioned medium, 2% retinal extract, 60% fresh medium, and 500 μg/ml heparin. Cells attached within 24 h, well-spread colonies
were present within 1 to 2 d, and cultures approached confluence within 2 to 3 d. Alkaline phosphatase staining confirmed
the microvascular origin of the material plated. Morphology, Factor VIII-related antigen staining and 1,1′-dioctacecyl-3,3,3′3,-tetramethyl-indocarbocyanine
perchlorate acetylated low density lipoprotein uptake suggested that MEC predominated. Cultures could be passaged and additionally
purified by sequential exposure to pancreatin and trypsin-EDTA. Pancreatin selectively removed MEC colonies leaving a relatively
homogeneous pericyte population. The relative ease with which such cultures can be produced should facilitate the in vitro
study of brain microvascular function and may also provide insights useful for growing MEC from other vascular beds.
This work was supported by grants from the Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine
(American Cancer Soc. IN97G) and from the National Institutes of Health (HL26895 and HL07224), Bethesda, MD. 相似文献
15.
Fatemeh Fazely Dorothy C. Moses Nada Ledinko 《In vitro cellular & developmental biology. Plant》1985,21(7):409-414
Summary Invasion of chick chorioallantoic membrane (CAM) organ cultures by rat 3Y1 cells transformed by the highly oncogenic human
adenovirus type 12 (3Y1/12-10 cells) was inhibited by several retinoids tested. The anti-invasive activity of the retinoids
was dependent on retinoid concentration and continuous (4d) exposure of the CAM. The 50% retinoid dose (dose effective in
achieving a response in half of the organ cultures) that inhibited invasion was 0.85 μg/ml of retinol palmitate, 0.39 μg/ml
of retinoic acid, or 0.16 μg/ml of retinol acetate. This dose was of the same order of magnitude as that which induced CAM
differentiation, and was three-to fourfold less than the dose that caused cytotoxic damage of CAM. In addition, the retinoids
inhibited 3Y1/12-10 cell growth by approximately 40% at levels over 10-fold higher than those needed for anti-invasion activity.
The findings suggest that the anti-invasive activity of retinoids was at least partly due to direct induction of cell differentiation
of the CAM host tissue.
This work was supported by National Cancer Institute Grant CA 13231 and by University of Akron Grant RG 832. 相似文献
16.
Karimullah A. Zirvi Darwin O. Chee George J. Hill 《In vitro cellular & developmental biology. Plant》1986,22(7):369-374
Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five
tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder
carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of
transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10−10
M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES
medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI
cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower
doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium
resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with
the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell
lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell
lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones
influence cell growth.
This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J.
Hill) and grant no. CA-37138 from the National Cancer Institute. 相似文献
17.
Richard I. Schwarz Deborah A. Farson Mina J. Bissell 《In vitro cellular & developmental biology. Plant》1979,15(12):941-948
Summary Primary avian tendon cells (PAT) maintain their embryonic state when cultured in medium F-12 with very low serum (0.2%) and
ascorbate (50 μg per ml); that is, they retain the potential for devoting 20–30% of their total protein synthesis to collagen.
However, if the cells are left at a confluent cell density or are derived from confluent cultures, this potential is irreversibly
decreased. This effect, along with poor medium formulations, probably accounts for the “dedifferentiation” process that occurs
when fibroblasts are cultured. In contrast, PAT cells kept at subconfluent cell densities retain the ability to synthesize
high levels of collagen. The one limitation in obtaining long-term cultures of high collagen-producing tendon cells in the
inability of serum at low concentrations to remain a potent mitogen after a few subcultures.
The quantitative loss of function has long been considered to be a cell culture artifact; however, we propose that this drop
in collagen synthesis is a reflection of the developmental programing of these cells. In separate series of experiments using
organ cultures, we show that tendon tissue from the embryo makes over 30% collagen, whereas, “young” tendons make 18% and
“older” tendons from the adult make less than 1%. Therefore, a quantitative drop in collagen synthesis would be expected if
normal development were to occur in culture. Our data are consistent with the idea that cultures of embryonic tendon cells
are triggered to mature by a mechanism that correlates with high cell density.
This investigation was supported in part by National Science Foundation Grant PCM 77-14982; in part by the Division of Biomedical
and Environmental Research of the Department of Energy under contract W-7405-ENG-48; and by a National Institutes of Health
Fellowship IF32 CA 05807-01, from the National Cancer Institute to R. I. S. 相似文献
18.
Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin 总被引:2,自引:0,他引:2
Janice E. Errick Katherine W. A. Ing Margaret C. Eggo Gerard N. Burrow 《In vitro cellular & developmental biology. Plant》1986,22(1):28-36
Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and
to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture
conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of
insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine
(10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present
at 10−9
M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system
provides a means to study the hormonal control of growth and differentiation in human thyroid cells.
This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto;
and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow. 相似文献
19.
Koichi Hirata Tadashi Oku Aaron E. Freeman 《In vitro cellular & developmental biology. Plant》1982,18(9):789-799
Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin
as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10%
bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10−8
M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began
to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin.
When 10−6
M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely
but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10−8
M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction
of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin
dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin
had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e.,
duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There
were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method.
This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded
by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice
were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute. 相似文献
20.
Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells 总被引:21,自引:0,他引:21
Summary Improved culture conditions have been developed that will support clonal growth of Swiss mouse embryo 3T3 cells at concentrations
of serum protein as low as 125μg/ml. Survival of the cells under completely protein-free conditions also is enhanced greatly.
The improvements that made these results possible include: (a) use of medium MCDB 402, which was developed specifically for
Swiss 3T3 cells by adjusting the concentrations of all components of Dulbecco's modified Eagle's medium to optimum values
for clonal growth with minimal serum protein and by adding other nutrients such as trace elements and “nonessential” amino
acids that were not in the original formula; (b) use of culture surfaces that are coated with a positively charged polymer,
poly-d-lysine; and (c) use of gentle low temperature trypsinization technique that minimizes cellular damage and the need to neutralize
residual trypsin.
Portions of this work were reported at the Thirtieth Annual Meeting of the Tissue Culture Association in Seattle, Washington.
This work was supported by Grant CA-15305 from the National Cancer Institute 相似文献