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1.
Terry L. Riss Betty H. Stewart David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1989,25(2):127-135
Summary The growth of GH4C1, GH3, GH1, and GH3C15 rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1∶1 (vol/vol) mixture
of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 μg/ml gentamicin supplemented with 10 μg/ml bovine insulin,
10 μg/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T3), 50 μM ethanolamine (Etn), and 500 μg/ml bovine serum albumin. Of the lines evaluated, only the GH1 failed to grow in TRM-1. Passage of the GH4C1 and GH3 lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher
rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations
of T3 (1.0 to 10 nM). After adaptation of the GH4C1 line in TRM-1 for ≤20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 μ/ml gentamicin, 10 μg/ml Tf, 10 nM T3, and 50 μM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was
lost progressively. When T3 was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only
slightly stimulated by T3. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary
cell populations that a) required both serum-factor(s) and T3 for optimum growth, b) required supraphysiologic concentrations of T3 without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented
only with Tf and nutrients without necessity of other serum factor(s) or T3.
This work was supported by grants CA-26617 and CA-38024 from the National Cancer Institute, Bethesda, MD, American Cancer
Society grant BC-255, and grant 2225 from the Council for Tobacco Research, Inc., USA. 相似文献
2.
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
3.
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
4.
Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium 总被引:2,自引:0,他引:2
Francois Gasser Philippe Mulsant Michel Gillois 《In vitro cellular & developmental biology. Plant》1985,21(10):588-592
Summary A new synthetic medium (referred to as GC3) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's
F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and
selenium (1×10−7
M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium)
also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading.
Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high
density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium. 相似文献
5.
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
6.
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
7.
Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium 总被引:1,自引:0,他引:1
Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells
or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine
serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells
for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication
of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing
the concentration of HC to 10 μg/ml (2.8×10−5
M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10−5
M putrescine, 10−5
M vitamin B12, or 3.7×10−6
M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition
of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v)
wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned
medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear
that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus,
factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects
of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life
span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either
on clonal growth or on cumulative multiplication potential of HK.
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
8.
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
9.
Summary
In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using
a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal,
which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m−2s−1) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented
with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with
0.5 μM zeatin and 1 μM gibberellic acid, A4 isomer (GA4). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5
μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA4 for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture. 相似文献
10.
T. Chakbavarty J. G. Norcini J. H. Aldrich R. S. Kalmbacher 《In vitro cellular & developmental biology. Plant》2001,37(5):550-554
Summary Creeping bluestem (Schizachyrium scoparium (Michx.) Nash var. stoloniferum (Nash) J. Wipff) embryogenic callus growing on solid medium was used to establish a cell suspension culture in Murashige
and Skoog (MS) basal medium supplemented with 1.5 mg l−1 (6.8 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg l−1 (0.88 μM) 6-benzylaminopurine (BA), 0.5 mg l−1 (1.4 μM) zeatin, 0.2 mg l−1 (0.58 μM) gibberellic acid (GA3), and 10% (v/v) of coconut water (CW). Pro-embryos from suspension culture matured on semi-solid MS medium in about 18 wk,
and were then cultured on semi-solid MS medium without growth regulators for 2–3 wk. Shoots were regenerated on MS basal medium
supplemented with 3.0 mg L−1 (13.6 μM) 2,4-D, 1.0 mg l−1 (4.4 μM) BA, 1.0 mg l−1 (2.9 μM) GA3, 0.5 mg l−1 (2.7 μM) 1-naphthaleneacetic acid (NAA), 500 mg l−1 easein hydrolysate, and 10% (v/v) CW. Rooted plantlets were successfully accelimatized to greenhouse and outdoor conditions.
Using this protocol, it would be possible to produce at least 1300 fully acclimatized plantlets annually. 相似文献
11.
Brigitte A. van der Haegen Richard G. Ham Tamiko Kano-Sueoka 《In vitro cellular & developmental biology. Plant》1989,25(2):151-157
Summary An improved serum-free medium has been developed that supports growth of rat mammary tumor line 64–24 with far less protein
supplementation and with a much smaller inoculum than previously possible. An initial survey showed that MCDB 202 supported
clonal growth with 1% dialyzed serum. The remaining serum was then replaced with 5 μg/ml insulin, 10 ng/ml epidermal growth
factor (EGF), 1 μg/ml hydrocortisone, 50 ng/ml ovine prolactin, and 5 μg/ml liposome B (a mixture of soy lecithin, sphingomyelin,
cholesterol, vitamin E, and vitamin E acetate in liposome form). Insulin and EGF are required and growth is improved by hydrocortisone
and prolactin. Estradiol is stimulatory in the absence of liposome B. With adequate iron supplementation, transferrin has
no effect. Liposome B increases growth rate substantially. Most of the growth stimulation can be replaced with phosphatidylethanolamine
or sphingomyelin.
This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by B.A.VdH.
in partial fulfillment of the requirements for the Ph.D. degree. This research was supported by grant CA-15305 to R.G.H. and
grant CA-30545 to T.K.-S., both from the National Institutes of Health, Bethesda, MD. 相似文献
12.
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
13.
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
14.
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
15.
Summary A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed
embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented
with 0.5 mg 1−1 (2.26 μM) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition,
gyratory shaker speed, various carbon sources, different cytokinins, and AgNO3 on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their
overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog
(MS) salts and vitamins supplemented with 40 g 1−1 (117 mM) sucrose, 0.05 mg 1−1 (0.22 μM) 6-benzylaminopurine, and 10 mg l−1 (58.8 μM) AgNO3. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective
carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development
of somatic embryos was promoted by AgNO3 at concentrations below 10 mg l−1 (58.8 μM). A semi-solid medium containing 1.5 g l−1 Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using
the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture. 相似文献
16.
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
17.
Ming-Sound Tsao Judith D. Smith Joe W. Grisham 《In vitro cellular & developmental biology. Plant》1985,21(5):249-253
Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor
medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca2+, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive
cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells
in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast,
phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells
in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal
cells, but such an effect does not seem to be a universal property of tumor promoters.
This research was supported by National Institutes of Health Grant CA 29323. 相似文献
18.
Andre P. Boulet Michel A. Fortier 《In vitro cellular & developmental biology. Plant》1987,23(2):93-99
Summary Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits.
Isolated cells were cultured in PRMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC)
at a plating density of 0.5×106 cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded
myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent
cultures. The addition of increasing doses of 17β-estradiol (E2) (0.1 nM to 1 μM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E2 (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was
due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein
was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell
pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E2 and P they should provide a useful model to study the regulation of myometrial contractility.
This research was supported by Natural Sciences and Engineering Research Council of Canada, Ottawa, Ontario, grant no. U-0389. 相似文献
19.
Dose-related influence of sodium selenite on apoptosis in human thyroid follicles in vitro induced by iodine, EGF, TGF-β, and H2O2 总被引:1,自引:0,他引:1
Lehmann P Rank P Hallfeldt KL Krebs B Gärtner R 《Biological trace element research》2006,112(2):119-130
Apoptosis of thyroid follicular cells is induced by high doses of iodide, epidermal growth factor (EGF), transforming growth
factor-β (TGF-β), as well as H2O2 and might be attenuated by antioxidants. Therefore, we examined the apoptotic index induced by these substances in selenium-treated
vs untreated human thyroid follicular cells. Reconstituted human thyroid follicles were incubated with sodium selenite (10
or 100 nM) for 72 h; controls received none. The follicles were then distributed to 24-well plates and incubated with potassium iodide
(5, 10, or 20 nM), EGF (5 ng/mL), TGF-β (5 ng/mL), or H2O2 (100 μM). Apoptosis was determined by a mitochondrial potential assay and the number of apoptotic cells counted by two independent,
experienced technicians and the glutathione peroxidase (GPx) activity was determined. A significant increase of apoptic cells
was obtained in control thyroid follicles treated with iodine (5, 10, or 20 μM), thyroid-stimulating hormone (TSH) 1, or 10 mU/mL in combination with 5 and 10 μM iodine, EGF (5 ng/mL) and TGF-β (5 ng/mL), or H2O2 (100 μM) (p<0.001). In contrast, in thyroid follicles preincubated with 10 or 100 nM sodium selenite, the apoptototic index was identical to the basal rate. In H2O2-treated follicles, the apoptotic index was still significantly elevated but 50% lower compared to control cells. The GPx
activity increased from 1.4±0.2 to 2.25±0.4 mU/μg DNA with 10 nM selenite and 2.6+0.4 mU/μg DNA with 100 nM selenite. Sodium selenite might increase the antioxidative potential in human thyroid follicles in vitro and therefore diminish
the apoptosis induced by TGF-β, EGF, iodide, and even H2O2 相似文献
20.
Elevated values of molar growth yield (Yx/s = 14–26 g mol–1) were obtained during exponential growth (μ > 0.4 h–1) of Zymomonas mobilis ATCC 29191 by using reduced concentrations of glucose (6.25–100 mM) and increased oxygen supply (E
h > 300 mV) in the growth medium, as compared to the Yx/s of anaerobic exponential growth (8–10 g mol–1). Aerobically grown cells showed an increased maximum growth rate (μmax), and a reduced specific glucose consumption rate (qs), and specific ethanol formation rate (qp), thus demonstrating a more pronounced energy-coupling growth under oxic conditions. These results can be neither explained
by the concept of a solely operating Entner-Doudoroff pathway as an ATP source in aerobically growing cultures of Z. mobilis nor considered to be consistent with existing data on the lack of the Pasteur effect in this bacterium. Therefore, the results
rather give evidence for the essential contribution of aerobic ATP generation under the reported conditions.
Received: 24 September 1996 / Accepted: 9 December 1996 相似文献