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1.
Constance Oliver 《In vitro cellular & developmental biology. Plant》1980,16(4):297-305
Summary Methods have been developed for isolating and maintaining differentiated rat exorbital lacrimal, parotid, and pancreatic acinar
cells for up to 1 month in culture. The dissociated cells retained their differentiated morphology when cultured as suspension
cultures at 35°C with the appropriate secretagogue (exorbital lacrimal, 10−6
M carbamyl choline; pancreas 10−5
M carbamyl choline; parotid, 10−6
M isoproterenol). Under these conditions the cells remained viable and differentiated for up to 4 weeks in culture and continued
to incorporate3H-leucine at rates similar to those of freshly isolated cells. If secretagogue was omitted from the medium, the cells rapidly
degenerated. These results indicate that differentiated from the medium, the cells rapidly degenerated. These results indicate
that differentiated exocrine gland acinar cells may be maintained in vitro and utilized as a model system for the study of
secretory processes. 相似文献
2.
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
3.
Alejandrina Robledo-Paz Víctor M. Villalobos-Arámbula Alba E. Jofre-Garfias 《In vitro cellular & developmental biology. Plant》2000,36(5):416-419
Summary Root apices from in vitro cultured garlic (Allium sativum) cloves of cvs. ABEN and GT96-1 were used as axenic explants for organogenic callus production and plant regeneration experiments.
Explants cultured in media based on those of Chu and co-workers (N6) or Murashige and Skoog (MS) could induce organogenic
callus after 8 wk culture in darkness. Both media were supplemented with 2,4-dichlorophenoxyacetic acid (2.2–4.5 μM), alone or combined with 6-furfurylaminopurine (kinetin, 2.3–4.6 μM). Shoots started to grow 3 wk after culturing in the presence of light and the addition to culture media of 4.4 μM N6-benzyladenine. Plants capable of producing microbulbs regenerated 6 wk later. Up to 170 plants g−1 FW callus were obtained when culturing was initiated in MS medium supplemented with 4.6 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid. 相似文献
4.
Yasseen Mohamed-Yasseen Suzanne Helene Costanza 《In vitro cellular & developmental biology. Plant》1996,32(2):100-102
Summary Two protocols for clonal propagation of kurrat (Allium ampeloprasum var.kurrat) using explants from the basal plates of mature plants are described. In direct formation, explants were cultured in Murashige
and Skoog (MS) medium and supplemented with either benzyladenine at 0.0 or 4.4 μM, or supplemented with 7.0 μM benzyladenine and 0.1 μM naphthaleneacetic acid. Shoots appeared after 4 wk of culture. In the two-step procedure, explants were cultured first on
MS medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid and 1.4 μM kinetin, and incubated in the dark for 4 wk. They were then transferred to MS medium supplemented with 4.4 μM benzyladenine for shoot formation. All shoots were rooted on MS medium containing 5 g·liter−1 activated charcoal. Normal viable plants were successfully established in soil. 相似文献
5.
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
6.
Summary Chili pepper (Capsicum annuum L., cv. Tampique?o 74) cell suspensions were employed to study the influence of phenylalanine and phenylpropanoids on the
total production of capsaicinoids, the hot taste compounds of chili pepper fruits. The effect of capsaicinoid precursors and
intermediates on the accumulation of lignin as an indicator of metabolic diversion was also investigated. Addition of 100
μM of either phenylalanine, cinnamic or caffeic acids to chili pepper cell cultures did not cause significant increases in total
capsaicinoids (expressed as capsaicin content, and calculated as averages of the measured values) during the growth cycle.
The highest total capsaicinoid content was recorded in cultures grown in the presence of vanillin (142.61 μg g−1 f.wt.), followed by cells treated with 100 μM vanillylamine (104.88 μg g−1 f.wt.), p-coumaric acid (72.36 μg g−1 f.wt.). and ferulic acid (34.67 μg g−1 f.wt.). Capsaicinoid content for control cells was 13.97 μg g−1 f.wt. Chili pepper cell suspensions cultured in the presence of 100 μM of either phenylalanine, or cinnamic, caffeic, or ferulic acids, or the same concentration, of vanillin and vanillylamine,
did not exhibit statistically significant differences in the content of lignin as compared with control cells. However, addition
of p-coumaric acid (100 μM) to the cultute medium significantly increased thelignin production (c. 10–15 times the contents of control cells). 相似文献
7.
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
8.
Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl−1 (9.8 μM) indolebutyric acid, 2.0 mgl−1 (10.74 μM) naphthaleneacetic acid, and 2.0 mgl−1 (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl−1 (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced
adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl−1 (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl−1 (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl−1 (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic
tissues to MS medium with 1.0 mgl−1 (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl−1 (4.14 μM) picloram or 1.0 mgl−1 (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl−1 (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation
of elite pineapple germplasms. 相似文献
9.
Suleiman A. Suleiman Jeffrey B. Stevens 《In vitro cellular & developmental biology. Plant》1987,23(5):332-338
Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of
oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study
were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan
blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing
132 μM O2, as compared to medium equilibrated with air (220 μM O2) or air + oxygen (298 μM O2). Cells cultured in 220 μM O2 (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material
remained. This loss of P-450 was minimized when cells were cultured in 163 μM O2 and abolished when cells were cultured in 132 μM O2. The 132 μM O2 exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in
cell respiration over time when the cells were cultured in either 220 μM O2 (air) or 298 μM O2 (air:O2). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid
peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N2 as compared to air or air:O2. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains
other functional characteristics of the cell.
This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School
of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health,
Bethesda, MD. 相似文献
10.
Summary Micropropagation of Arnica montana L. using Murashige and Skoog (MS) medium supplemented with N6-[2-isopentenyl]adenine (2iP), zeatin and α-naphthaleneacetic acid (NAA) in different concentrations does not ensure the formation
of a high number of regenerated plants; a maximum of 3.2 neoplantlets per explant were obtained. After 4 wk of culture on
medium with zeatin (4.5 μM) and NAA (5.3 μM), plants were 3.06 cm in length. The following step was to improve the clonal propagation of this species. Micropropagation
of Arnica montana L., initiated from nodal segments using semisolid media (4 g l−1 agar), was obtained. Explants were inoculated on MS medium supplemented with NAA (5.3 μM), 2iP (5.0 μM), maize extract (1.0 ml l−1), phloroglucinol (0.6 mM) or adenine sulfate (0.2 mM). Only 3 wk after the inoculation, plant multiplication as well as induction of roots were obtained, the optimal variant
being that containing NAA (5.3 μM), 2iP (5.0 μM) and maize extract (1.0 ml l−1). Six weeks after the inoculation plants were transferred to Perlite, with 80% plant survival being obtained. By isoesterase
pattern we concluded that we have obtained the clonal propagation of Arnica montana, because the pattern of several individuals belonging to different clones was the same. Only one region with esterase activity
that is present in all individuals has been identified. 相似文献
11.
Marián López José Pacheco Roberto Rodríguez Ricardo J. Ordás 《In vitro cellular & developmental biology. Plant》1996,32(2):109-114
Summary Plantlet regeneration of salgare?o pine (Pinus nigra Arn. ssp.salzmannii (Dunal) Franco) was achieved from cotyledons. The data showed that the best differentiation response occurred when cotyledons,
from 1-d-old embryos germinated in darkness, were cultured on Murashige and Skoog medium (half-strength macroelements) with
22 μM N6-benzyladenine (BA) and 0.05 μM α-naphthaleneacetic acid (NAA) for 2–3 wk. Shoot development was obtained by subculturing treated explants on the same medium
without growth regulators. Shoots were successfully micropropagated by sequential subculturing them on medium containing growth
regulators (5 μM BA and 0.005 μM NAA) and on hormone-free medium for 2 and 12 wk, respectively. To induce adventitious roots, shoots were treated with 3 μM NAA, and 8 μM indole-3-butyric acid for 2 wk, followed by transfer to Murashige and Skoog medium (1/4-strength macroelements, 20 g·L−1 sucrose) without growth regulators. After 3–4 wk, almost all the rooted shoots (65%) could be successfully transplanted and
acclimatizated in the greenhouse, where the plants exhibited normal growth habit. 相似文献
12.
Summary Culture media, environmental and genotypic factors affecting regeneration from multi-shoot cultures derived from corn seedling
apical explants were investigated. The frequency of shoot regeneration was highes for seedlings that were 4–5 cm in length.
Flow cytometry was used to show that the most responsive culturs contained a high proportion of cells in the G1 phase. Proline
in the multi-shoot induction medium (MSI) significantly increased the shoot induction frequency. Continuous low light (30–40
μEm−2s−1) stimulated multi-shoot induction. The highest number of multi-shoots developed in medium containing 4 gl−1 proline, 2 mgl−1 (8.8 μM) 6-benzylaminopurine (BA), and 1 mgl−1 (4.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Multi-shoots were induced in this culture system from 44 of 45 corn genotypes and
approximately 70% of the genotypes exhibited a high to moderate response (greater than 20 shoots per explant in 4 wk of culture).
This culture procedure is an efficient and widely applicable method for corn regeneration that may be a useful target for
transformation. 相似文献
13.
J. Y. Choi H. J. Kim C. H. Lee J. M. Bae Y. S. Chung J. S. Shin N. I. Hyung 《In vitro cellular & developmental biology. Plant》2001,37(2):274-279
Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture
vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious
shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants
in Erlenmeyer flasks with medium containing 4 g l−1 agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained
with 5 mg l−1 (22.8 μM) zeatin and 0.1 mg l−1 (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l−1 (45.6 μM) zeatin and 0.1 mg l−1 (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to
9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully
influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’,
it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free
MS1/2N demium after having been dipped for 30 s in 250 mg l−1 (1.1. mM) IBA solution. 相似文献
14.
Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud
Gerardo Armando Aguado-Santacruz José Luis Cabrera-Ponce Víctor Olalde-Portugal M. A. Rosario Sánchez-González Judith Márouez-Guzmán Luis Herrera-Estrella 《In vitro cellular & developmental biology. Plant》2001,37(2):182-189
Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated
from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid
(2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N6-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations
containing 1 mg l−1 (4.52 μM) 2,4-D plus 0.5 mg l−1 (2.22 μM) BA. and 2 mg l−1 (8.88 μM) BA plus 1 mg l−1 (4.14 μM) Picloram with or without 40 mg l−1 (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient
treatments for induction of embryogenic callus contained 2 mg l−1 (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l−1 (2.22 μM) BA, or 1 mg l−1 (4.52 μM) 2,4-D with 0.50 mg l−1 (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus
1 mg l−1 (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg
l−1 (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l−1 (8,28 μM) Picloram, 1 mg l−1 (4.44 μM) BA and 40 mg l−1 (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds. 相似文献
15.
Masayoshi Ono John W. Perry Takami Oka 《In vitro cellular & developmental biology. Plant》1981,17(2):121-128
Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant
mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein
accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka,
T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml
prolactin, 2.8×10−9
M to 2.8×10−7
M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition
of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase
in α-lactalbumin accumulation attained in the presence of 1.4×10−8
M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin.
Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing
a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured
tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved
an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated
in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin.
The maximal increases were obtained in the presence of 2.8×10−6
M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration,
of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be
different from that involved in casein synthesis. 相似文献
16.
Y. C. Yen-Chow S. Y. Chow W. S. S. Jee D. M. Woodbury 《In vitro cellular & developmental biology. Plant》1984,20(9):677-684
Summary The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was −10.2±0.20 mV (n=390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal
preparations, such as dibutyryladenosine 3′,5′-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10−5 to 10−4
M), hydrocortisone (10−7 to 10−6
M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl− concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein
contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined
by [14C] dimethyloxazolidine-2, 4-dione, and3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activiities of Na+, K+-, HCO3
−-, and Ca++, Mg++-ATPases in these cultured cells were 19.0±2.1, 13.6±2.1, and 6.6±1.2 nmol pi/mg protein per minute, respectively, and the
carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte
concentrations observed in this study indicate that Na+, K+, Cl−, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl−, and H+ are actively transported out of the cells and K+ into the cells.
This study was supported by Grant AM20935 from the NIAMDD, NIH, Bethesda, Maryland, and National Aeronautics & Space Administration
NASA-Ames Grant NAG 2-108 and U.S. Department of Energy Contract DE-AC02-76-EV-00119. D. M. W. is the recipient of a Research
Career Award (5-K6-NB-13838), NINCDS, NIH. 相似文献
17.
Chandra P Lecluyse EL Brouwer KL 《In vitro cellular & developmental biology. Animal》2001,37(6):380-385
Summary This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives
on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes
were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagencoated 60-mm dishes, overlaid
with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were
conducted in Dulbecco's modified Eagle's medium (DMEM)+1.0μM dexamethasone (DEX)+5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h
with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2
ml collagen and incubated in DMEM+0.1μM DEX, DMEM+0.1μM DEX+5% FBS, Williams' medium E+0.1μM DEX+1% ITSΘ+, DMEM +1.0μM DEX, DMEM+1.0 μM DEX+5% FBS, or modified Chee's medium (MCM)+0.1 μM DEX+1% ITSГ+. [3H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca2+-free HBSS was measured, and the biliary-excretion index (BEI: percentage of accumulated TC localized in the canalicular compartment)
was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular
morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary
excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI=0.649 [0.1 ml collagen]; BEI=0.659 [0.2 ml collagen])
compared with those overlaid at 0 h after seeding (BEI=0.538 [0.1 ml collagen]; BEI=0.517 [0.2 ml collagen]), although the
differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI=0.396).
Differing DEX concentration (0.1 μM versus 1.0 μM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC. 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.
Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range
of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid,
α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency
was obtained with MS medium containing 2.0 mg l−1 (8.88 μM) BA and 0.5 mg l−1 (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within
4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were
transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk. 相似文献
20.
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