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1.
W. J. Hendelman N. de Savigny K. C. Marshall 《In vitro cellular & developmental biology. Plant》1985,21(2):129-134
Summary The purpose of this study was to compare the development of organotypic cultures in defined medium versus nutrient containing
serum and embryo extract (EE). Explant cultures of cerebellum with or without locus ceruleus were grown in the Maximow system
and monitored in the living state and with histological stains. Thinner explants, fibronectin and a more frequent feeding
schedule were required to overcome the growth differences encountered using a defined medium. The final medium formulation
was arrived at by evaluation of living cultures and consisted of a basal medium (Dulbecco's minimal essential medium), a number
of hormones and other supplements, and a final glucose concentration of 750 mg %. Using a Golgi stain and histofluorescence,
it was shown that the three major types of neurons—Purkinje, deep nuclear, and locus ceruleus—developed similarly in the defined
medium and in serum-EE cultures. Myelination occurred in virtually all cerebellar cultures in defined medium and the onset
was earlier than in serum-EE cultures. These results indicate that differentiation of oligodendroglia and maturation of neurons
occur in a defined medium. Elimination of thyroid hormone delayed the maturation of the cultures, both neurons and myelin,
by 3–4 days.
This project was supported by a grant from Supply and Services (Canada) and from the Department of Health and Welfare (Canada).
The findings and opinions are the sole responsibility of the authors.
EDITOR'S STATEMENT This article describes adaptations of serum-free cell culture methods previously developed by other laboratories
to the organ culture of central nervous system tissues. Although it is difficult to develop reliable procedures for quantitative
analyses in cultures of this type, organ cultures provide unique advantages in the study of development, regeneration and
response to damage, organismal and cellular senescence and genetic abnormalities of the nervous system. Observations reported
here regarding effects of thyroid hormone on cellular maturation in this culture system may be valuable in future studies
in these areas. 相似文献
2.
Carl Monder Alena Hatle Coufalik 《In vitro cellular & developmental biology. Plant》1979,15(8):579-586
Summary Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of
decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the
culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases
from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked
through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of
proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight—soluble malate dehydrogenase, glutamate
dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine,
aminotransferase, and alanine aminotransferase—were unchanged or increased. Glycogen synthetase, aspartate aminotransferase,
pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence
on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents
of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that
fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro.
This investigation was supported by grants from the National Institute of Child Health and Human Development (RO 1 HD09715),
National Cancer Institute (CA 14194), and United States Public Health Service General Research Support Grant RR 5589. 相似文献
3.
Lone Rønnov-Jessen Bo van Deurs Maja Nielsen Ole W. Petersen 《In vitro cellular & developmental biology. Animal》1992,28(4):273-283
Summary Myofibroblasts from human breast carcinomas were identified and experimentally generated in culture, and a possible function
was examined. The frequency ofα-smooth muscle actin immunoreactive cells was evaluated as a measure of myofibroblast differentiation in primary culture.
Few or noα-smooth muscle actin-positive stromal cells (6.1 ± 8.4%) were identified in primary cultures from normal breast tissue (n=9). In contrast, high frequencies (68.8 ± 15.1%) were observed in primary cultures from carcinomas (n=19). The frequencies of myofibroblasts in primary cultures were almost identical to those obtained in the corresponding cryostat
sections (69.1 vs. 68.8%). A possible precursor cell to the myofibroblast was looked for among typical fibroblasts and vascular
smooth muscle cells. Purified blood vessels containing both fibroblasts and vascular smooth muscle cells were embedded in
collagen gel and incubated with medium conditioned by breast epithelial cells. Fibroblasts rather than smooth muscle cells
were recruited from the blood vessels. In medium conditioned by carcinoma cell lines or in co-cultures of carcinoma cell lines
and purified fibroblasts,α-smooth muscle actin and the typical myofibroblast phenotype were induced in otherwiseα-smooth muscle actin-negative fibroblasts. The effect of myofibroblasts on cellular movement—essential to neoplastic cells—was
analyzed. Spontaneous motility of tumor cells (MCF-7) was entirely suppressed in a collagen gel assay. Under these conditions
tumor cell motility was selectively mediated by direct cell-to-cell interaction between tumor cells and myofibroblasts. Under
chemically defined conditions, interaction was dependent on the presence of plasminogen. Anti-plasminogen, soybean trypsin
inhibitor, and anti-fibronectin partly neutralized the effect of plasminogen. It is concluded that elements of myofibroblast
differentiation and function may be studied in culture. 相似文献
4.
Mária Bučková Gabriela Vizárová Alexandra Šimonovičová Mária Chalanyová 《Acta Physiologiae Plantarum》2000,22(2):179-184
The typical soil micromycetes Aspergillus niger and Cladosporium cladosporioides from the family moniliaceae were investigated with emphasis on production of ABA into the culture medium. The both fungi
were cultivated in a static liquid Czapek — Dox medium and agar Czapek — Dox medium. Aspergillus niger and Cladosporium cladosporioides showed ability to produce ABA. Analytical detection of ABA from the culture medium was performed by TLC combinated with biotest
and HPLC with spectroscopy. 相似文献
5.
Per E. Schwarze Anne E. Solheim Per O. Seglen 《In vitro cellular & developmental biology. Plant》1982,18(1):43-54
Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number
of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both
as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein
degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations,
and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at
least a week without any significant net loss of cells or cellular protein.
The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities.
An erratum to this article is available at . 相似文献
6.
Indra K. Vasil 《In vitro cellular & developmental biology. Plant》2008,44(5):365-372
The International Association for Plant Biotechnology (IAPB) was founded in 1963 at the first truly international conference
on plant tissue culture, which was organized by Philip R. White. White was a devoted internationalist and was strongly committed
to global scientific cooperation. He felt that the time had come for the international tissue culture community to organize
so that it could meet regularly and provide a forum to its members for the exchange of ideas and information of mutual interest
and use. The various activities of the IAPB since its founding—the publication of its newsletter, its journal, and the proceedings
of its quadrennial congresses—faithfully document the remarkable advances in plant biotechnology that were made possible by
the successful integration of tissue culture and molecular biology. In particular, the congress proceedings serve as time
capsules, providing a wealth of information about the best of science and the most prominent scientists of the time. The history
of the IAPB is indeed the history of plant biotechnology. 相似文献
7.
A total of 11 tissue culture media and one synthetic medium were screened to determine whether they could support the growth of two strains of Acholeplasma. Two similar chemically defined media were found in which Acholeplasma laidlawii Strain A (Buchanan & Gibbons 1974) could grow. This is the first report of a fully defined medium for the growth of this organism (Greenaway 1981). 相似文献
8.
Mei-Chun Lu 《Plant Cell, Tissue and Organ Culture》2004,78(1):93-96
High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium
with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l−1) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l−1) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength
of Murashige—Skoog (MS) medium with 0.5 mg l−1 TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5
mg l−1 2,4-D and 0.5 mg l−1 TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These
calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true
leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study. 相似文献
9.
Pankaj K. Sikka Kenneth E. McMartin 《In vitro cellular & developmental biology. Animal》1996,32(5):285-291
Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys
and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high
yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency
in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1
to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces
than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified
Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal
growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing
and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the
RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The
cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate
filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence,
the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture
model will be a valuable tool for substrate uptake and nephrotoxicity studies. 相似文献
10.
Nandivada H Villa-Diaz LG O'Shea KS Smith GD Krebsbach PH Lahann J 《Nature protocols》2011,6(7):1037-1043
The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells. 相似文献
11.
Rubén Mallón Juan Rodríguez-Oubiña María Luz González 《Plant Cell, Tissue and Organ Culture》2010,101(1):31-39
The effect of different cytokinins on multiple shoot regeneration from shoots of Centaurea ultreiae was studied. The culture system consisted of solid basal half-strength Murashige and Skoog medium supplemented with one of
four cytokinins [6-benzyladenine (BA), zeatin, kinetin, or N6-(2-isopentyl) adenine (2-iP)] at each of five different concentrations. The highest multiplication rate (5.52 shoots per
explant) was obtained in the medium supplemented with 4.44 μM BA. Shoots were successfully rooted (91% success) by dipping
the basal end into a solution containing 10 M 1-naphthaleneacetic acid for 30 s. Genetic stability of the regenerated plants
was assessed by random amplified polymorphic DNA (RAPD) analysis and flow cytometry. In the initial randomly selected plant
material (control) and 20 of its regenerants, 2,688 bands were generated by RAPD with 12 different primers, and the same banding
profiles were exhibited. Molecular and cytological analyses did not reveal genomic alterations in any of the regenerated plants
obtained on medium containing 4.44 μM BA. The success of acclimatization to environmental conditions—100% of plants were successfully
acclimatized—suggests that the micropropagation system described is a reliable method for propagation of C. ultreiae. 相似文献
12.
Bernd Fischer Manfred Lambertz Christa Hegele-Hartung 《In vitro cellular & developmental biology. Animal》1992,28(5):337-347
Rabbit morulae and blastocysts were cultured in conventional culture media [Ham’s F10 or BSM II supplemented with bovine serum
albumin (BSA) or serum] or in Ham’s medium supplemented with synchronous or asynchronous uterine flushings, mostly for 2 days,
and afterwards investigated by light and electron microscopy and by autoradiography. Ultrastructure and cell proliferation
differed considerably between cultured embryos and noncultured controls. Cultured embryos displayed more dead cells. They
were developmentally retarded (predominance of smooth endoplasmic reticulum rather than the age-specific rough endoplasmic
reticulum, mitochondria still round to ovoid shaped) and showed nonspecific signs of cells damage (swollen mitochondria and
Golgi complex vesicles, increased number of lysosomes). All these features were also present in embryos grown in uterine flushing-supplemented
media, but were less pronounced. Cell damage and impaired cell proliferation had affected trophoblast cells more than embryoblast
cells. Endoderm could be differentiated only if culture had been started with blastocysts—not with morulae—and seems to require
uterine secretions. No significant ultrastructural differences were observed between embryos cultured in synchronous or in
asynchronous uterine flushings. Present results indicate that cultured preimplantation rabbit embryos deviate clearly from
those grown in vivo and maintain, for some time, a better cellular structure—and probably function —in the presence of uterine
flushings than in conventional culture media. Specific abnormal morphologic features related to a particular medium could
not be identified. 相似文献
13.
Sandra Regina B. R. Sella Belquis P. Guizelini Luciana P. S. Vandenberghe Adriane B. P. Medeiros Carlos Ricardo Soccol 《Applied microbiology and biotechnology》2009,82(6):1019-1026
Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes
and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support
sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect
of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation.
Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental
designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation
time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses
and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time.
Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%),
initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the
control and no significant difference in dry heat resistance was observed. 相似文献
14.
Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N6-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines
were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry.
Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine
and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine
free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence
of cytokinin.
Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was
characterized by a lower level of each polyamine in comparison to chlorophyll-less callus.
Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase
the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine)
in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an
inhibitor of S-adenosylmethionine decarboxylase depressed the greening process.
Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated
through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic
apparatus is discussed. 相似文献
15.
P. I. P. Perera D. M. D. Yakandawala V. Hocher J-L. Verdeil L. K. Weerakoon 《Plant Cell, Tissue and Organ Culture》2009,96(2):171-180
The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators
at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram)
and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic
acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination
with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental
effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos
when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113
and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced
from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y3 medium containing 66 μM 2,4-D), followed by maturation medium (Y3 medium without growth regulators) and germination medium (Y3 medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA3), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%). 相似文献
16.
Bolibok H Gruszczyńska A Hromada-Judycka A Rakoczy-Trojanowska M 《Cellular & molecular biology letters》2007,12(4):523-535
This study was conducted in order to identify quantitative trait loci (QTLs) for the in vitro culture response of winter rye (Secale cereale L.) immature embryos and immature inflorescences. A genetic linkage map comprising 67 SSRs, 9 ISSRs, 13 SAMPLs, 7 RAPDs,
2 SCARs and one EST marker was created based on the analyses of 102 recombinant inbred lines from the cross between lines
L318 (which has a good response in tissue cultures) and L9 (which is unable to regenerate plants from somatic tissues and
anthers). The map spans 979.2 cM, and the average distance between markers is 9.9 cM. Two characteristics were evaluated:
callus induction (CI) and somatic embryogenesis ability (SE). They were expressed as the percentage of immature embryos/inflorescences
producing callus (designated ECI/ICI) and the percentage of explants producing somatic embryos (ESE/ISE). All the analysed
traits showed continuous variation in the mapping population but a non-normal frequency distribution. We identified nine putative
QTLs controlling the tissue culture response of rye, explaining up to 41.6% of the total phenotypic variation: two QTLs for
ECI — eci-1, eci-2; 4 for ESE — ece-1, ese-2, ese-3, ese-4; 2 for ICI — ici-1, ici2; and 1 for ISE — ise-1. They were detected on chromosomes 1R, 4R, 5R, 6R and 7R. 相似文献
17.
Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes 总被引:11,自引:0,他引:11
Norimasa Sawada Akito Tomomura Carol A. Sattler Gerald L. Sattler Hynda K. Kleinman Henry C. Pitot 《In vitro cellular & developmental biology. Plant》1987,23(4):267-273
Summary Some effects of culturing adult rat hepatocytes on each of four different substrates—laminin (LN), collagen type I (C-I),
collagen type IV (C-IV), and fibronectin (FN)—have been investigated under defined conditions. No differential effect on the
attachment of the cells to the various substrates was noted; however, the spreading of hepatocytes shortly after initial plating
was most strikingly enhanced by FN, whereas LN exhibited little or no such enhancement. The two collagen substrates enhanced
the spreading of hepatocytes more than did LN, but less than FN. The different substrates had no differential effect on the
induction of tyrosine aminotransferase by dexamethasone and glucagon for at least the first 10 d in culture. The longevity
of the hepatocytes was not changed significantly by any of the substrates, at least through the 14th d of culture. During
the culture periods the hepatocytes at high cell density were maintained as confluent monolayers, regardless of the substrate
on which they had been cultured. After 14 d of culture, γ-glutamyltranspeptidase activity was highest in cells cultured on
C-IV, and lowest in those on FN. DNA synthesis in cultured hepatocytes at a low cell density was highest in cells cultured
on FN, with decreasing levels of this parameter in cells cultured on C-IV, C-I, and LN, respectively. These results demonstrate
that specific components of the extracellular matrix modulate both differentiated functions and the replication of hepatocytes
cultured in serum-free medium.
This work was supported in part by grants (CA-07175, CA-09135, CA-22484) from the National Cancer Institute, Bethesda MD.
N. Sawada was supported by a Cancer Research Campaign Grant D (U.K.) from the International Union Against Cancer. 相似文献
18.
E. N. Kaparullina N. V. Doronina L. V. Trilisenko V. M. Vagabov Yu. A. Trotsenko 《Applied Biochemistry and Microbiology》2009,45(5):498-502
The obligate destructor of ethylene diamine tetraacetate—a culture of Chelativorans oligotrophicus LPM-4—did not grow on a medium with glucose, but it was good to use it under cultivation on a mixture with EDTA after considerable
decrease of the EDTA concentration in the medium (two-phase growth). Strong inhibition of hexokinase and glucose 6-phosphate
dehydrogenase in cell exracts 4 mM EDTA was revealed. Using EDTA, cells accumulated polyphosphates whose rate decreased during
glucose utilization phase. High activities of polyphosphate biosynthesis ferments (adenylat kinase and polyphosphate kinase)
were distinguished during the first phase of the cultivation; considerable decrease of them and increase of polyphosphate
glucokinase were found during the second phase of the cultivation. This points to the possible participating of polyphosphates
in glucose metabolism as a supplementary energy source. 相似文献
19.
D. Hellenbroich U. Valley T. Ryll R. Wagner N. Tekkanat W. Kessler A. Roß W.-D. Deckwer 《Applied microbiology and biotechnology》1999,51(4):447-455
A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases
were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex
medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two
different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and
followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell
retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor,
supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized
nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present
in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using
complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations.
Received: 24 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998 相似文献
20.
Michael H. Simonian Mark L. White David A. Foggia 《In vitro cellular & developmental biology. Plant》1987,23(4):247-252
Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium
and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life
span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings
in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability
to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and
also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared
to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized
for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with
8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines
produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were
similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented
medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and
its relation to differentiated function for this cell culture system.
This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes
of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health
(grant HL07485). 相似文献