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1.
Metanephric development in serum-free organ culture 总被引:4,自引:0,他引:4
Ellis D. Avner Demetrius Ellis Thomas Temple Ronald Jaffe 《In vitro cellular & developmental biology. Plant》1982,18(8):675-682
Summary A new mouse metanephric organ culture system has been developed to study mammalian renal development. The system permits in
vitro organotypic differentiation in a serum-free, hormone supplemented medium consisting of Dulbecco’s minimal essential
medium (MEM) and Ham’s F12 medium supplemented with insulin, 5 μg/ml; PGE1, 25 ng/ml; T3, 3.2 pg/ml; hydrocortisone, 5 μg/ml; and transferrin, 5 μg/ml. In this system, metanephric development continues morphologically
beyond the S-shaped tubule stage. A well differentiated proximal tubule forms with a well defined brush border, specialized
intercellular connections, and an apical endocytic network. In addition, a unique devascularized glomerulus, with highly differentiated
podocytes surrounding areas of basement membrane, forms entirely from epithelial elements.
The present organ culture model goes beyond the limitations of previously described systems in that it does not require separation
of nephrogenic blastema from ureteric bud, nor require animal serum or nonspecific tissue extracts for metanephric development.
The model is thus suited for morphological, biochemical, and endocrinological study of normal and abnormal renal organogenesis. 相似文献
2.
The development of the permanent mammalian kidney, or metanephros, depends on mesenchymal-epithelial interactions, leading to branching morphogenesis of the ureteric bud that forms the collecting ducts and to conversion of the metanephric mesenchyme into epithelium that forms the nephrons. Rat metanephric organ culture in which these interactions are maintained is a valuable in vitro model system for investigating normal and abnormal renal organogenesis. Methods were designed to evaluate either the capacity of the ureteric bud to branch or that of the mesenchyme to form nephrons. Both are based on specific staining of the ureteric bud and the glomeruli with lectins. Using this approach, we have shown that retinoids are potent stimulating factors of nephrogenesis, acting through an increase in the branching capacity of the ureteric bud. On the other hand, several drugs such as gentamicin and cyclosporin A were found to reduce the number of nephrons formed in vitro. While gentamicin affects the early branching pattern of the ureteric bud, cyclosporin may affect the capacity of the mesencyme to convert into epithelium. This methodology therefore appears a potentially useful tool for toxicological studies new drugs. 相似文献
3.
Normand Brière Pierre Chailler 《In vitro cellular & developmental biology. Animal》1994,30(4):269-274
Summary Human fetal kidney explants can be maintained during 5 days in Leibovitz’s L15, a basic serum-free medium. Because culture
conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes
thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of
fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to
growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an
antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20μg/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation.
Nor did it change the activities of alkaline phosphatase and γ-glutamyltransferase, two enzymic markers of brush border differentiation.
After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus
transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still
present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The
effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor.
These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline
does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term
fetal kidneys. 相似文献
4.
Hamster early two-cell embryos developed to the expanded blastocyst stage within the isolated mouse ampulla maintained in organ culture system. Mouse ampullae isolated at different times after treating the mice with human chorionic gonadotropin (hCG) (0–72 h) or pregnant mare's serum gonadotropin (PMSG) (30–32 h) were flushed with culture medium, and hamster early two-cell embryos were introduced into these ampullae. Mouse ampullae isolated at 14–32 h after hCG injection were more favorable for the development of the embryos than those isolated at 70–72 h. When mouse ampullae were isolated 30–32 h after hCG or PMSG treatment, 39% of the cultured eggs developed, some of them to the expanded blastocyst stage after additional culture for 65–70 h. These results indicate that unknown oviductal factors stimulate the development of hamster early two-cell embryos, and these factors are under the control of hCG or PMSG. In addition, these factors are common to the mouse and hamster. 相似文献
5.
Joseph T. M. Koumans Jean-yves Sire 《In vitro cellular & developmental biology. Animal》1996,32(10):612-626
Summary To develop a serum-free, chemically definedin vitro organ culture system enabling the study of epithelial-mesenchymal interactions in development and growth of fish dermal skeleton,
we investigatedin vitro continuation of scale regeneration in the cichlid fishHemichromis bimaculatus. The culture medium in our system is based on Leibovitz medium (L-15) supplemented with vitamin C, additional amino acids
and HEPES. With this basis medium, we examined the effects of all trans-retinoic acid, dexamethasone, and prostaglandin-E2
(PG-E2), factors known to exert an effect on development and growth of teeth and bone in mammalian culture systems, on thein vitro regeneration of scales. These effects were compared with those obtained by supplementation of the basis medium with newborn
and fetal calf serum. To evaluate our culture system, the medium that allowed to mimick in the best possible way thein vivo regeneration of scales (i.e., the basis medium plus dexamethasone and PG-E2) was also tested on thein vitro development of teeth in the same fish species.
Our serum-free, chemically defined organ culture system enablesin vitro development and growth of both scales and teeth. With this model culture system, it is possible to evaluate thein vitro effects of hormones, growth factors, and other substances on growth and development of dermal skeleton in fish. 相似文献
6.
Long-term organ culture of mouse mammary gland 总被引:1,自引:0,他引:1
J. W. Harbell P. D. Bowman J. M. Shannon C. W. Daniel 《In vitro cellular & developmental biology. Plant》1977,13(8):490-496
Summary A method for maintaining mouse mammary gland in organ culture for periods of at least 30 days is described. Strips of the
number four mammary glands were cultured in individual tubes while fully submerged in Medium 199 supplemented with insulin,
aldosterone, ovine prolactin and bovine growth hormone. Exchange processes were aided by slowly rotating the tubes during
culture. Mammary tissue from midpregnant BALB/c and virgin GR/A mice was induced to undergo lobulo-alveolar development, secrete
and remain differentiated and metabolically active for the period of culture. Cells of both the ductal and alveolar epithelium
continued to synthesize DNA and divide. The submerged roller-tube culture allows the use of larger pieces of tissue than can
be accommodated in static culture, and the technique may prove applicable to the culture of a variety of tissues. 相似文献
7.
Hiroyoshi Hoshi Yuji Takagi Keizo Kobayashi Masakazu Onodera Taneaki Oikawa 《In vitro cellular & developmental biology. Animal》1991,27(7):578-584
Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated
culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2),
lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent
cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of
fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or
BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed
in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After
granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling
level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations
(14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation
of bovine granulosa cells. 相似文献
8.
Whole retinae from Midas cichlids Cichlasoma citrinellum were maintained successfully in superfusion culture for 21 days post-removal and continued to exhibit normal light- and circadian-driven cone movements. 相似文献
9.
Violet Albert David Barkla Graeme P. Young 《In vitro cellular & developmental biology. Animal》1994,30(7):443-449
Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling
rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory
hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in
serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when
cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640
plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium.
Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture.
However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after
48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the
rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II,
des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity
of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat
jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological
responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None
of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal
epithelium. 相似文献
10.
James A. McAteer Orion D. Hegre 《In vitro cellular & developmental biology. Plant》1978,14(9):795-803
Summary A method of perfusion organ culture is described in which explants cultured at the airmedium interface are bathed by a continuous
flow of nutrient medium. Morphological studies on the fetal rat lung indicate that explant development in this system is comparable
to that obtained using standard organ-culture dishes. Medium supply is easily manipulated and continuous sampling of the effluent
stream is possible without disturbing the immediate explant environment. The basic design facilitates secretory-response studies
on cultured organ explants as demonstrated by a study of glucose-stimulated insulin release by the neonatal rat pancreas.
This work was supported by U. S. Public Health Service Training Grant No. GM 00114. 相似文献
11.
James H. Resau Ph.D. Kosaku Sakamoto John R. Cottrell Carnell Newkirk 《Cell biology and toxicology》1986,2(3):401-415
Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.Abbreviations 4F-1G
4% formaldehyde 1% glutaraldehyde fixative
- LDH
lactate dehydrogenase
- OsO4
osmium tetroxide
- PAS/PLH
periodic acid, periodic acid Shiff lead hematoxylin stain 相似文献
12.
13.
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
14.
Douglas G. Matsell Tracey Bennett 《In vitro cellular & developmental biology. Animal》1998,34(2):138-148
Summary We have developed a unique human fetal kidney explant model to study the role of the insulinlike growth factor (IGF) system
in metanephric development. Kidneys from 10–18 wk gestation human abortuses were maintained in serum-free conditions and defined
medium, which was shown to support the induction and differentiation of the viable metanephric blastema. Histologically the
tissue remained viable to 192 h of serum-free culture, while metanephric differentiation, reflected by a shrinking nephrogenic
zone and the formation of maturing S-shape and glomerular forms, was accelerated and occurred between 48 and 96 h. In the
nephrogenic zone, a significant decrease in IGF-II gene expression occurred, which reflected the differentiation of the metanephric
blastema cell mass. IGF-II expression persisted, however, in the expanded interstitial mesenchyme. With differentiation over
48 h an increase in IGFBP-2 and WT1 gene expression by Northern blot analysis occurred, and was localized by in situ hybridization to the differentiating glomerular epithelial cell mass. Analysis of the explant-conditioned media by Western
ligand blot demonstrated an increase in the rate of IGF binding protein (IGFBP)-2 peptide production by the differentiating
explant, consistent with an increase in IGFBP-2 gene expression and with metanephric differentiation. This pattern of temporal
and spatial gene expression closely approximates that of normal in vivo fetal renal development and of glomerular epithelial cell differentiation. 相似文献
15.
William E. Sweeney Jr. Ellis D. Avner 《In vitro cellular & developmental biology. Animal》1998,34(2):189-194
Summary Several lines of evidence suggest that hepatocyte growth factor (HGF), a soluble protein secreted by mesenchymal cells, may
elicit a morphogenic response in the developing metanephros. We investigated the role of HGF at three different stages of
murine metanephric development utilizing serum-free organ culture. Cultures were initiated at E-13, E-15, and E-17; treated
with exogenous HGF or antibodies to HGF (to block endogenous HGF) for 120 h in vitro; and evaluated for growth and differentiation in comparison to control explants cultured for 120 h in basal medium. HGF treatment
of E-13 explants resulted in a reduction of growth and differentiation compared to control explants. Treatment of E-13 explants
with antibodies to HGF produced explant growth and differentiation indistinguishable from control explants. In contrast to
the results of E-13 cultures, explants initiated at E-15 and E-17 demonstrated an increased growth and differentiation profile
when treated with HGF compared to controls. Treatment of E-15 and E-17 explants with antibodies to HGF resulted in a decrease
growth and differentiation profile compared to control or HGF-treated explants. These data demonstrate that HGF has differential
effects on renal morphogenesis at progressive developmental stages of metanephric development. 相似文献
16.
A. A. J. J. L. Rutten B. G. A. G. G. Béquet-Passelecq H. B. W. M. Koëter 《In vitro cellular & developmental biology. Plant》1990,26(4):353-360
Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA
insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas
the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture
model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI
1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially
similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture.
The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the
intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter
it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between
single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used
to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore,
within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model
because the rabbit skin can be cultured for 7 d.
The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the
Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse
Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid. 相似文献
17.
Summary The effect of variations in culture conditions on survival of fragments of mouse and rat descending colon in organ culture
was studied by morphological and functional criteria. A combination of conditions demonstrated to be beneficial permitted
maintenance for at least 35 days. These included: a gaseous environment of 95% O2:5% CO2, an attachment matrix consisting of a Metricel GA-4 membrane (pore size, 0.8 μ), intermittent exposure to the gas and fluid
phases by rocking in 5 ml medium and supplementation of the medium with 1.0 μM dexamethasone and 10% FBS. During this time,
the crypt structure of the mucosal epithelium was well preserved, and DNA synthesis in the crypts and mucin production in
the crypts and superficial epithelium continued. In addition, the synthetic hormone, pentagastrin, stimulated DNA synthesis
in the mucosal epithelium of mouse colon fragment in short-term organ culture.
This work was supported by Environmental Protection Agency Grant R803998-01-1 and National Cancer Institute Contract N01-CP-75952. 相似文献
18.
David Cossar Jeanne Bell Malcolm Lang Robert Hume 《In vitro cellular & developmental biology. Animal》1993,29(4):319-324
Summary In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes.
Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial
immunoreactivity to cytokeratins CK 7,8, and 18 decreases with differentiation both in utero and in organ culture, whereas
reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace
of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0±7.3 wk. In organ culture, capillary
blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct
invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests
which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar
crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal
maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost
synchronous in human lung in utero but clearly dissociate in organ culture. 相似文献
19.
J. Feng A. H. Melcher D. M. Brunette H. K. Moe 《In vitro cellular & developmental biology. Plant》1977,13(2):91-99
Summary The method of Deutsch and Weeks was modified to provide a reliable and reasonably quick method for assaying the L-ascorbic
acid content of culture medium. The modified method was used to determine the decay of L-ascorbic acid under various conditions
of culture and the concentration of the vitamin in commercially prepared media. The half-life of L-ascorbic acid in a modified
New circulator gassed with 95% O2+5% CO2 was 1.5 hr.; and when gassed with 20% O2+5% CO2+75% N2, about 2 hr. In Petri dishes gassed with 20% O2+5% CO2+75% N2, the half-life of L-ascorbic acid was 0.9 hr. About 4% of the L-ascorbic acid was lost per day when medium was stored at
0°C and about 9% per day when stored at 5°C. When medium with an initial content of 300 μg per ml was stored at room temperature,
the half-life was found to be 15.5 hr. The L-ascorbic acid in five commercially available media, which contain the vitamin
in their formulations, was assayed immediately after their delivery to the laboratory. The values of L-ascorbic acid measured
in these media were in all cases far lower than prescribed. A continuous-flow organ culture system has been designed which
allows the provision of a relatively constant level of L-ascorbic acid to an explant by taking advantage of the slow oxidation
of L-ascorbic acid at 0°C. 相似文献
20.
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