共查询到20条相似文献,搜索用时 93 毫秒
1.
Merrill S. Babcock Maria R. Marino William T. Gunning III Gary D. Stoner 《In vitro cellular & developmental biology. Plant》1983,19(5):403-415
Summary The clonal growth and serial propagation of rat esophageal epithelial cells in low serum-containing medium has been achieved
without feeder layers or conditioned medium. To date, a total of four lines have been developed and maintained for as many
as 40 passages in culture. Growth of the cells was possible only after modifying the culture medium (PFMR-4) by reducing the
calcium concentration from 1 to 0.1 mM, and by adding low levels of dialyzed fetal bovine serum and seven growth factors; i.e. epidermal growth factor, hydrocortisone,
ethanolamine, phosphoethanolamine, insulin, transferrin, and cholera toxin. Cell lines have been developed from both explant
outgrowths and enzyme dissociated esophagi. The epithelial nature of the cells was confirmed by electron microscopy and immunological
methods. Clonal growth studies revealed that optimal cell growth occurred in medium containing 2.4% dialyzed fetal bovine
serum and 0.1 mM calcium. Calcium levels of 0.3 mM or higher caused the cells to stratify and undergo terminal differentiation. Coating the culture dishes with collagen, or
a combination of collagen, fibronectin, and bovine serum albumin, increased both the cell growth rate and the colony forming
efficiency. The successful long term culture of rat esophageal epithelial cells permits their use as models in studies concerned
with esophageal differentiation and carcinogenesis.
This investigation was supported by U.S. Public Health Service Grant CA 28950, awarded by the National Cancer Institute, Bethesda,
MD. 相似文献
2.
Petra M. de Jong Marianne A. J. A. van Sterkenburg Johanna A. Kempenaar Joop H. Dijkman Maria Ponec 《In vitro cellular & developmental biology. Animal》1993,29(5):379-387
Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies
obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies)
using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation
and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells
were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged,
low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell
cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under
submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an
appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six
layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged
and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed
in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the
culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was
not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia
appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial
cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function. 相似文献
3.
Jose Russo Megan J. Milis Michelline J. Moussalli Irma H. Russo 《In vitro cellular & developmental biology. Plant》1989,25(7):643-649
Summary In experimental animal models the susceptibility of the mammary gland to neoplastic transformation is related to its degree
of development and proliferative activity; this observation led us to determine whether the human breast epithelium also exhibits
development-related differences, and whether these differences could be detected in an in vitro system. Normal breast tissue
obtained from reduction mammoplasties of 9 patients ranging in age from 18 to 56 years were characterized in both whole mount
preparations and organoids obtained after collagenase-hyaluronidase digestion by their degree of development based upon the
types of lobules present. Lobules were classified into type 1 (Lob 1), composed of approximately 11 alveolar buds, the less
developed; lobules type 2 (Lob 2), of moderate development, composed of approximately 47 ductules each, and lobules type 3
(Lob 3), composed of 80 ductules each, represented the highest level of development. Epithelial organoids obtained after digestion
were plated in DMEM:F12 medium supplemented with hydrocortisone, cholera toxin, insulin and 5% horse serum with a calcium
concentration of 1.05 mM Ca++. Following attachment, the medium was replaced by medium containing 0.040 mM Ca++. The percentage of attachment of organoids to the flask was greater in cells from Lob 1 (89–99%) and Lob 1+2 (79–100%) than
in cells from Lob 3, which had a 53–67% attachment. The total yield of cells after 7 weeks in culture was also greater in
cells derived from Lob 1 and Lob 1+2 than in cells from Lob 3. The total yield of cells obtained from primary cultures was
not related to the number of organoids plated, but to the degree of development of the gland. The DNA-labeling index (DNA-LI)
in intact breast tissue correlated with that in primary cultures; it was greater in Lob 1 and Lob 1+2 than in Lob 3. By flow
cytometry, the highest percentage of cells in S-phase was seen in cells with the highest DNA-LI. We concluded that the growth
characteristics of mammary epithelial cells in vitro in a low Ca++ medium is modulated by the degree of development and differentiation of the gland.
Supported by PHS Grant CA-38921 awarded by the National Cancer Institute, DHHS, and an Institutional Grant from the United
Foundation of Greater Detroit. 相似文献
4.
Niels C. Krejci Lynne Smith Rebecca Rudd Robert Langdon Joseph McGuire 《In vitro cellular & developmental biology. Animal》1991,27(12):933-938
Summary To investigate the regulation of epithelial differentiation, normal human epidermal keratinocytes were cultured floating on
the surface of culture medium without attachment to a solid substrate. Keratinocytes spread out on the surface of the medium,
proliferated and differentiated either into several flat lacy sheets 1 to 3 cells thick (on medium containing 0.15 mM calcium) or formed one single aggregate of cells from 5 to 15 cells in thickness on medium containing 1.15 mM calcium. The cell aggregates demonstrated a pattern of ordered epithelial differentiation. Levels of progressive differentiation
resembling the structure of normal human epidermis were identified by light microscopy, immunohistochemistry, and electron
microscopy. Differentiation proceeded from cells at the air side toward cells at the medium side with basal appearing cells
on the air side and keratinocytes expressing filaggrin and involucrin on the side toward the medium. These results demonstrate
that organized epithelial differentiation can occur in the absence of extracellular matrix. In contrast with other air-liquid
interface cultures, epithelial differentiation in the absence of extracellular matrix progresses from air towards medium. 相似文献
5.
Summary During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of
thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation
in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5mM) calcium (Ca2+) in the absence or presence of thyroxine (10−7
M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05
mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When
tissues were exposed to high levels of calcium (0.5mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence
of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of
skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca2+ concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca2+ leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca2+ concentrations (0.5mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and
differentiation inRana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells. 相似文献
6.
Mark R. Rosenberg Stephen C. Strom George Michalopoulos 《In vitro cellular & developmental biology. Plant》1982,18(9):775-782
Summary Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture.
The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth
of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that
corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10−5
M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media.
Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10−5
M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated
by hydrocortisone and nicotinamide.
This study was supported by NIH Grant CA30241-01. 相似文献
7.
Summary The sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures
grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those
grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence
in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was
clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of
regulation at the level of mRNA accumulation or translation rather than posttranslational modification.
This research was supported by Public Health Service grant AR 27130 from the National Institute of Arthritis, Musculoskeletal
and Skin Diseases, Bethesda, MD, and National Research Service postdoctoral fellowship ES 05336 from the National Institute
of Environmental Health Sciences, Research Triangle Park, NC. 相似文献
8.
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
9.
Barbara E. Kurth Debra J. Hazen-Martin Mary Ann Sens Donald A. Sens 《In vitro cellular & developmental biology. Plant》1988,24(6):593-600
Summary The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to
improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this
and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and
CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as
severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in
the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the
study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated
only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering,
desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth
medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo
ductlike differentiation.
This work was supported by Public Health Service grant AM 11028, Department of Health and Human Services, Washington, DC. 相似文献
10.
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
11.
Eric W. Johnson Susan F. Meunier Christopher J. Roy Nancy L. Parenteau 《In vitro cellular & developmental biology. Animal》1992,28(6):429-435
Summary We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported
establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and
rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium
containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased
particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium
cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion
of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting
effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into
a morphologically differentiated epidermis. 相似文献
12.
Josiah Ochieng Larry Tait Jose Russo 《In vitro cellular & developmental biology. Plant》1990,26(4):318-324
Summary Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to
a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca2+ medium (1.05 mM). To clarify the mechanism by which Ca2+ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent
to high Ca2+ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca2+ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike.
When the cells were switched to high Ca2+ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical
localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca2+-grown cells did not display a network of tubulin fibers, whereas high Ca2+-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments
designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca2+ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatement of the cells with the calcium ionophore A23187 in low
calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment
of the cells with cyclic-AMP in low Ca2+ medium had no observable effect on cell morphology. These results indicate that high levels of Ca2+ either favor tubulin polymerization or stabilize the polymerized state.
This research was supported by NCI grant CA-38921 from the National Cancer Institute, Bethesda, MD, and by an institutional
grant from the United Foundation of Greater Detroit. 相似文献
13.
Summary Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium.
The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis
and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal
and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered
Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial
cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific
keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival
tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression
was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca2+ is shown by the expressionof K13.
This work was supported by Biomedical Research grant RR05346, National Institutes of Health grant DE04660, University of Washington
Graduate Fund and Hack Foundation Fund, Department of Periodontology, University of Washington. 相似文献
14.
Kristina Sundqvist Yun Liu Kristina Arvidson Kari Ormstad Lennart Nilsson Rune Toftgård Roland C. Grafström 《In vitro cellular & developmental biology. Animal》1991,27(7):562-568
Summary Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial
outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins
5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings
per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in
buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit
a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope
formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca2+ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area,
involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly
derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal
epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably
respond to several agents shown to modulate growth of cells that originate from other types of epithelia.
This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research
Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments,
the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg?rden, Stockholm, Sweden. 相似文献
15.
Linda L. Marden Charles R. Crawford Robert E. Bryant 《In vitro cellular & developmental biology. Plant》1982,18(6):550-556
Summary Cells of the cultured hamster cell line V79 were labeled with tritiated adenosine and incubated for up to 30 min in the presence
of inhibitors of glycolysis and oxidative phosphorylation. These inhibitors were (a) 5 mM KCN plus 5 mM iodoacetate, (b) 5 mM KCN plus 5 mM KF, and (c) 15 mM KCN plus 15 mM KF. The fate of the tritium label was examined during incubation with inhibitors and also during subsequent incubation in
growth medium in the absence of inhibitors. The tritiated ATP pool was found to decrease in cells incubated in the presence
of any of the inhibitor combinations, but only in the presence of 15 mM KCN plus 15 mM KF was this pool decreased below the level of detection. After cells were incubated with KCN plus KF, a high level of ATP
was recovered when the inhibitors were removed. Cells incubated with KCN plus iodoacetate retained depletion levels of ATP.
Plating efficiency and trypan blue staining showed that KCN-KF treated cells retained viability, whereas KCN-iodoacetate treated
cells did not. Cells were examined for ability to take up tritiated uridine before, during, and after depletion of ATP by
incubation in the presence of 15 mM KCN plus 15 mM KF. These cells were found to have a variation in uridine uptake that was related directly to intracellular ATP level. Cells
in which the ATP was very low exhibited little or no uridine uptake, whereas cells in which the ATP level was near normal
exhibited normal uridine uptake.
This work was supported in part by Grant GM24271 from the National Institutes of Health, Bethesda, Maryland. 相似文献
16.
H. Barton Grossman Gary Wedemeyer Judith Stein 《Cancer immunology, immunotherapy : CII》1988,26(3):269-272
Summary The autologous serologic reactivity of 13 patients with bladder cancer was evaluated using cell lines derived from each individual's own tumor as targets. Protein A and immune adherence assays were employed to determine antibody binding to the tumor targets at varying passage numbers. Autologous reactivity was found in 6 of the 13 cell lines tested. However, the titer was usually low regardless of the passage number. Seven autologous serum/cell line combinations were tested using both low and high passage cells as targets. In six of these combinations, the degree of antibody binding was similar with both low and high passage target cells. The incidence of autologous reactivity in the 12 patients with urothelial tumors was 50%.This investigation was supported by PHS Grant number CA36933, awarded by the National Cancer Institute, DHHS. 相似文献
17.
Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium 总被引:6,自引:0,他引:6
Summary Gingival tissue from healthy adult human donors was used as a source of epithelial cells for culture. An overnight incubation
of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective
tissue. This step minimized culture contamination with fibroblasts. The epithelium was then trypsinized to prepare a single
cell suspension. The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated
at 37° C and 5% CO2 in a humid atmosphere. Primary cultures grew in small islands that coalesced at confluency. Immunohistochemistry demonstrated
uniform staining of the cells with antibodies to keratins of stratified squamous epithelium. Ultrastructurally, the cells
contained distinct intermediate filaments. When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes. However, when cells were grown under
physiologic calcium (1.2 mM), desmosomes were prominent and well developed. Cells were maintained in culture for over 100 d (7 passages).
This work was supported by Biomedical Research grant RR 05346 from the National Institute of Health, Bethesda, MD, and the
University of Washington Graduate School Research Fund. 相似文献
18.
Summary Homeostasis of intracellular calcium ([Ca++]i) and pH (pHi) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain
normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes
of [Ca++]i and pHi on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure
[Ca++]i with fura-2 and pHi with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pHi in these cells was 7.37±0.05 (n=20 cells) and resting [Ca++]i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH4Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca++]i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH4Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium
stores. On removal of NH4Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pHi decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca++]i, but produced a biphasic change in pHi. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial
values. When [Ca++]i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pHi decreased by 0.35±0.02 units. We conclude that an increase in pHi leads to an increase in [Ca++]i in rabbit corneal epithelial cells; however, a decrease in pHi leads to minor changes in [Ca++]i. The ability of CE cells to maintain proper calcium homeostasis when pHi is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification,
which occur during prolonged eye closure. 相似文献
19.
Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates 总被引:1,自引:0,他引:1
Yoshinobu Kubota Eugene B. Gehly Karl H. Link Charles Heidelberger 《In vitro cellular & developmental biology. Plant》1981,17(11):965-978
Summary Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These
cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone,
epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca++-and Mg++-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate
tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor
for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of
both cell lines. These cell lines have hypotetraploid chromosome numbers and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis.
This work was supported in part by Grant CA-21, 746, and by the Electron Microscope Core Facility on Grant CA-14,089, from
the National Cancer Institute, National Institutes of Health, Bethesda, MD. 相似文献
20.
A simplified method for passage and long-term growth of human mammary epithelial cells 总被引:8,自引:0,他引:8
Herbert D. Soule Charles M. McGrath 《In vitro cellular & developmental biology. Plant》1986,22(1):6-12
Summary A method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations
and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue
are plated in medium containing 1.05 mM Ca++ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca++ to overcome “renewal inhibition” and to stimulate growth. In low Ca++ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental
requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca++, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca++, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of
(linear) growth. Cells released from monolayer were greater than 90% viable and yielded 105 cells/cm2 of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin
for dissociation. Long-term free-floating cells were typical mammary epithelium: (a) they formed domes and exhibited renewal
inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments,
and (d) they were diploid. 相似文献