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1.
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
2.
Lawrence E. Shapiro Neil Wagner 《In vitro cellular & developmental biology. Plant》1988,24(4):299-303
Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium
is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum
containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these
two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium
was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the
absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium
from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free
culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells.
This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes
of Health grants CA 24604-09 and CA 16463-14. 相似文献
3.
Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium 总被引:19,自引:1,他引:18
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Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived. 相似文献
4.
A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures 总被引:4,自引:0,他引:4
A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function. 相似文献
5.
S. N. W. Mohamed R. Holmes C. R. Hartzell 《In vitro cellular & developmental biology. Plant》1983,19(6):471-478
Summary A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac
cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham’s F12 and
Dulbecco’s modified Eagle’s medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine
serum albumin, hydrocortisone (HC),l-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free
medium. Cells grow in this medium exhibited a higher beating rate and were maintained for a longer time compared to those
cells grown in serum.
The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population
were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to
200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented
medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show
that some hormones affect growth, whereas others affect function. 相似文献
6.
Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium 总被引:4,自引:1,他引:3
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An epithelial cell line from pig kidney (LLC-PK1) with properties of proximal tubular cells can be maintained indefinitely in hormone-supplemented serum-free medium. Continuous growth requires the presence of seven factors: transferrin, insulin, selenium, hydrocortisone, triiodothyronine, vasopressin, and cholesterol. The hormone-defined medium (a) supports growth of LLC-PK1 cells at a rate of approaching that observed in serum-supplemented medium; (b) allows vectorial transepithelial salt and fluid transport as measured by hemicyst formation; and (c) influences cell morphology. The vasopressin dependency for growth and morphology can be partially replaced by isobutylmethylxanthine or dibutyryl cyclic AMP. The medium has been used to isolate rabbit proximal tubular kidney epithelial cells free of fibroblasts. 相似文献
7.
Tai C. Chen Norman P. Curthoys Carl F. Lagenaur Jules B. Puschett 《In vitro cellular & developmental biology. Plant》1989,25(8):714-722
Summary Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by percoll gradient centrifugation.
Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal
cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s
F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E1. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent
monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further
characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid
hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules.
The cells also retained significant levels of 25-hydroxyvitamin D3-lα-hydroxylase, alkaline phosphatase, and ψ-glytamyltranspeptidase, three enzymes that are primarily associated with the
proximal tubule. The cultured epithelial cells also exhibit a Na+-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic
of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within
this segment of the rat nephron.
This work was supported in part by the Veterans Administration (JBP), Washington, DC, by grant DK-37124 (NPC) from the National
Institutes of Health, Bethesda, MD, and by grant BNS-86-17004 (CFL) from the National Science Foundation, Washington, DC. 相似文献
8.
Yasuhiro Tomooka Stephen E. Harris John A. McLachlan 《In vitro cellular & developmental biology. Plant》1985,21(4):237-244
Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained
in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically,
immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number
was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10
ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required
eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin,
and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free
media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides
a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells. 相似文献
9.
Di Lorenzo Teresa P. De Maro Joseph A. Pumo Dorothy E. 《In vitro cellular & developmental biology. Plant》1989,25(10):909-913
Summary A serum-free culture system was used to compare the nutritional requirements of mouse mammary cells transformed by bovine
papillomavirus type 1 (ID13 cells) and the uninfected parent line (C127 cells). The serum-free, chemically defined medium
used for this study was an MCDB 151-based medium (MCDB 151+S+I), supplemented with epidermal growth factor, transferrin, hydrocortisone,
ethanolamine, phosphoethanolamine, retinoic acid, trace metals, and insulin. Proliferation of either cell type in serum-free
culture required the addition of 250 μg/ml of insulin. ID13 cells have a doubling time of greater than 96 h in MCDB 151+S+I,
whereas C127 cells have a doubling time of 60 h. This is in sharp contrast to the growth characteristics of the two cell types
in 10% fetal bovine serum, where doubling times for the ID13 and C127 cells are 24 and 30 h, respectively. Culture of the
cells in a serum-free medium has therefore revealed that the papillomavirus-transformed cells have more stringent growth requirements
than the uninfected parent line.
This work was supported in part by grant #1-P01 NS19214 from the National Institutes of Health, Bethesda, MD, NSF grant #R11-8217798
from the National Science Foundation, Washington, DC, and by a grant from the Otolaryngology Foundation. 相似文献
10.
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
11.
Paul B. Hatzinger James L. Stevens 《In vitro cellular & developmental biology. Plant》1989,25(2):205-212
Summary A culture system is described in which rat kidney proximal tubule epithelial cells (RPTE) can be prepared with good yield
and high viability and grown in culture under serum-free conditions. The cells require EGF, insulin, cholera toxin and either
1% dialyzed serum or a complex of bovine serum albumin with oleic acid (BSA/OA). The cells can be maintained for long periods
of time and express several markers for RPTE. The cells have both alkaline phosphatase and γ-glutamyltransferase activity
and respond to parathyroid hormone but not vasopressin. The specific activity of γ-glutamyltransferase decreases when the
cells begin to grow, but increases when they reach confluence. Extracellular calcium plays a role in the induction of γ-glutamyltransferase
in confluent cells. Cells grown in media containing low calcium, i.e. less than 0.4 mM, have reduced specific activity of
γ-glutamyltransferase. Extracellular calcium also alters the morphology of the cells in that cells grown in low calcium are
single cells or loose clusters suggesting poor cell-cell contact. When the calcium is raised to 1.0 mM, the cells change their
shape and organization to adopt the morphology of cells maintained continuously in 1.0 mM calcium. The cells can be passaged
onto plastic surfaces which have been coated with collagen but cannot be subcultured on uncoated or serum coated plastic.
This culture system will be a useful model for the investigation of renal carcinogenesis and the role of cell proliferation
in that process.
This work was supported by grants CA48197 and DK38925 from the National Institutes of Health
Editor's Statement This paper reports a method for isolation and, uniquely, multipassage culture of rat proximal tubule epithelial
cells. The authors use this culture system to explore some aspects of the physiology of these cells, demonstrating the value
of the model. 相似文献
12.
The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium. 相似文献
13.
Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media 总被引:1,自引:0,他引:1
J H Miller 《Journal of cellular physiology》1986,129(2):264-272
Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell. 相似文献
14.
Lawrence E. Shapiro Neil Wagner 《In vitro cellular & developmental biology. Plant》1989,25(7):650-654
Summary We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free
culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the
absence and presence of [35S]methionine. A 1∶4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell
numbers per assay dish from 1.59±0.12×105 to 3.35±0.34×105 after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P<0.05) less growth (2.14±0.41×105 cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned
medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [35S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet
of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate
that 29.1±1.2 ng of transferrin was secreted per 106 cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P<0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35
cells into serum-free culture is due to transferrin.
This work was funded by a feasibility grant from the American Diabetes Association, as well as by grants CA 24604-09 and CA
16463-14 from The National Institutes of Health, Bethesda, MD. 相似文献
15.
Summary A serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal
rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of
immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the
Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbecco's modified Eagle's
medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10
ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over
a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the
alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like
projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin,
progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing
in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network
with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development
occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane.
In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this
model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells.
This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health,
Bethesda, MD. 相似文献
16.
Maintenance of proximal and distal cell functions in SV40-transformed tubular cell lines derived from rabbit kidney cortex 总被引:1,自引:0,他引:1
A Vandewalle B Lelongt M Geniteau-Legendre B Baudouin M Antoine S Estrade F Chatelet P Verroust R Cassingena P Ronco 《Journal of cellular physiology》1989,141(1):203-221
This paper reports the preparation and describes the properties of three renal tubular cell lines derived using SV40 infection of primary cultures of rabbit kidney cortical cells, enriched in proximal cells. RC.SV1 was initially derived from cultures grown in the presence of fetal calf serum exhibiting a low degree of proximal differentiation. The cells were subsequently adapted to grow in serum-free hormonally defined medium and display basic properties of proximal tubule cells including well-developed apical microvilli, strong expression of brush-border hydrolases, Na+-coupled glucose uptake, and increased cyclic AMP production when exposed to PTH. The other two cell lines were derived from cultures in serum-free hormonally defined medium and propagated in the same medium. They are characterized by some common properties including rare and short microvilli, low expression of apical hydrolases, and low or undetectable Na+-dependent glucose uptake, but differ by their abilities to respond by an increase in cAMP to various hormonal stimuli. RC.SV2 cells are sensitive to calcitonin and to a lesser extent to isoproterenol and PTH, suggesting that they may originate from the thick ascending limb of Henle's loop and the bright portion of the distal tubule. RC.SV3 responds essentially to isoproterenol and arginine vasopressin, suggesting a more distal origin (late distal and initial collecting tubule). Emergence of distal cell lines from cultures exhibiting proximal characteristics may be related to distal cell overgrowth as suggested by analysis of growth kinetics and increased Na+/H+ exchanger activity in RC.SV2 compared with RC.SV1. 相似文献
17.
Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel
Timothy Turner Howard A. Bern Peter Young Gerald R. Cunha 《In vitro cellular & developmental biology. Plant》1990,26(7):722-730
Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved
in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with
bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol,
putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells
occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture.
Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics.
Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated
and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male
mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal
or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258,
epithelial cells of mouse origin were distinguishable from stromal cells of rat origin.
Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship
GM08730 to T. T. 相似文献
18.
Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial
(RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue
culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone,
cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors
that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS
requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum
levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin
(BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum,
and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with
EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing
medium.
This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda,
MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD. 相似文献
19.
A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium 总被引:10,自引:0,他引:10
P. Briand O. W. Petersen B. Van Deurs 《In vitro cellular & developmental biology. Plant》1987,23(3):181-188
Summary A new, nontumorigenic human breast epithelial cell line, HMT-3522, has been established from fibrocystic breast tissue. Cells
were explanted and propagated in chemically defined medium including insulin, transferrin, epidermal growth factor, hydrocortisone,
estradiol, prolactin, and Na-selenite. The epithelial nature of the cell line was established by immunocytochemical detection
of cytokeratins. Moreover, electronmicroscopy revealed monolayers of polarized cells connected by desmosomes and provided
with apical microvilli. Milk fat globule membrene antigen, specific for the apical membrane domain of normal, luminal breast
epithelial cells, was expressed only in confluent cultures where some cells overlaid others, indicating “stem cell”-like properties.
After 25 to 30 passages, the cells are diploid with a few marker chromosomes and loss of chromosomes in the D-group. The cells
are nontumorigenic in athymic mice; they lack estrogen receptors, and estradiol does not stimulate growth. The HMT-3522 cell
line may represent a useful model for the study of brest cell differentiation and carcinogenesis in vitro.
This work was supported by a grant from the Danish Cancer Society. 相似文献
20.
Christine Halleux Yves-Jacques Schneider 《In vitro cellular & developmental biology. Animal》1991,27(4):293-302
Summary Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain
largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon
adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells).
Cells are cultivated in a serum-free medium, containing 1μg/ml insulin, 1 ng/ml epidermal growth factor, 10μg/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen. Cells rapidly
adhere, grow, and form confluent monolayers; after 15 days, scanning electron microscopy reveals numerous uniform microvilli.
Domes, which develop on nonporous substrata, are absent on high porosity membranes. Culture medium from upper and lower compartments
of microplate inserts and cell lysates were immunoprecipitated after labeling with [3H]glucosamine and leucine; analysis was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed
by autoradiography. [3H]transferrin is found mainly in the lower compartment and in cells; [3H]apolipoprotein B is released in both compartments, and fibronectin almost entirely recovered in the lower compartment; [3H]transferrin receptors and ferritin are only present in cell lysates. Binding experiments also show that transferrin receptors
are accessible from the lower compartment. These results suggest that CaCo2 cells, cultivated in synthetic medium on membranes
of appropriate porosity, could provide an in vitro model of the intestinal barrier, with the upper compartment of the culture
insert corresponding to the apical pole facing the intestinal lumen and the lower one to the basal pole in contact with blood.
This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (grant 3.4551.88) and the Walloon Region. 相似文献