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1.
J. H. Carver E. P. Salazar M. G. Knize 《In vitro cellular & developmental biology. Plant》1983,19(9):699-706
Summary Reducing serum concentrations in media provides a potential cost advantage. To determine whether such media could be used
for applied mutagenesis assays, we measured cloning efficiency and growth parameters in suspension of Chinese hamster ovary
cells cultured in reduced serum with or without additives (1 μg/ml insulin, 3×10−7
M linoleic acid, 1×10−8
M H2SeO3) or bovine serum albumin (BSA, 1% wt/vol). With the additives and ≤0.5% fetal bovine serum (FBS), Ham’s F12 medium (without
hypoxanthine and thymidine) was more optimal than alpha Eagle’s minimum essential medium (MEM) (without ribosides and deoxyribosides)
for low density cloning and high density suspension growth. Acceptable cloning efficiencies were obtained with 2% FBS plus
BSA without additives in either medium; the addition of BSA resulted in improved colony size and more compact colony morphology.
In alpha MEM, satisfactory growth rates and maximum saturation densities in suspension culture were obtained only with 5%
FBS; in Ham’s F12, 1% FBS+deoxycytidine+BSA yielded satisfactory suspension growth. Spontaneous mutant frequencies were compared
for each medium containing 10% dialyzed FBS (DFBS), 1% FBS plus BSA, or 2% FBS plus BSA. The spontaneous frequency of azaadenine-resistant
phenotypes (mutant at theaprt locus) in 1% FBS plus BSA was significantly lower than the frequency observed in 2% FBS plus BSA or 10% DFBS. Frequencies
of spontaneous mutants resistant to thioguanine (hgprt locus) or fluorodeoxyuridine (tk locus) were similar with 10% DFBS, 1% FBS plus BSA, or 2% FBS plus BSA. Compared to alpha MEM with 10% DFBS, frequencies
of drug-resistant mutants induced by ethyl methanesulfonate or mitomycin C (MMC) were not significantly lower in alpha MEM
with 2% FBS plus BSA; observed mutant frequencies induced by dimethyl-nitrosamine or benzo(a)pyrene seemed to be decreased at lower survival levels.
This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory
under Contract W-7405-ENG-48 and supported by the Environmental Protection Agency under Interagency Agreements IAG-D5-E681-AN
and AO. 相似文献
2.
Elizabeth Mendiaz Michael Mamounas John Moffett Ellis Englesberg 《In vitro cellular & developmental biology. Plant》1986,22(2):66-74
Summary Insulin, FeSO4, or transferrin are major requirements together with HEPES buffer and selenium for the growth of CHO-K1 (CCL 61) in a modified
F12 medium (M-F12). Insulin stimulates growth at 1 ng/ml to 10 μg/ml. In the defined medium minus insulin, CHO-K1 grows slowly
as elongated, elliptical cells in parallel arrays typical of normal diploid fibroblasts in contrast to round-to-cuboid cells
in loosely overlapping arrays in the presence of serum or insulin. During prolonged incubation in the absence of insulin the
cells gather up into a large spherical cluster of viable cells. Insulin “independent” mutants have been isolated whose growth
rate during exponential phase in the absence of insulin (48 h to 84 or 96 hrs) is 2.7 to 3.6 times that of the parental culture.
Insulin stimulates the growth of these variants only during the first 48 h and is inhibitory at 50 to 500 ng/ml during the
exponential phase. Insulin induction of the A system of amino acid transport occurs in about 8 h and requires both protein
and RNA synthesis.
This work was supported in part by National Science Foundation Grant PCM 7903242 and by the University of California. 相似文献
3.
Karimullah A. Zirvi Darwin O. Chee George J. Hill 《In vitro cellular & developmental biology. Plant》1986,22(7):369-374
Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five
tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder
carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of
transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10−10
M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES
medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI
cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower
doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium
resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with
the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell
lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell
lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones
influence cell growth.
This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J.
Hill) and grant no. CA-37138 from the National Cancer Institute. 相似文献
4.
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
5.
Terry L. Riss' David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1989,25(2):136-142
Summary The effect of 17β-estradiol (E2) on growth of GH4C1 rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum.
Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T3), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E2 responsive only when growth was retarded by reducing the T3 concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E2 to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with
charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E2 to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing
and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects
in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors.
This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society
grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc. 相似文献
6.
Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor 总被引:2,自引:0,他引:2
Summary BC3H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified
Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin,
and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a
rate equal to that of serum-supplemented medium, and at low cell input (103 cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell
survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency
of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF. 相似文献
7.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
8.
The serum-free growth of primary cultures of normal human epithelial-like cells from amniotic membranes was accomplished. The synthetic medium consists of a 1 : 1 basal nutrient mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 supplemented with 2.5 μg/ml insulin, 50 ng/ml epidermal growth factor (EGF), 5 μg/ml transferrin, and 0.1 ng/ml triiodothyronine (T3). EGF is the primary mitogen and is essential for cell proliferation in this system. 相似文献
9.
Summary We assessed the potential role of all-trans-retinoic acid on the developing chick pancreas, specifically with regard to the proportions of insulin cells. The endodermal
component of the dorsal pancreatic bud of 5-d-old chick embryos was cultured on Matrigel. Retinoic acid (10−6 or 10−5
M) was added to a standard serum-free medium, Ham's F12 containing insulin, transferrin and selenium (F12.ITS). Control grafts
were cultured in F12.ITS alone or in F12.ITS with DMSO (the diluent for retinoic acid). After 7 d the explants were retrieved,
freeze-dried, vapor-fixed, and embedded in resin. Endocrine cell types were identified by immunocytochemistry. The numbers
of insulin cells were expressed as a proportion of the sum of insulin plus glucagon cells. Retinoic acid had a dose-related
effect; the proportion of insulin cells in explants treated with the lower dose of retinoic acid (10−6
M) was more than twice the proportion of insulin cells in explants treated with the higher dose (10−5
M) of retinoic acid and more than three times that of the control grafts. 相似文献
10.
Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media 总被引:3,自引:0,他引:3
David Danielpour Terry L. Riss Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(1):42-52
Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated
DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When
ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate
was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect
on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors
in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal
growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium
equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth
rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion
may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.
This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255. 相似文献
11.
Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions 总被引:1,自引:0,他引:1
Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/106 cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/106 cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/106cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media. 相似文献
12.
Both retinoic acid (RA) and transforming growth factor (TGF)-beta1 are known to be influential in the development of insulin cells. Respectively, they increase and decrease the proportion of insulin cells when added to cultures of embryonic chick dorsal pancreatic buds. The aim of this study was to define the action of RA in the presence of decreased levels of TGF-beta1, as are found in growth factor-reduced Matrigel (GFRM), on the proportion of insulin cells. The endodermal component of 5-d chick dorsal pancreatic buds was explanted on to GFRM. Retinoic acid (10(-6) M) was added to Ham's F12 culture medium containing insulin (5 microg/ml), transferrin (5 microg/ml), and selenium (10(-10) M) (F12.ITS). Control explants were cultured in F12.ITS alone or in F12.ITS containing dimethyl sulfoxide (DMSO). After 7 d in culture, insulin and glucagon cells were localized immunocytochemically; changes in numbers of insulin cells were expressed as a percentage of insulin plus glucagon cells. Medium containing RA or DMSO increased the proportion of insulin cells significantly compared with the proportion in the explants cultured in F12.ITS medium alone. 相似文献
13.
Ananth Parampalli Kent Eskridge Leonard Smith Michael M. Meagher Mark C. Mowry Anuradha Subramanian 《Cytotechnology》2007,54(1):57-68
A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate
reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared,
which was similar in composition to HAM’s F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture.
The vitamin concentration of the basal medium was twice that of HAM’s F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody
grew from 2 × 105 cells to maximum cell density of 1.04 × 106 cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction
among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino
Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two
controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration
showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2×)
EAA, 1.4 mM (0.5×) NEAA, 1× ITS supplement, 0.7× Lipids supplement. The maximum viable cell density attained in the optimized
medium was 1.4 × 106 cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 ± 3.4 μg/mL, a 50% improvement. 相似文献
14.
Terry L. Riss Betty H. Stewart David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1989,25(2):127-135
Summary The growth of GH4C1, GH3, GH1, and GH3C15 rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1∶1 (vol/vol) mixture
of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 μg/ml gentamicin supplemented with 10 μg/ml bovine insulin,
10 μg/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T3), 50 μM ethanolamine (Etn), and 500 μg/ml bovine serum albumin. Of the lines evaluated, only the GH1 failed to grow in TRM-1. Passage of the GH4C1 and GH3 lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher
rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations
of T3 (1.0 to 10 nM). After adaptation of the GH4C1 line in TRM-1 for ≤20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 μ/ml gentamicin, 10 μg/ml Tf, 10 nM T3, and 50 μM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was
lost progressively. When T3 was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only
slightly stimulated by T3. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary
cell populations that a) required both serum-factor(s) and T3 for optimum growth, b) required supraphysiologic concentrations of T3 without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented
only with Tf and nutrients without necessity of other serum factor(s) or T3.
This work was supported by grants CA-26617 and CA-38024 from the National Cancer Institute, Bethesda, MD, American Cancer
Society grant BC-255, and grant 2225 from the Council for Tobacco Research, Inc., USA. 相似文献
15.
Jay S. Morrow Bruce C. Weintraub Saul W. Rosen 《In vitro cellular & developmental biology. Plant》1981,17(5):421-426
Summary The HeLa-S3 cell strain grown in Ham's F12 medium supplemented with insulin, transferrin, cortisol, epidermal growth factor, fibroblast
growth factor, and trace elements, but containing no serum, continued to produce the common α-subunit of the glycoprotein
hormones for the 10 d study. The amounts of α-subunit secreted into the medium during the first 4 d were indistinguishable
from those in F12 medium supplemented with 10% fetal bovine serum. During the remainder of the experiment the amounts of α-subunit
reached 50 to 80% those in the serum-supplemented medium. 相似文献
16.
Delano V. Young Michael C. Dean Peter Heit Stewart D. Chipman 《In vitro cellular & developmental biology. Plant》1980,16(11):949-957
Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated
on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that
has recently been characterized as containing as its major agent, biotin.
To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological
concentrations (200 to 500 ng/ml) and transferrin (5×10−8
M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are
nearly as growth enhancing as 10% serum.
The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization.
Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr
population doubling time, 1×106 cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density
(1.5×106 cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth
individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME
supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation
(14-versus 12-hr population doubling time, 1 to 2×106 versus 2 to 3×106 cells final cell density).
This work was supported by NIH Grant CA 20040. 相似文献
17.
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
18.
Frank C. Praeger Vincent J. Cristofalo 《In vitro cellular & developmental biology. Plant》1986,22(6):355-359
Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated
Ca2+ concentrations was investigated. At 5 mM CaCl2, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number
at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl2 or epidermal growth factor (1 mM CaCl2) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor
and CaCl2 resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth
in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to
elevated Ca2+ concentrations. In fact, elevated Ca2+ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their
response to the combined treatment of insulin, transferrin and dexamethasone.
This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378.
Editor's Statement This paper rigorously dissects the interplay among external Ca2+ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan 相似文献
19.
Various polypeptide growth factors, culture substrates, basal media, sera and further supplements were assayed for improvement of growth of human vascular endothelial cells from umbilical cord veins. The resulting optimized medium consisted of gelatinized culture substrates, a mixture (1:1) of Iscove's MDM and Ham's F12 basal media supplemented with 20% newborn calf serum, 500 ng/ml crude fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 g/ml transferrin, 5 g/ml insulin and 10 g/ml heparin. The medium allowed long term cultivation of HUVEC up to 45 generations with maximal cell densities of about 105 cells per cm2 and a minimal doubling time of about 14 hours at low cell densities.Abbreviations HUVEC
Human Endothelial Cells From Umbilical Cord Veins
- FGF
Fibroblast growth factor
- EGF
Epidermal Growth Factor
- FCS
Fetal Calf Serum
- NCS
Newborn Calf Serum
- HBS
HEPES-Buffered Saline
- ECM
Extracellular Matrix
- LHM
Peptide PyroGlu-His-Ser-Phe-Thr-Ile-Lys-Ile-ThrCONH2
- IF
1:1 mixture of Iscove's MDM and F12 basal media 相似文献
20.
Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age)
have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained
in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics
(penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml;
fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO2: 95% air.
The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning
and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and
interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria,
rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive
staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.
This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute. 相似文献