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1.
Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum 总被引:5,自引:0,他引:5
Richard G. Ham Judy A. St. Clair Cecelia Webster Helen M. Blau 《In vitro cellular & developmental biology. Plant》1988,24(8):833-844
Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized
nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin,
dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS)
as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final
medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo
or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both
SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer
growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little
differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of
increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's
medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were
always those of muscle cells.
This research was supported by a grant from the Muscular, Dystrophy Association.
Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements
for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well
as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy. 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.
Development of a new serum-free medium,USC-HC1, for growth and normal phenotype in postembryonic chicken growth plate chondrocytes 总被引:1,自引:0,他引:1
Laura V. Hale John E. Hale Mary Lynn S. Kemick Yoshinori Ishikawa Roy E. Wuthier 《In vitro cellular & developmental biology. Plant》1986,22(10):597-603
Summary A serum-free medium for postembryonic chicken epiphyseal growth plate chondrocytes has been developed from 104 MCDB medium.
To enable these fastidious cells to survive, grow, and express normal phenotype, a substantial increase over MCDB 104 in the
level of many of the amino acids was required, as well as a change in the buffer system and the addition of SerXtend, a defined,
serum-free product containing various growth factors, including fibroblast growth factor. Also required was the provision
of cell attachment factors, either by coating culture surfaces with type II collagen, or better, by allowing the freshly released
cells to recover for several hours in a medium supplemented with 10% fetal bovine serum before plating. Ths new serum-free
medium, which we call USC-HC1, supports growth and replication, the retention of normal polygonal morphology, the expression
of significant levels of cellular alkaline phosphatase activity, the production of sulfated proteoglycans, type II collagen,
and the formation of alkaline phosphatase-rich matrix vesicles by the chondrocytes. The major advantage of USC-HC1, however,
is that it will provide for the first time an opportunity to examine the effects of various defined growth and hormonal factors
on the phenotypic expression and differentiation of growth plate chondrocytes, in the absence of the variable (stimulatory
and inhibitory) factors present in fetal bovine serum.
This work was supported by grant AM18983 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases,
Bethesda, MD. 相似文献
4.
Dr. Donna M. Peehl Richard G. Ham 《In vitro cellular & developmental biology. Plant》1980,16(6):526-540
Summary A survey of commercially availabla media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal
keratinocytes (HK) when supplemented with 10 μg/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather
than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal
clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 μg/ml
HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly
from MCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth
of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of
calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal
growth of HK occurs at a very low level of Ca2+ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium.
However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the
Ca2+ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDB 151 with
10 μg/ml HC and 1.0 mg/ml FBSP.
This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna
M. Peehl in partial fulfillment of the requirements for the Ph.D. degree.
This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute
on Aging. 相似文献
5.
David G. Chilton Betty H. Johnson Laurence Danel-Moore Simon Kawa E. Brad Thompson 《In vitro cellular & developmental biology. Plant》1990,26(6):561-570
Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously
cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium
of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine
serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar
to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for
3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell
morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived
lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited
increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine
synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be
completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially
sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone
in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results
suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid
hormone action may be pursued more precisely in a clearly defined culture medium.
This work was conducted in conjunction with the Walls Medical Research Foundation. 相似文献
6.
The requirements for the serum-free culture of PC13 murine embryonal carcinoma cells were determined. Supplementation of a 50:50 mixture of Dulbecco's modified Eagles medium and MCDB104 with transferrin (5 μg/ml), human high-density lipoprotein (HDL) (100 μg/ml), and human low-density lipoprotein (LDL) (50 μg/ml) supported growth comparable to that observed with 5% foetal calf serum. Media supplementation with lipoproteins apparently substitutes for the effects of insulin, desoctapeptide insulin (DOP), or multiplication-stimulating activity (MSA) on EC cell multiplication. Clonal growth of PC13 EC cells in this serum-free medium could only be achieved in the presence of suitable feeder cell monolayers. These observations demonstrate that PC13 EC cells do not have an absolute requirement for exogenous mitogens to support multiplication. 相似文献
7.
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
8.
Protein-free medium for C-1300 mouse neuroblastoma cells 总被引:2,自引:0,他引:2
Peter C. Agy Gary D. Shipley Richard G. Ham 《In vitro cellular & developmental biology. Plant》1981,17(8):671-680
Summary An optimized medium, designated MCDB 411, has been developed for mouse neuroblastoma cells. At cell densities of 1×104 cells/cm2 or greater, several different clones of C1300 mouse neuroblastoma cells can be cultured serially in Medium MCDB 411 with
no serum or other undefined supplementation and with a doubling time of about 24 h. At clonal densities it is necessary to
supplement the medium with 1.0 μg/ml insulin. Alternately, good clonal growth can be obtained without direct supplementation
by coating the culture dishes with insulin and rinsing off all that is not tightly bound. Primary cultures of cells from serially
transplanted C1300 tumors that have never been cultured previously in vitro can be established directly in unsupplemented
Medium MCBD 411 with rapid initiation of multiplication and no apparent crisis or selection for minority cell types. The availability
of a synthetic medium that supports growth of neuroblastoma cells without supplementation should facilitate the use of these
cells as model systems for the study of neuronal function and differentiation.
This work was supported by Grant CA-15305 from the National Cancer Institute. 相似文献
9.
Linda J. Loretz Catherine A. Reznikoff 《In vitro cellular & developmental biology. Plant》1988,24(4):333-342
Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells
(HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency
(CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded
in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×105 lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum.
Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions
in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining
for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder
cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone
supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented
MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%.
This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral
fellowship. 相似文献
10.
Hiroyoshi Hoshi Yuji Takagi Keizo Kobayashi Masakazu Onodera Taneaki Oikawa 《In vitro cellular & developmental biology. Animal》1991,27(7):578-584
Summary We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated
culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2),
lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent
cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of
fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or
BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed
in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After
granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling
level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations
(14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation
of bovine granulosa cells. 相似文献
11.
Modification of MCDB 110 medium to support prolonged growth and consistent high cloning efficiency of diploid human fibroblasts 总被引:6,自引:0,他引:6
In preparation for studies on the growth factor requirements of normal and transformed human fibroblasts, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human fibroblasts and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment factors instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal growth factor or platelet-derived growth factor. In this modified serum-free medium, which we have designated McM+SR1, diploid human fibroblasts grew for 21 days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., 21 population doublings). During the next 20 days, they underwent 15 population doublings which was 75% of the rate of cells growing in the medium containing serum. 相似文献
12.
Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum 总被引:8,自引:0,他引:8
Summary An optimized basal nutrient medium, MCBD 131, has been developed that supports clonal growth of human microvascular endothelial
cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth
factor (EGF) and 1 μg/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized
for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment
of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of
MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most
unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg2+ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can
be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS.
This research was supported by grant CA 15305 from the National Cancer Institute, Bethesda, MD. 相似文献
13.
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
14.
C. H. Uittenbogaart Y. Cantor J. L. Fahey 《In vitro cellular & developmental biology. Plant》1983,19(1):67-71
Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm
(up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with
bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM.
Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained
their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines
JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components
should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.
This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los
Angeles, and CA 09120 (C. U.) 相似文献
15.
Masatoshi Togami Daphne Blazka Jun Hayashi 《In vitro cellular & developmental biology. Plant》1988,24(7):699-704
Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined
medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite),
H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing
culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various
hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid
hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert
their effect on angiotensinogen production had little or no effect.
This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD. 相似文献
16.
17.
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
18.
We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state. 相似文献
19.
A cloned rat thymic epithelial cell line established from serum-free selective culture 总被引:6,自引:0,他引:6
Arthur Piltch Paul Naylor Jun Hayashi 《In vitro cellular & developmental biology. Plant》1988,24(4):289-293
Summary A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells. The growth
media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor. Cultures
have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established. These
cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin α markers for thymic
endocrine cells.
This work was partly supported by grant PCM-834 0582 from the National Science Foundation, Washington, DC, and grant P01 CA
37589-2 from the National Cancer Institute, Bethesda, MD. 相似文献
20.
Asish C. Nag Mei Li Lee James R. Kosiur 《In vitro cellular & developmental biology. Plant》1990,26(5):464-470
Summary A culture system for adult rat cardiac muscle cells has been established without exposure of cells to serum at any step of
the procedure. The methodology has been standardized and optimized to obtain better quality and high yield of cells and culture.
Subsequent to enzyme perfusion, the release of myocytes from enzyme-perfused tissues was carried out in enzyme-free Joklik's
medium instead of exposing cells to proteolytic enzyme(s) as done previously. Approximately 5 million cylindrical muscle cells
per ventricle were obtained. The culture medium contained Eagle's minimum essential medium with Earle's salts, basic fibroblast
growth factor, epidermal growth factor, insulin, transferrin, selenium, norepinephrine, triiodothyronine (T3), bovine serum albumin, nonessential amino acids, and ascorbic acid. The plating efficiency of the experimental cultures
was comparable to that of the control cultures grown in the presence of serum. The cells in the serum-free medium contained
myofibrillar and myosin isoforms characteristics of the adult myocytes. The cells underwent cellular reorganization comparable
to that of the controls. The initial phase of reorganization involved the breakdown of myofibrils and extrusion of mitochondria,
degraded myofibrils, and other cellular organelles. The latter phase of reorganization included myofibrillogenesis and organellogenesis
resulting in the development of myofibrillar apparatus with cellular organelles. Myocytes were contractile throughout the
culture period. Cardiac myocytes grown, in serum-free medium expressed the predominant myosin isoform V1 similar to their
counterparts in vivo. T3 is essential for the expression of isomyosin V1. This study demonstrates that adult cardiac muscle cells can be maintained
in long-term serum-free culture from seeding to termination. The cells in serum-free conditions maintain at least two differentiated
characteristics of adult myocytes investigated, namely, abundant organized myofibrils and predominant myosin isoform V1.
This work is supported by grant DCB-8709594 from the National Science Foundation, Washington, DC 相似文献