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1.
The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.Abbreviations BSA
bovine serum albumin
- CS
calf serum
- DMEM
Dulbecco's modified Eagle's medium
- ELISA
enzyme-linked immunosorbant assay
- McAb
monoclonal antibody
- PEG
polyethylene glycol
- SFM
serum-free medium 相似文献
2.
A serum-free medium that supports the growth of cultured skeletal muscle satellite cells 总被引:2,自引:0,他引:2
Ronald E. Allen Michael V. Dodson Lynda S. Luiten Linda K. Boxhorn 《In vitro cellular & developmental biology. Plant》1985,21(11):636-640
Summary A serum-free medium has been devised that supports the proliferation and differentiation of primary cultures of rat skeletal
muscle satellite cells for up to 4 d. The medium consists of a mixture of Dulbecco's modified Eagle's medium and MCDB-104
plus insulin, dexamethasone, pituitary fibroblast growth factor, Deutsch fetuin, and linoleic acid. In addition to promoting
the formation of myotubes from satellite cells, a decrease in fibroblast contamination of these cultures was observed when
cultures grown in serum-free medium were compared to cultures grown in serum-containing medium.
This work was supported by the Arizona Agriculture Experiment Station, Project No. R11, U.S. Public Health Service Grant R01
AG03393, Lilly Research Laboratoires, and Merck Institute for Therapeutic Research. This communication is Arizona Agriculture
Experiment Station Journal Paper No. 3966. 相似文献
3.
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
4.
Masatoshi Togami Kosei Yasumoto Tokujiro Yano Teruyoshi Ishida Genki Kimura Keizo Sugimachi Kikuo Nomoto 《Cytotechnology》1991,6(1):39-47
We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (3H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM
Iscove's Modification of Dulbecco's Medium
- rIL-2
recombinant Interleukin
- LAK
Lymphokine-Activated Killer
- RLNL
Regional Lymph Node Lymphocytes
- PBL
Pheripheral Blood Lymphocytes
- PBS
Phosphate-Buffered Saline
- RBC
Red Blood Cells
- RPMI-AB
RPMI 1640 medium supplemented with 10% human AB-type serum
Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan 相似文献
5.
Martine Chessebeuf Prudent Padieu 《In vitro cellular & developmental biology. Plant》1984,20(10):780-795
Summary Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids
adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver
functions; induction ofl-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary
bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and α-muricholic acid specific of the rat bile. 相似文献
6.
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
7.
Adaptation of mammalian cells to growth in serum-free media 总被引:5,自引:0,他引:5
A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage
to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of
a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated
cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series
of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density
conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances
released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells. 相似文献
8.
D. Gaillard R. Négrel G. Serrero-Davé C. Cermolacce G. Ailhaud 《In vitro cellular & developmental biology. Plant》1984,20(2):79-88
Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike
cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin,
transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium
is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet
extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other
established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular
fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these
cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert
to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition
of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements
for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described.
This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant
4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la
Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006). 相似文献
9.
J. Fantini J. P. Galons B. Abadie P. Canioni P. J. Cozzone J. Marvaldi A. Tirard 《In vitro cellular & developmental biology. Plant》1987,23(9):641-646
Summary Human colonic adenocarcinoma cells have been successfully grown on polystyrene microcarriers by modifying the culture conditions
used in monolayer culture. The method can be divided into two culture phases: a) a phase of spreading, wherein cells were
seeded in presence of serum-supplemented medium; b) a phase of active growth wherein spread cells on the beads were allowed
to grow in a serum-free medium. Under these conditions, optimal spreading and growth of HT 29 and HRT 18 cells on the microcarriers
were obtained. A differential propagation was observed between HT 29-D4 and HT 29-D9 cells (both clonal populations derived
from HT 29 cells) on the microcarriers that is tentatively related to the discrepancy observed in the spreading efficiency
of these clonal cells on serum-coated culture flasks. An index of spreading efficiency (IS index) has been defined to quantify
the efficiency of spreading of each cell line on microcarriers. These data gave the opportunity to develop serum-free, scale-up
methods to culture cells like HT 29 which release potentially useful products.
This work was supported by CNRS (U.A. 202 and U.A. 1186), Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC),
INSERM (CRE, no 847006), CNAMTS-INSERM (8386), MRT (GBM 85M0564) and l'Association pour la Recherche sur le Cancer (ARC 86-234). 相似文献
10.
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
11.
A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of
the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine
and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone
supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free
long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences
in final cell density compared to controls cultivated with serum.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
12.
A simple medium for the study of hepatocyte growth in culture under defined conditions 总被引:3,自引:0,他引:3
Tor-Erik Sand Thoralf Christoffersen 《In vitro cellular & developmental biology. Plant》1988,24(10):981-984
Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable
conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated
by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and
cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation
and plating.
This work was supported by the Norwegian Cancer Society. 相似文献
13.
Michael H. Simonian Mark L. White David A. Foggia 《In vitro cellular & developmental biology. Plant》1987,23(4):247-252
Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium
and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life
span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings
in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability
to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and
also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared
to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized
for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with
8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines
produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were
similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented
medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and
its relation to differentiated function for this cell culture system.
This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes
of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health
(grant HL07485). 相似文献
14.
骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用 总被引:2,自引:0,他引:2
本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。 相似文献
15.
Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium 总被引:11,自引:0,他引:11
Robert E. Lanford Kenneth D. Carey Larry E. Estlack G. Con Smith Rick V. Hay 《In vitro cellular & developmental biology. Plant》1989,25(2):174-182
Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability
and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed
that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating
cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and
numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium
by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [35S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling
of cells with [35S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being
secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and
low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B
h
, E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation
of lipoprotein production by hepatocytes.
This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972
from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of
Health (HL 15062), the American Heart Association, and the Louis Block Fund. 相似文献
16.
A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells 总被引:1,自引:0,他引:1
Louise Malan-Shibley P. Thomas Iype 《In vitro cellular & developmental biology. Plant》1983,19(10):749-758
Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium
(SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth
factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media
facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would
attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed.
Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free
medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented
medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings
per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM
may be useful in studies of the regulation of cell proliferation and differentiation.
This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc.
The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services,
nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government. 相似文献
17.
Methylcellulose was found to protect serum-free cultured cells from the deleterious effects of freezing and thawing. We have formulated a simple medium suitable for freezing serum-free cultured cells that consists of 0.1% methylcellulose, 10% dimethylsulfoxide, and MEM or any other serum-free culture medium. 相似文献
18.
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
19.
Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity. 相似文献
20.
We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS. 相似文献