共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
In order to develop a new protein-free cell culture system, microcarriers immobilized with insulin were synthesized. For the synthesis, glass and polyacrylamide beads were treated for the introduction of amino groups on the surface, and insulin was immobilized on the surface by using several method. Anchorage-dependent cells. mouse fibroblast cells STO and fibroic sarcoma cells HSDM(1)C(1), and the anchorage-independent cells, mouse hybridoma cells SJK132-20 and RDP 45/20 were cultivated on the microcarriers immobilized with insulin. The insulin-immobilized microcarriers did not have any effect on the proliferation of the anchorage independent cells but promoted the growth of anchorage-dependent cells remarkably. The activity of immobilized insulin was larger than that of free or adsorbed insulin. The repeated use of the insulin-immobilized microcarrier was possible, and the promotion activity in the the repeated use was greater than that in the use. (c) 1992 John Wiley & Sons, Inc. 相似文献
3.
Serum-free culture of rat keratinocytes 总被引:2,自引:0,他引:2
Hirosuke Oku Chikara Kumamoto Tomoyuki Miyagi Takanori Hiyane Junichi Nagata Isao Chinen 《In vitro cellular & developmental biology. Animal》1994,30(8):496-503
Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn
rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally
for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes,
bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine
serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support
rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors
and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2
mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth
of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and
were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately
the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study
focusing on lipid metabolism or hormonal regulation of rat keratinocytes. 相似文献
4.
Tracy A. Tebb Shiao-Wen Tsai Veronica Glattauer Jacinta F. White John A. M. Ramshaw Jerome A. Werkmeister 《Cytotechnology》2006,52(2):99-106
A method for the preparation of bioresorbable collagen beads with an open porous structure is presented. These beads were
prepared from collagen-alginate composite beads by removal of the alginate component. These collagen beads were suitable for
rapid proliferation of chondrocytes in a dynamic, spinner culture system. Histology and immuno-histology showed that biochemical
markers of chondrocytes are present during this cell proliferation, indicating that there was control of phenotype and that
cell de-differentiation had not occurred. Unlike other 3-D scaffolds that have been used, the cells were amplified from very
low cell densities and were able to proliferate freely without loss of phenotype.
Tracy A. Tebb, Shiao-Wen Tsai, Veronica Glattauer and Jacinta F. White have made equal contributions to this study. 相似文献
5.
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
6.
Yoshiko Myoken Yoshinari Myoken Tetsuji Okamoto Mikio Kan J. Denry Sato Kazuaki Takada 《In vitro cellular & developmental biology. Animal》1994,30(11):790-795
Summary A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast
growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular
mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity
FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [125I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense
oligonucleotides at 25 μM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent
and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was
suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 μg/ml. These results demonstrate
that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1
binding sites on Nakata cells. 相似文献
7.
Violet Albert David Barkla Graeme P. Young 《In vitro cellular & developmental biology. Animal》1994,30(7):443-449
Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling
rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory
hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in
serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when
cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640
plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium.
Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture.
However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after
48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the
rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II,
des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity
of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat
jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological
responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None
of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal
epithelium. 相似文献
8.
A packed bed bioreactor was investigated as means for the cultivation of mammalian cells. The packed bed is comprised of porous ceramic particles with pores sufficiently large for cell immobilization as well as for intraparticle convective flow. In this way, the transport of limiting nutrients such as oxygen can be significantly enhanced, allowing maintenance of cell viability and productivity in an environment protective of adverse shear effects. The extent of intraparticle convective medium flow was experimentally quantified relative to the reactor operating conditions, and was found to be the dominant mechanism of nutrient transport to cells immobilized in the particle interior. An approximate linear relationship was obtained between overall reactor productivity and the extent of intraparticle convection. As the latter can be controlled at the single-particle level through total flow rate control, this relationship is a useful scale-up tool for the design of bioreactors. The high cell densities and the high volumetric productivities achieved by using small lab-scale reactors underline the potential of this simple bioreactor configuration for large-scale cell culture applications. (c) 1993 John Wiley & Sons, Inc. 相似文献
9.
10.
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
11.
The in vitro culture of human hematopoietic cells has many research and therapeutic applications. Traditionally, human hematopoietic cultures have been conducted using serum-containing media. The disadvantages inherent in the use of serum could be eliminated by the use of serum-free media. In this review, we summarize and discuss the current status of serum-free media for both mature and immature human hematopoietic cells. The mature hematopoietic cells discussed are of lymphoid (e.g., lymphokine activated killer cells and tumor infiltrating lymphocytes) and myeloid origin (e.g., monocytes/macrophages). The cultures of immature hematopoietic cells discussed are clonogenic and long-term cultures. In addition, we briefly review the types of human hematopoietic cells, their clinical applications, and the basic strategies and components used to formulate serum-free media, Finally, we outline future requirements and directions in the development of serum-free media for primitive hematopoietic cells. 相似文献
12.
Serum-free culture of AtT 20 pituitary cells: A system for neuroendocrine studies under defined conditions 总被引:2,自引:0,他引:2
Helmut Vedder 《In vitro cellular & developmental biology. Plant》1990,26(11):1068-1072
Summary The growth of the mouse pituitary cell line AtT 20 was studied under different in vitro conditions. A completely defined,
serum-free culture medium supported the survival of cells for a period of more than 2 mo. The medium, designed SFI, consisted
of basal medium supplemented with transferrin, insulin, putrescine, and selenium. For maintenance of cells during long-term
culture, no additional compounds were necessary. The time-dependent increases in cell number during culture with fetal bovine
serum (FBS) and under serum-free conditions showed similar properties. Analysis of the effects of different substrata on cell
growth demonstrated that polylysine supported adhesion and initial growth of cells to a greater extent than untreated plastic
or FBS adsorbed to culture dishes. Synthesis and regulation of proopiomelanocortin (POMC)-mRNA, the precursor-mRNA of adrenocorticotropin
(ACTH), could be detected by Northern blot analysis under basal conditions and after incubation with steroids and corticotropin-releasing
hormone (CRH), indicating the serum-independent expression of important cellular properties. 相似文献
13.
Sakae Satoh Junji Kobayashi Junzo Mizoguchi Makoto Nogawa Masaru Otani 《Cytotechnology》1991,5(Z2):95-114
For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 107 cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established. 相似文献
14.
Shiqing Wang Sandra Z. Haslam 《In vitro cellular & developmental biology. Animal》1994,30(12):859-866
Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of
mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined
serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells,
fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit
good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In
contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone.
In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells.
These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The
media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard
to hormone and growth factor regulation of their growth and differentiation. 相似文献
15.
Hormonal growth control of cells in culture 总被引:15,自引:0,他引:15
Summary Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide
hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH3, can grow in serum-free medium if the medium is supplemented with 3,3′,5-triiodothyronine, TSH-releasing hormone, transferrin,
parathyroid hormone, insulin and three isoelectric focusing fractions of blood meal. The blood-meal components can be replaced
by fibroblast growth factor and somatomedin C. The growth rate of GH3 cells in hormone-supplemented serum-free medium is equal to that in serum-supplemented medium, and subculture in such medium
is also possible. These results indicate that the replacement of the serum component is complete in the GH3 system. The hormonal requirements of GH3 cells and those of HeLa and mouse melanoma, M2R, were compared. Two generalizations could be made: (a) All three cell lines
require insulin and transferrin. (b) There is a requirement for a hormone which localizes in the nucleus for each cell line.
These generalizations seem to hold true for most of the other cell lines for which the hormonal requirements have been partially
worked out. Since insulin is one of the universally required hormones, its effects on GH3, HeLa and M2R were compared. Insulin stimulates glycogen synthesis in all three cell lines and facilitates fatty-acid synthesis
in GH3 and M2R. However, there is a difference in the effect of insulin on growth among the three cell lines. Insulin is an absolute
requirement for GH3 cells without which the cells cannot survive, whereas this is not the case for HeLa and M2R. The most stringent requirement
for HeLa cells is for hydrocortisone, and for M2R, it is for transferrin. These results indicate that even though the necessity
for some hormones is common, the degree of requirement may vary from one cell line to another. Whether this difference reflects
the difference in the primary mode of action of the hormone on each cell type needs further investigation.
Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture
Association, New Orleans, Louisiana, June 6–9, 1977.
This work was supported by NIH Grant GM 17019. J. Larner was supported by Josiah Macy Foundation 相似文献
16.
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
17.
Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture,
and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free
medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous
growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine
growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced
it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal
cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free
medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and
large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
18.
Hodaka Ikeda 《In vitro cellular & developmental biology. Plant》1990,26(2):193-200
Summary Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete
medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared
with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced
larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but
no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic
agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period
of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen
gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about
three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without
collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium
on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary
culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen
gel matrix. 相似文献
19.
20.