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1.
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
2.
《Cytotherapy》2014,16(7):915-926
BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes. 相似文献
3.
Lin-Chang Chiang Janet Silnutzer James M. Pipas David W. Barnes 《In vitro cellular & developmental biology. Plant》1985,21(12):707-712
Summary NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth
factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL
results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with
SV40, the polyomavirus middle T antigen gene, the activated humanras gene, and the mouse c-myc gene.
This work was supported by grants ES-00-210 from the National Institutes of Health (NIEHS) and MV-182 from the American Cancer
Society.
Dr. L.-C. Chiang is a Visiting Research Scientist from PPG Industries, Inc.
Editor’s statement This paper represents a new approach to the identification of oncogenes that would escape the screening
methods currently in use. Inherent in the method is the assignment of function to oncogenes. 相似文献
4.
The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can b e carried out without the use of any serum.The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can be carried out without the use of any serum. 相似文献
5.
Angie Rizzino 《In vitro cellular & developmental biology. Plant》1984,20(10):815-822
Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of
non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to
grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background
activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot
used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases
the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of
the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free
medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and
high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains
insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no
colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus,
the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors,
including serum factors, for TGF activity.
Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The
techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined
detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also
should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent
conditions. D. W. Barnes 相似文献
6.
Hamster tracheal organ culture in serum-free media: A quantitative comparison of in vitro epithelial morphology with that of in vivo controls 总被引:1,自引:0,他引:1
Robert E. Sigler Raymond T. Jones John R. Hebel Elizabeth M. McDowell 《In vitro cellular & developmental biology. Plant》1987,23(2):100-110
Summary The epithelial morphology of the hamster trachea in serum-free organ culture was compared with that of age-matched in vivo
control tissues by collecting and statistically analyzing several quantifiable parameters. By this technique it was possible
to detect both subtle and dramatic epithelial alterations. Midtracheal tissues from 6-wk-old male Syrian golden hamsters were
used as the explants. Explants were placed on Gelfoam sponges and cultured for 1, 2, and 3 wk in CMRL 1066 alone and in CMRL
1066 to which seven factors were added: insulin and transferrin (5 μg/ml); hydrocortisone (5×10−7
M); epidermal growth facotr (5 ng/ml); bovine pituitary extract (0.5%); and phosphoethanolamine and ethanolamine (5×10−5
M). The following data were collected and statistically analyzed for each tracheal ring: number of epithelial cells; proportion
and number of each cell type; basement membrane length; linear density of epithelial cells; epithelial height; and mitotic
index.
Compared to controls, ciliated cells decreased by 52% during washes in Leibovitz (L15) medium and tissue manipulation performed
before culture and this loss persisted after cutlure for 1 wk. Explants culturedwithout the factors showed marked changes after 2 and 3 wk including epithelial thickening and folding, which was associated with
increased linear density. Many cells in these specimens could not be categorized by type (22% were unidentifiable after 3
wk). Epithelial migration onto the outside of the explant was inhibited. In contrast, explants culturedwith the factors maintained a morphology similar to controls at 2 and 3 wk and epithelial migration onto the outside of the explant
was supported. This study shows that explants in CMRL 1066 with the seven factors provide a useful biological model for the
in vitro study of the mucociliary respiratory epithelium.
This work was supported by grant HL24722 from the National Institutes of Health, Bethesda, Maryland. 相似文献
7.
Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary
to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems
used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have
been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The
Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale
production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been
evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target
protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information
needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3,
an SFM developed in our laboratory, as a CHO culture medium. When CHO cells were cultured in MED-3 instead of an SSM, several
genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like
growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several
key supplements-including insulin, transferrin, ethanolamine, and selenium-were removed from MED-3, theIGF expression was consistently down-regulated and cell growth decreased proportionately. Based on these results, we concluded
that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain
a high level ofIGF expression. The data presented in this study, therefore, might provide useful information for the design and development
of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally
for protein production in pharmaceutical and biomedical research. 相似文献
8.
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
9.
Richard Fike Barbara Dadey Richard Hassett Robert Radominski David Jayme David Cady 《Cytotechnology》2001,36(1-3):33-39
To overcome limitations of conventional milling technology, we investigated the application of fluid bed granulation for the
production of dry-form nutrient media. Serum-free, protein-free and chemically-defined specialty media were produced in granulated
format and compared with identical formulations manufactured by conventional methods. HPLC analysis of multiple lots of granulated
materials demonstrated that biochemical constituents were precisely and homogeneously distributed throughout the granules
and that nutrient levels were comparable to conventional formats. Comparison of medium performance in cell proliferation and
biological production assays demonstrated equivalence with reference media. The fluid bed granulation process meets pharmaceutical
quality requirements and may be applied to a broad range of nutrient formulations required for bioproduction applications.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
11.
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
12.
研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。 相似文献
13.
Summary A new culture method was used to study increases in wet and dry weight and soluble protein during normal development of the
transparent lens. Seven different media with more than ten different additives were tested for their effects on cultured lens
transparency.In vivo, rat lenses increased 53% in soluble protein content between 3 and 5.5 days of age. Only HL-1 serum-free medium containing
15 μg/ml insulin plus 1–2 ng/ml BB platelet-derived growth factor (PDGF), or 5–7 ng/ml epidermal growth factor (EGF) allowed
similar growthin vitro, during the same time period. Normal lens grwoth occurred in culture when fresh medium was delivered to lenses as a pulse
every 4–6 hours. Lenses decreased in dry weight and soluble protein content, and became opaque when the same medium was delivered
continuously. Lenses increased only 26% and 32% in soluble protein content when delivered pulses of HL-1 medium containing
BB PDGF or EGF in the absence of insulin. We suggest that pulsatile delivery of medium containing insulin and PDGF or EGF
stimulates lens growth during developmentin vitro. This pulsatile organ culture system is presented as a new approach for studying the effects of growth factors on cell proliferation,
differentiation, and receptor regulation in a developing tissue.
This work was supported by grants from EY-07031 and EY-04542 from the National Eye Institute and a grant from the Oculon Corporation.
Editor's statement This report documents an in vitro system that may mimic lens development and response to growth regulators
and hormones. The system may be useful for application to other organs and provide a foundation for cell and molecular level
analysis. 相似文献
14.
人外周血活化淋巴细胞survivin基因的转录激活和表达相关的信号转导通路研究 总被引:4,自引:0,他引:4
Survivin是与细胞增殖和细胞凋亡调控密切相关的重要功能蛋白质,也是肿瘤临床诊断的分子标志物及治疗研究的理想靶标.由于发现人外周血活化淋巴细胞存在Survivin蛋白的显著表达,故以植物血凝素(PHA)和IL-2共刺激培养的正常人外周血单个核细胞为实验材料,采用RT-PCR、蛋白质印迹以及细胞周期分析等实验方法,观察淋巴细胞活化与Survivin蛋白表达之间的相互关系,并利用3种激酶抑制剂探索了淋巴细胞活化所致Survivin蛋白表达相关的信号转导通路.实验结果表明:人外周血单个核细胞在刺激培养36 h前后出现survivin基因的明显转录激活和蛋白质的起始表达,表达产物的水平随培养时间的延长而增加,且具有明显的细胞周期和周期时相依赖性.JAK2的抑制剂AG490阻断细胞周期的运行,且强烈抑制survivin基因的转录激活和蛋白质表达,PI3K和MEK的抑制剂Wortmannin和PD98059也有一定程度的抑制作用.所得实验结论是:survivin基因的转录激活和蛋白质表达与淋巴细胞活化相关联,代表细胞处在增殖状态.JAK2介导的JAK-STAT信号通路是淋巴细胞活化,Survivin蛋白表达必需和最重要的信号转导通路,PI3K和MEK介导的信号通路也具有一定的影响作用. 相似文献
15.
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
16.
Jack Litwin 《Cytotechnology》1992,10(2):169-174
Vero cell lines, usually considered anchorage-dependent, could be grown as cell-aggregates in suspension culture with serum-free media. Several different combinations of base media gave growth results above 106 cells/ml (NCTC 135:SFRE 199-1; NCTC 135:Waymouth MB 752/1; NCTC 135:RPMI 1640). Insulin was not essential for growth and Bovine Serum Albumin could be diluted out of the media if linoleic acid was present. The size and density of the aggregates formed varied depending on the media used. 相似文献
17.
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
18.
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
19.
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
20.
Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium 总被引:6,自引:0,他引:6
M. E. Kaighn R. F. Camalier F. Bertolero U. Saffiotti 《In vitro cellular & developmental biology. Plant》1988,24(8):845-854
Summary Mouse keratinocytes cultures readily develop into established cell lines without undergoing a “crisis” in a newly-developed
serum-free medium, LEP/MK2. LEP/MK2 consists of calcium-free MEM with non-essential amino acids supplemented with 8 factors.
Two lines, MK1 and MKDC4, have been isolated and have now doubled more than 400 and 200 times respectively. In MK1 cells,
Giemsa banding has revealed significant karyotypic changes as early as the 4th passage, leading to a near-tetraploid karyotype
with random loss and gain of individual chromosomes. Minute chromosomes, but no stable markers have been observed. After these
initial changes, examination of cultures at several passage levels has shown that the karyotype has remained essentially stable.
The MKDC4 line, also sub-tetraploid at the 7th passage, had 4 marker chromosomes by the 47th passage. The rapid increase in
chromosome number may have contributed to the “immortalization” of these lines.
The response of these established keratinocyte lines to growth factors and serum-derived inhibitors changed with increasing
passage level. Most notable of these changes were a reduction in the requirement for bovine pituitary extract (an absolute
requirement for growth of secondary MK1 cells) and a decreased sensitivity to serum and serum-derived inhibitors, e.g., transforming
growth factor-β. The established lines, like primary and secondary keratinocytes, remain responsive to calcium-induced terminal
differentiation and are non-tumorigenic in athymic, nude mice. This serum-free system is suitable for transformation studies
with oncogenes and chemical carcinogens.
Editor's Statement Keratinocytes are useful and important models for studies of carcinogenesis and tumor promotion and differentiation.
This paper provides a solid in vitro basis for examination of the cellular endocrinology of these phenomena in vitro, and
implicates TGF beta as a regulator of these cells. 相似文献