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1.
The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B. 相似文献
2.
In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI)
to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes
were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI
transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method).
The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression
levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the
ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several
different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained
with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA
concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared
to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared
to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1
(CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression
in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production
of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments,
however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
3.
W. F. Hink 《In vitro cellular & developmental biology. Animal》1991,27(5):397-401
Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for
fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell
viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching
cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts
of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented
medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium. 相似文献
4.
Lüdemann I Pörtner R Schaefer C Schick K Srámkova K Reher K Neumaier M Franěk F Märkl H 《Cytotechnology》1995,19(2):111-124
A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c
substrate or product concentration mmol l–1
- c0
substrate or product concentration in the feed mmol l–1
- cGlc
glucose concentration mmol l–1
- cGln
glutamine concentration mmol l–1
- cAmm
ammonia concentration mmol l–1
- cLac
lactate concentration mmol l–1
- cFAB
concentration of Fab# 10 antibody fragment g l–1
- cMAb
monoclonal antibody concentration mg l–1
- D
dilution rate d–1
- q
cell-specific substrate uptake or metabolite production rate mmol cell–1 h–1
- qGlc
cell-specific glucose uptake rate mmol cell–1 h–1
- qGln
cell-specific glutamine uptake rate mmol cell–1 h–1
- qMAb
cell-specific MAb production rate mg cell–1 h–1
- q*
volume-specific substrate uptake or metabolite production rate mmol l–1 h–1
- q*FB
volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol lFB
–1 h–1
- q*FB,Glc
volume-specific glucose uptake rate related to the fixed bed volume mmol lFB
–1 h–1
- q*FB,Gln
volume-specific glutamine uptake rate related to the fixed volume mmol lFB
–1 h–1
- q*FB,MAb
volume-specific MAb production rate related to the fixed volume mg lFB
–1 h–1
- q*FB,02
volume-specific oxygen uptake rate related to the fixed bed volume mmol lFB
–1 h–1
- t
time h
- U
superficial flow velocity mm s–1
- V
medium volume in the conditioning vessel of the fixed bed reactor l
- VFB
volume of the fixed bed l
- xv
viable cell concentration cells ml–1
- yAmm,Gln
yield of Ammonia from glutamine
- yLac,Glc
yield of lactate from glucose
-
specific growth rate h–1
- d
specific death rate h–1 相似文献
5.
Yasuhiro Tomooka Stephen E. Harris John A. McLachlan 《In vitro cellular & developmental biology. Plant》1985,21(4):237-244
Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained
in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically,
immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number
was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10
ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required
eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin,
and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free
media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides
a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells. 相似文献
6.
Michel Moenner Elissavet Hatzi Josette Badet 《In vitro cellular & developmental biology. Animal》1997,33(7):553-561
Summary The requirement of serum in cell culture is a major limitation for studies on secreted ribonucleases (RNases) because serum
contains a high amount of ribonucleolytic activity. Defined culture condition is thus of interest to improve our knowledge
of the RNase biology. We report here that cells from three different types and origins, Chinese hamster lung fibroblasts,
bovine smooth muscle cells, and human endothelium-derived EA.hy926 cells, proliferate consistently in the presence of a basal
medium supplemented with bovine serum albumin, high-density lipoproteins, basic fibroblast growth factor, insulin, and transferrin.
Using a new quantitative radio-RNase inhibitor assay, two distinct ribonucleolytic assays, and a radioimmunoassay against
angiogenin, it is shown that RNases became apparent in media conditioned by cell monolayers. Both the hamster lung fibroblast
and the EA.hy926 cell lines secreted larger amounts of RNase inhibitor-interacting factors and RNase activity than normal
smooth muscle cells. The serum-free medium represents an alternative way to grow these cells and allows investigation of biosynthesis
and functions of RNases in culture. It should be useful to identify and quantitate unambiguously specific members of the RNase
family secreted by normal versus tumor cells in culture. 相似文献
7.
Fetal rhesus monkey lung cells can be grown in serum-free medium for the replication of dengue-2 vaccine virus 总被引:1,自引:0,他引:1
Barbara Malewicz Lloyd E. Anderson Karan Crilly Howard M. Jenkin 《In vitro cellular & developmental biology. Plant》1985,21(8):470-476
Summary Serum-free media were developed to grow diploid fetal rhesus monkey lung (DBS-FRhL-2) cells and to propagate dengue-type 2
virus vaccine strain PR-159 (dengue-2 vaccine virus). Vitamins, amino acids, growth factors, hormones and other organic compounds,
and inorganic salts were substituted for fetal bovine serum. The composition of the medium that was optimal for growth of
DBS-FRhL-2 cells differed from medium optimal for the propagation of dengue-2 vaccine virus. Insulin, epidermal growth factor,
fibroblast growth factor, and platelet-derived growth factor were required for DBS-FRhL-2 cell proliferation in serum-free
medium but were inhibitory for virus propagation. Adenosine, cytidine, guanosine, uridine, and thymidine, each at 0.01 mM concentration, were necessary as medium supplements to obtain a high yield of dengue-2 vaccine virus in DBS-FRhL-2 cells
under serum-free conditions. DBS-FRhL-2 cells grown in serum-free medium produced dengue-2 vaccine virus with yields similar
to those of cells grown in the presence of serum. Dengue-2 vaccine virus obtained under serum-free conditions retained its
phenotypic markers such as temperature sensitivity and small plaque size.
This investigation was supported by Contract DAMD-17-81-C1029 from the U.S. Army Medical Research and Development Command,
and by The Hormel Foundation. 相似文献
8.
Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen 总被引:4,自引:0,他引:4
Nozomu Nishi Yuhsi Matuo Takahisa Nakamoto Fumio Wada 《In vitro cellular & developmental biology. Plant》1988,24(8):778-786
Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC
404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated
from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached
to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for
cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were
higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more
than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted
by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest
that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively,
of prostate epithelial cells in vivo. 相似文献
9.
Development of embryonic chick insulin cells in culture: Beneficial effects of serum-free medium, raised nutrients, and biomatrix 总被引:1,自引:0,他引:1
Summary A previous finding that insulin cells do not survive or differentiate in explants of embryonic avian pancreas cultured in
collagen gel with a serum-containing medium has provided a model system for identification of conditions favorable for development
of these cells. To this end, we here modify the substrate and the medium. The epithelial component of dorsal pancreatic buds
of 5-d chick embryos was cultured for 7 d on Matrigel in serum-containing and in serum-free medium, the latter incorporating
insulin, transferrin, and selenium, Endocrine cell types were distinguished by immunocytochemistry; insulin cell counts were
expressed as a proportion of insulin plus glucagon cells. With serum-containing medium, Matrigel stimulated a significant
increase in this proportion as compared with collagen gel—3.1% as against 0.2%; the serum-free medium further increased this
proportion to 17.3%. Raising the level of essential amino acids approximately fivefold increased the latter figure somewhat
(to 18.9%), but it was more than doubled (to 37.4%) by raising the glucose concentration from 10 mM to 20 mM. Raising the levels of amino acids and glucose simultaneously yielded a lesser increase (to 31.8%). Some cultures grown in
collagen gel and serum-containing medium for 7 d were transferred to Matrigel and serum-free medium for a further 7 d. Insulin
cell development recovered, indicating that progenitor cells had survived and were stimulated to develop by the improved conditions.
This study indicates that components of the biomatrix and the medium (in particular, a raised glucose concentration) are important
for the survival and differentiation of embryonic insulin cells. 相似文献
10.
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
11.
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
12.
Growth and differentiation of periodontal ligament-derived cells in serum-free defined culture 总被引:4,自引:0,他引:4
Tetsuji Okamoto Nobuhiro Yatsuzuka Yoshiharu Tanaka Mikio Kan Takenori Yamanaka Akihiko Sakamoto Takashi Takata Yasumasa Akagawa Gordon H. Sato J. Denry Sato Kazuaki Takada 《In vitro cellular & developmental biology. Animal》1997,33(4):302-309
13.
Carsten Ropke Bo van Deurs Ole W. Petersen 《In vitro cellular & developmental biology. Plant》1990,26(7):671-681
Summary Thymic epithelial cells were grown in defined medium without unknown serum factors and without concurrent growth of other
cell types. Thymic tissue was obtained from 1- to 4-wk-old mice, disaggregated, and incubated in a mixture of collagenase-dispase-DNAse.
The resulting organoids were seeded on collagen-coated flasks. The culture medium consisted of DME-F12 with low or high concentration
of Ca2+ supplemented with insulin, epidermal growth factor, cholera toxin, hydrocortisone, and transferrin. Under these conditions,
explants attached to the substrate within 2 d, and expanding epithelioid monolayer islets emerged from the organoids during
the following days. [3H]Thymidine incorporation revealed a growth fraction of the cells close to 5%. By omitting either epidermal growth factor,
insulin, or cholera toxin from the medium, pronounced reduction in sizes of islets and in [3H]thymidine incorporation was found. Throughout the culture period, the islets appeared as continuous sheets of polygonal
cells. The epithelial nature of the expanding cell islets was confirmed by demonstration of cytokeratins and of desmosomes.
Ultrastructural evaluation of early cultures revealed clusters of epithelial cells intermixed with lymphocytes, and late cultures
showed a typical pattern of stratified keratinizing epithelium. However, squamous metaplasia was avoided by the use of low
Ca2+ medium, which also proved essential for cell transfer. MHC class II antigen was detected on the majority of the cultured
cells, and culture supernatants contained co-mitogenic activity for thymocytes and GM-colony stimulating activity.
This work supported by The Danish Research Council, grant 12-8148. 相似文献
14.
以悬浮适应的表达尿激酶原CHO工程细胞为研究对象,在100mL的摇瓶中进行无血清悬浮培养,以细胞密度、细胞活力、Pro-UK活性、葡萄糖比消耗速率(qglc)、乳酸比生产速率(qlac)、乳酸对葡萄糖的得率系数(Ylac/glc)为观察指标,同时以细胞有血清悬浮培养作为参照,考察CHO工程细胞无血清悬浮培养生长和代谢特征。观察结果表明,CHO工程细胞在无血清及有血清悬浮培养条件下表现为大致相似的细胞生长和代谢特征。在此基础上,依据实际检测的数据,应用MATLAB软件对细胞对数生长期的细胞生长、乳酸生成及葡萄糖消耗的模型参数进行非线性规划,获得全局性收敛的最优参数估计值,建立了细胞在无血清培养条件下的生长及代谢动力学模型。 相似文献
15.
Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(9):911-920
Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed
of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate.
Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained
MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA,
insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors
in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml
concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic
for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA.
This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells
without interfering activities known to be present in serum.
This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American
Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc. 相似文献
16.
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
17.
Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media 总被引:3,自引:0,他引:3
To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H2O2 exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H2O2 to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required. 相似文献
18.
David G. Thomassen 《In vitro cellular & developmental biology. Plant》1989,25(11):1046-1050
Summary The colony-forming efficiency of rat tracheal epithelial (RTE) cells was determined in serum-free media containing different
types of commercially available bovine serum albumin (BSA): crude fraction V, essentially globulin-free, essentially fatty-acid-free,
and essentially globulin- and fatty-acid-free BSA. RTE cells exhibited a concentration-dependent increase in colony-forming
efficiency in response to crude fraction V BSA. Similar results were obtained using essentially globulin-free BSA. However,
deletion of cholera toxin from the medium resulted in a decrease in the colony-forming efficiency for cells plated in high
concentrations (>2 mg/ml) of globulin-free, but not one type of fraction V, BSA. Essentially fatty-acid-free or essentially
fatty-acid- and globulin-free BSA stimulated RTE cell colony formation at low concentrations (less than 2.5 to 5 mg BSA/ml)
but resulted in concentration-dependent decreases in colony-forming efficiency at higher concentrations. The response of cells
to these BSAs was not dependent on cholera toxin. Finally, commerically available fraction V BSA prepared by heat shock, dialysis,
charcoal treatment, and deionization was stimulatory at low concentrations but inhibitory at high concentrations. These data
suggest that impure preparations of BSA can, under different conditions, stimulate or inhibit cell proliferation and that
the expression, of these activities is affected by the method of BSA preparation, the concentration of BSA used, and, in some
cases, by the presence or absence of cholera toxin.
Research conducted with support from the Office of Health and Environmental Research, U.S. Department of Energy, Washington,
DC, under contract no. DE-AC04-76EV01013 in facilities fully acredited by the American Association for Laboratory Animal Care. 相似文献
19.
To investigate the effects of factors secreted by different cell lines on human monoclonal antibody (MAb) integrity, 600 mg of a human MAb, which specifically binds to human erythrocytes, were produced in a perfusion process. After purification by protein A affinity chromatography, the MAb was used for integrity testing in supernatants of several cell lines to investigate their potential to degrade the antibody in the extracellular environment. One insect cell line (IPLB-SF-21 AE) and four mammalian cell lines [CHO K1, BHK-21 (C13), C1271, P3-X63-Ag8.653], all of them commonly used for the production of recombinant proteins, and the human-human-mouse heterohybridoma cell line itself (H-CB-hahE), were adapted to serum-free culture media. For integrity testing all cell lines were cultivated in spinner flasks using serum-free media supplemented with 30 mug mL(-1) of purified MAb. MAb integrity was assayed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, both followed by Western blotting, and an antigen binding assay. None of the mammalian cells showed any detectable effects on antibody stability and integrity during exponential growth, whereas isoelectric focusing of monoclonal antibody taken from IPLB-SF-21 AE culture supernatants revealed a new band indicating a partial modification of the MAb by secreted factors of these cells. This observation did not correlate with the total proteolytic activity, which was measured in all supernatants and found to be lowest in the insest cell cultures. For mammalian cell cultures, it could be concluded from these findings that shifts of the antibody microheterogeneity pattern, which can be found normally as a result of variations in different production parameters, are not caused by extracellular factors once the product has been secreted into the supernatant. In addition to their well-known advantages in posttranslational modifications (e.g., formation of complex type N-glycans), mammalian cells appear to be more suitable as expression systems for human monoclonal antibodies to be used in vivo when compared with baculovirus-infected insect cells. (c) 1995 John Wiley & Sons, Inc. 相似文献
20.
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