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151.
A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8
Mouse myeloma cell line P3-X63-Ag8.653
- BME
Basal Medium Eagle
- BSA
Bovine Serum Albumin
- DMEM
Dulbecco's Modified Eagle's Medium
- EDTA
Ethylenediaminete-traacetic Acid
- e-PC
Phosphatidyl choline from egg yolk
- FCS
Fetal Calf Serum
- FGF
Fibroblast Growth Factor
- GHL
Glycyl-histidyl-lysine
- HDL
High Density Lipoprotein
- HPL
Human Plasma Lipid
- IF
1:1 mixture of IMDM and Ham's F12
- IMDM
Iscove's Modified Dulbecco's medium
- LDL
Low Density Lipoprotein
- NS1
Mouse myeloma cell line NSI-1-Ag4-1
- PBS
Phosphate Buffered Saline
- s-PC
Phosphatidylcholine from soy beans
- s-PE
Phosphatidylethanolamine from soy beans
- s-lecithin
lecithin from soy beans 相似文献
152.
Matuo Y Nishi N Muguruma Y Yoshitake Y Masuda Y Nishikawa K Wada F 《Cytotechnology》1988,1(4):309-318
Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM
Alpha Modification of Eagle's Minimal essential medium
- CHAPS
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate
- CHAPSO
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate
- CS
Calf Serum
- EGF
Epidermal Growth Factor
- FGF
Fibroblast Growth Factor
- HPLC
High-Performance Liquid Chromatography
- NGF
Nerve Growth Factor
- NOG
1-O-n-octyl--D-glucopyranoside
- NP-40
Nonidet P-40
- PBS
Phosphate-Buffered Saline
- SB 12
3-(dodecylmethylammonio)-1-propane sulfonate
- SDS
Sodium Dodecyl Sulfate
- TGF- and
Transforming Growth Factor type and 相似文献
153.
Summary In order to study mitogenic control during axolotl limb regeneration, we have developed a primary blastema cell culture as a very sensitive bioassay for blastema mitogens. Transferrin, an iron-binding glycoprotein which has been shown to be the neurotrophic factor for muscle cells, is the mitogen which has been analysed in the present report. Addition of approximately 2 g human transferrin/ ml of serum-free culture medium enhances blastema cell proliferation 11-fold over control levels and 2-fold over that produced by the addition of nerve extracts or purified growth factors extracted from nerve tissues (basic and acidic fetal growth factor, FGF). At a higher concentration (20 g/ml), transferrin alone has no mitogenic effect unless the medium is also supplemented with FeCl3 (100 M). The results are discussed with regard to the sensitivity of the blastema cell culture bioassay and in the context of the neurotrophic theory of urodele limb regeneration. 相似文献
154.
用金桔茎段为外植体,培养在附加1.0毫克/升BA和0.l毫克/升IBA的MS培养基上,诱导愈伤组织和芽形成。观察了愈伤组织和芽形成过程中的组织细胞学变化。培养一周后,在茎组织切口两端开始膨大,细胞增大和开始分裂。培养两周后,开始形成瘤状愈伤组织。在愈伤组织中有形成层状分生组织、维管组织结节和分生细胞团。培养四周后,表层的分生细胞团分化形成大量芽原基,同时愈伤组织深层也出现分生细胞团。带节茎段可从切口两端的愈伤组织分化形成芽,亦可从叶腋的潜伏芽直接形成芽。 相似文献
155.
以[~(35)S]-Na_2SO_4为示踪物,观察培养的人脐静脉内皮细胞(EC)合成及分泌的蛋白聚糖(PG),经DEAE-Sephacel离子交换及Sepharose6B凝胶滤柱层析分析发现细胞层及培养液均含有三种PG单体,即硫酸乙酰肝素蛋白聚糖(HS-PG)、硫酸软骨素蛋白聚糖(CS-PG)及硫酸皮肤素蛋白聚糖(DS-PG)HS-PG又可分为大小两种,前者(HS-PG_L)位于V_o处,后者(HS-PG_s)Kd=0.53(sepharose6B);CS-PG/DS-PG分为三个峰,峰Ⅰ位于V_0处,峰Ⅱ、峰Ⅲ的Kd值分别为0.26及0.52(sepharose6B)。汇合前后细胞层及培养液中各种PG的含量不同。细胞层PG总量汇合前低于汇合后,无论是细胞层还是培养液汇合前HS-PG_L均低于汇合后,HS-PG_L与HS-PG_s比值亦为汇合前低于汇合后,而CS-PG/DS-PG含量则高于汇合后。汇合前后EC合成及分泌PG的差异与文献报道的EC损伤及正常者类似。 相似文献
156.
Päivi Heikkilä Arvi I. Kahri Christian Ehnholm Petri T. Kovanen 《In vitro cellular & developmental biology. Plant》1988,24(9):936-942
Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture,
fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence
of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria
were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets
were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated
zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three
media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells
with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes
were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased
in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased
secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids
secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol
is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without
exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented
medium.
This work was supported by Finnish Culture Foundation. 相似文献
157.
A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organogenesis 总被引:8,自引:0,他引:8
Herman H. Vandenburgh 《In vitro cellular & developmental biology. Plant》1988,24(7):609-619
Summary A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum
in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell
Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity
controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of
simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts
proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under
static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing
the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel
to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling
orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly
affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in
vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous
bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation
for subsequent functional work.
Supported by grants NS16753, AR36266, and RR05818 from the National Institutes of Health, Bethesda, MD. 相似文献
158.
Terry L. Riss Kenneth P. Karey B. Daniel Burleigh David Farker David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(11):1099-1106
Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant
insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on
poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's
modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin,
100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free
growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold
more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The
concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like
growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin,
ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth
factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures.
This work was supported by a contract from IMCERA Bioproducts, Inc. 相似文献
159.
Cyprian Weaver Robert L. Sorenson Brian Kobienia 《In vitro cellular & developmental biology. Plant》1988,24(2):108-116
Summary The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient
rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied
because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas
undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied
glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within
which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the
lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent
to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated,
as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue
forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit
aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA
synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing
cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic
polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography
of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may
serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating
effects on islet function.
This work was supported by a research grant from the Diabetes Research and Education Foundation. 相似文献
160.
M. Pilar López M. Jose Gómez-Lechón Jose V. Castell 《In vitro cellular & developmental biology. Plant》1988,24(6):511-517
Summary This study examines the factors involved in the rapid glycolysis and glycogenolysis that occur during the first stages of
hepatocyte culture: a) Shortly after seeding glycolysis, estimated as lactate released to culture medium, increased 10 times
in comparison to that reported in vivo. By 8 to 9 h of culture, hepatocytes were nearly glycogen-depleted even in the presence
of insulin. b) 6-Phosphofructo-2-kinase remained 100% active during this period. The proportion of the initial active phosphorylase
(87%) decreased to 57% by 7 h of culture. c) Fructose 2,6-bisphosphate content was initially similar to that found in liver
of fed animals, decreased after seeding and increased thereafter up to four times the initial concentration. In spite of changes
in the concentration of this activator, the glycolytic rate remained high and constant. d) ADP and AMP increased sharply after
cell plating, reaching values 1.7 and 3.5 times higher. The rise in AMP levels may be involved in the activation of glycolysis
and glycogenolysis, because this metabolite is known to act as an allosteric activator of phosphofrucktokinase and glycogen
phosphorylase. This metabolic situation resembles that of cells under hypoxia.
Part of this work was presented at the 38th Annual Meeting of the Tissue Culture Association, Washington, DC, May 1987. 相似文献