Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.
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PMID:Heterogeneity in the hormonal responsiveness of clones derived from the 13762NF rat mammary tumor. 300 10

Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.
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PMID:Renal cell culture using autopsy material from children with cystinosis. 669 73

Tissue culture of gall-bladder was attempted in the following media: Dulbecco, Eagle's minimum essential medium, NCTC 135, medium 199 and Ham's F12. Growth occurred in all of them for up to 2 weeks assessed by light microscopy. No enhancement of growth was induced by collagenase trypsin insulin or hydrocortisone. Scanning electron microscopy confirmed that new cells colonised the free surface of the explant. Transmission electron microscopy showed good preservation of the original tall epithelial cells for the period of study. The new migrating cells were flatter, but retained the morphological features of the columnar cells. Secretory granules were absent after 1 day in culture but increased amounts of glycogen and lipid began to appear in the epithelium.
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PMID:Tissue culture of guinea-pig gall-bladder epithelium. 686 70

Large quantities of differentiated mammalian chondrocytes from normal hyaline cartilage were isolated after digestion of foetal bovine tracheas with collagenase. Incubation of the newly isolated cells for 1 day in the presence of dextran sulphate inhibited formation of cell aggregates during subsequent subculture in the absence of dextran sulphate. After incubation with dextran sulphate, the cells were plated in Ham's F12 medium with or without foetal calf serum on hydrophilic or hydrophobic Petri dishes. Chondrocytes cultured on hydrophilic substrates in the presence of serum attached to the substrate and showed cytoplasmic spreading. The cells did not attach to hydrophobic substrates in the presence of serum, but remained in suspension as single cells. In the absence of serum the chondrocytes attached to either substrate, but did not show any cytoplasmic spreading. By using labelling with [35S]sulphate and [3H]-thymidine it was shown that glycosaminoglycan synthesis did not require the presence of serum, whereas DNA synthesis required serum factors. Extracellular glycosaminoglycans were recovered in two pools: the medium pool and the pericellular pool, the latter being isolated by proteolytic digestion. The kinetics of these pools differed, depending on the presence or absence of serum and the type of substrate used. The turnover of the pericellular pool was studied in a pulse-chase experiment. At the end of the chase (72 h), only 60% of the material in the pericellular pool had been metabolized.
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PMID:Isolation and culture techniques of foetal calf chondrocytes. 732 90

In reconstructive head and neck surgery, there is a great need for cartilage transplants. Sufficient autologous graft is often not available. Heterologous cartilage is used frequently, although there is danger of transmitting viral infections and resorption rates are high. We have developed a three-dimensional model for the formation of cartilage in vitro. The aim of this study was to characterize the collagen synthesis under these culture conditions. Human chondrocytes were isolated by digesting septal cartilage matrix in the presence of type II collagenase, hyaluronidase, and Dnase II in Ham's F12 medium. The resulting cells were kept in monolayer culture for one week and then suspended in 2% ultra-low-melting agarose (1:1). The cell-agarose conglomerate was encapsulated with a 3% ultra-low-melting agarose solution and placed in a perfusion culture chamber. A permanent flow of fresh medium (Ham's F-12 supplemented with 50 micrograms/ml ascorbic acid and 2% fetal calf serum) was provided by a peristaltic pump which delivered 1 ml/h with on/off intervals of 30 min. Samples were recovered after two weeks. Using electron microscopy abundant collagen fibril formation was shown. The collagen fibrils were identified histologically as cartilage specific type II collagen. No mRNA expression of collagen type X was observed using in situ hybridization. The cells appeared in a round cell shape with round nucleus and only slight variations in form and size. The present results indicate that the chondrocytes maintain their differentiated phenotype and continue to synthesize typical matrix products in this three-dimensional perfusion culture chamber.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Cultivation of human cartilage tissue in a 3-dimensional perfusion culture chamber: characterization of collagen synthesis]. 749 39

Growth cartilage were cultured in order to determine whether cartilage growth could replace bone loss and generate autologous bone capable of further growth. An abundant quantity of mature functional chondrocytes is required after culture. Chondrocytes were isolated from a cartilage fragment by enzyme digestion. Trypsin, then collagenase were used as a function of the number of chondrocytes obtained per gram of growth cartilage. The optimal culture conditions were determined on the basis of three parameters: the density of cell seeding and the type and composition of the culture medium. The kinetics of chondrocyte growth and their fixation on different support media as well as their functional capacity was determined in different media according to the following conditions: seeding at 200,000 or 300,000 chondrocytes/cm2 on type 1 collagen and in Hamm-F12 medium without supplementation with growth factors. The first trials of cryopreservation of the chondrocytes after culture gave promising results. Yields, expressed as the percentage of viable cells varied from 91 to 98% depending on the cryoprotective solution used.
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PMID:[Growth cartilage in rabbits. Culture and cryopreservation]. 772 15

Stromal-vascular (S-V) cells isolated from adipose tissue of newborn pigs (NBPC) and mature pigs (MPC) by collagenase digestion were used to evaluate differences in preadipocyte culture and development. Cells were seeded at a density of 3 x 10(4) cells/cm2 on six-well (35-mm) tissue culture plates in 3 mL of DMEM/HAM's F12 medium plus 10% fetal calf serum and cultured at 37 degrees C under a humidified atmosphere of 95% air:5% CO2 for 24 h. Cells were then washed thoroughly in DMEM/HAM's F12 medium without fetal calf serum and maintained in serum free (SF) medium or SF medium supplemented with 2.5% newborn pig serum (NBPS) or mature pig serum (MPS) for 12 d. After 1 d, more NBPC adhered to the culture plates, as indicated by DNA values. After 12 d, protein per culture well was not significantly different, but DNA concentration per well remained higher (P < .05) in cultures of NBPC than in the MPC cultured in the same medium, indicating fewer MPC. Protein:DNA ratios were higher (P < .05) in cultures of MPC regardless of the medium, reflecting larger cell size. More cells containing fat deposits were seen with NBPC in all conditions in comparison with MPC, and more fat was deposited in NBPC in SF than in SF plus NBPS or MPS. The NBPC had higher (P < .05) sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) per protein than MPC regardless of the medium. For both cell types, GPDH activity in either serum was less than activity of cells grown in SF.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of age on the differentiation of porcine adipose stromal-vascular cells in culture. 773 Jan 75

In the field of otolaryngology cartilage grafting is commonly performed to reconstruct skeletal defects. Knowledge of chondrocyte growth and differentiation can now be used to engineer cartilage tissue for grafting. The first condition is that chondrocytes maintain their differentiated phenotype besides being able to produce a new cartilage matrix. The target of this study was to develop a three-dimensional culture system for in-vitro formation of vital cartilage transplants. Chondrocytes were isolated by digesting the cartilage matrix with collagenase and hyaluronidase. After embedding in "low-melting" agarose, the chondrocytes were placed into a perfusion culture chamber to provide a constant supply of nutrients to the cultures. The peristaltic pump was operated with on/off intervals of 30 min. Ham's F12 supplemented with 2% FCS and 50 micrograms/ml ascorbic acid was employed as culture medium. Monoclonal antibodies specific to collagens type I and type II were used to characterise cells and matrix synthesis. Synthesis of proteoglycans and collagens was achieved using toluidine blue and azan staining. Under the described culture conditions, the chondrocytes maintained a differentiated phenotype (expression of collagen type II) with synthesis of collagens and proteoglycans. An accumulation of matrix products was achieved pericellularly. After 2-8 weeks the obtained tissue exhibited an excellent histological appearance showing the typical features of cartilage tissue. The results show that the perfusion chamber allows a quick in-vitro fabrication of a piece of pure cartilage tissue for transplantation.
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PMID:[Culture of human cartilage tissue using a perfusion chamber]. 781 42

The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) medium was used for studying the influence of the cAMP-effectors epinephrine (100 microM), norepinephrine (100 microM), glucagon (1 microM) and isoproterenol (1-1000 microM) as well as the synthetic cAMP-analogon dibutyryl-cAMP on the metabolism of metallothionein. Liver parenchymal cells isolated by a two-step collagenase perfusion were incubated with DMEM/F12 containing 5% (v/v) fetal calf serum (FCS) and 20 microM zinc in Petri dishes. Experiments were initiated after a 24 h equilibration period by adding the agonists for 18 h. MT in hepatocyte homogenates was quantified by the 109Cd-hemoglobin-binding assay. Cell viability was assessed by the activity of the cytosolic enzyme lactate dehydrogenase (LDH) liberated into the culture medium and by trypan blue exclusion. Isoproterenol and glucagon produced a significant increase of cytosolic MT about 50%. In contrast, incubation with epinephrine and norepinephrine did not lead to any significant effects in the amount of hepatic metallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (500 microM) did not affect the content of hepatic metallothionein. To examine transcriptional and translational regulatory effects supplementation of cycloheximide (0.1-500 microM) and actinomycin D (0.1-100 microM) showed a total inhibition of the agonist induced amounts. Particularly in combination with isoproterenol low LDH activities reflected a high viability of hepatocytes. In conclusion, in primary hepatocyte cultures cAMP-mobilizing-agonists like isoproterenol and glucagon indicate an independent effect on the MT-metabolism. This is possibly due to the de novo synthesis of the protein because suppression by actinomycin D can be observed. However, cAMP-effectors do not seem to be involved in the induction of metallothionein because theophylline and dibutyryl-cAMP did not affect MT-metabolism by suppressing the phosphodiesterase or by stimulating the cAMP-cascade.
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PMID:Influence of cAMP-effector-agonists on the synthesis of metallothionein in rat primary hepatocytes. 858 45

An original and reliable technique to culture growth plate chondrocytes was developed to obtain an abundant amount of mature and functional chondrocytes. Growth plates were provided from the epiphysis of 3 week old rabbits. The isolation of the chondrocytes was optimized by the use of trypsin and collagenase. The culture was realized according to the following conditions: seeding at 20,000 or 30,000/cm2 on type I collagen substrate and in Ham F12 medium without a supplementation of glucose or growth factors. After 7 days of culture, the implantation was to be carried out. Different implantation substrates were evaluated in vivo. Agar turned out to be the only substrate to provide strong and healthy chondrocytes 21 days after the grafting. In an other experimentation, the culture was implanted into surgically created defects in the growth plate area. In this case, the culture did not avoid the occurrence of an epiphysiodesis. However, the 6 weeks post operative histologic examination, showed that the implant remained viable, continued to maintain a proteoglycan rich matrix, and began to organize in ordered columns of mature chondrocytes.
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PMID:[In vitro culture of growth cartilage and in situ reimplantation]. 894 14


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