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At least 73 records · Page 4Linked to original sources
PubMed2004

[Establishment of an in vitro model of brain-blood barrier].

OBJECTIVE: To establish an in vitro model of brain-blood barrier (BBB) using cultured mouse brain microvascular endothelial cells (BMVEC). METHODS: Mouse BMVEC were seeded on micro-pore membrane of gelatin-coated cell culture insert and cultured to confluence. The establishment of BBB was preliminary judged by a 4 h water-leaking test. The tight junctions between BMVEC were demonstrated by scanning and transmission electron microscope. The transendothelial electrical resistance(TEER) over BMVEC was measured. The permeability of Horseradish peroxidase (HRP) through the BBB was analyzed and the effect of RMP-7 on permeability of the BBB was investigated. RESULTS: The 4 h water-leaking test became positive when BMVEC were cultured to confluence. By scanning and transmission electron microscope, the tight junctions were demonstrated on confluent BMVEC. The TEER over BMVEC monolayer increased 3.2 and 7.68 times and the permeability rates for HRP were 13.4% and 6.7% respectively, as compared with sub-confluent BMVEC and human umbilical vein endothelial cell monolayer(HUVEC). The HRP permeability rate in the model of BBB increased 2.7 times after treatment with RMP-7. CONCLUSION: The established in vitro model of BBB has basic characteristics of BBB in vivo, and is suitable for central nervous system (CNS) drug research over BBB.

Animals↗
PubMed2001

Cell proliferation in cancer prevention; effects of preventive agents on estrogen-related endometrial carcinogenesis model and on an in vitro model in human colorectal cells.

Proto-oncogenes such as c-fos, c-jun and c-myc are known to relate to cell proliferation and differentiation. Some oriental herbal medicines like Glycyrrhizae radix or Juzen-taiho-to were found to suppress estradiol-17 beta (E2)-induced expression of c-fos/jun in uterine corpus and inhibited N-methyl-N-nitrosourea and E2-induced endometrial carcinogenesis in mice. It is suggested that the effects of such oriental drugs are exerted probably through suppression of estrogen-induced c-fos/jun expression and they are promising preventing agents for endometrial cancers. In the combined in vitro assay for cell proliferation (MTS assay) and apoptosis (DNA fragmentation) in human colorectal cancer cells (Colo 320), a number of naturally occurring chemopreventive agents such as curcumin, quercetin, auraptene, 1'-acetoxychavicol acetate (ACA) and indole-3-carbinol were shown to generate apoptosis as well as to inhibit cell proliferation. The results suggest a mode of action of these chemopreventive agents and also imply that such in vitro short term assay is useful for detection of new agents for cancer prevention.

Adenocarcinoma↗
PubMedDate not supplied

Keratocyte-populated collagen gel as an in vitro model of excimer laser keratectomy.

BACKGROUND: To develop an in vitro model to study the effects of excimer laser keratectomy on corneal stromal cells, we evaluated two types of collagen gel populated with keratocytes. METHODS: Keratocyte-populated collagen gels were prepared with type I collagen in 6-well plates or in culture plate inserts, the bottom of which consisted of a nitrocellulose membrane, contained within 6-well plates. The gels were ablated by the 193-nm excimer laser, set to ablate 50, 100, or 200 microns deep, and was observed under a phase-contrast microscope for 2 days. RESULTS: Keratocytes cultured in collagen gel developed cytoplasmic processes and formed networks of interconnected cells. Cells within the ablated area in the 6-well plates began to lose their cytoplasmic processes and became round approximately 3 hours after excimer laser ablation. These cellular changes were more prominent in the gels ablated to a depth of 200 microns. Cells outside of the ablation zones in the 6-well plates and the culture plate inserts remained intact. CONCLUSIONS: These results suggest the use of keratocyte-populated collagen gel as an in vitro model of cellular response to excimer laser keratectomy and also suggest that gel prepared in culture plate inserts is the preferred method.

Animals↗
PubMed1988

Neoplastic transformation of osteogenic cells: quantitative morphometric analysis of an in vitro model for osteosarcoma.

Previously we have reported the development of a model in vitro system for the study of osteosarcoma. In this system, when chick periosteal explants are infected with Fujinami sarcoma virus (FSV), osteosarcoma-like tissue is formed. In the present study, a series of histopathologic parameters of neoplastic transformation and osteogenesis were quantitated, at a single cell level, by computer-assisted morphometry. Most significantly, it was found that compared to uninfected (control) cultures, in the FSV-infected (experimental) cultures, the bone to osteoid ratio per unit area was decreased due to a relative decrease in the area of bone and an increase in the area of osteoid. The cellularity of the FSV-infected tissues was significantly increased due to an increase in the number of unlabeled and [3H]thymidine-labeled cells, while the proportion of alkaline phosphatase (AP) positive cells decreased. Double-label immunohistochemistry (with anti-P140gag-fps) and histochemistry for AP activity was performed, to demonstrate production of the oncogene-encoded protein, and osteoblastic differentiation respectively. In an in vitro transformation assay, single cells derived from control, uninfected cultures did not grow, while those derived from FSV-infected cultures formed colonies in semisolid medium. Some of these colonies demonstrated AP staining. Taken together these data show that in this in vitro system (i) neoplastic transformation of osteogenic cells does occur, (ii) changes in osteoid and bone production are related to neoplastic transformation, and (iii) osteosarcoma-like changes can be quantitated at the individual cell level.

Animals↗
PubMed1995

The Syrian hamster embryo (SHE) cell transformation system: a biologically relevant in vitro model--with carcinogen predicting capabilities--of in vivo multistage neoplastic transformation.

Neoplastic transformation is a multistep process that can be modeled in vitro using Syrian hamster embryo (SHE) cells. SHE cells multistage transformation involves several intermediate stages, including morphological transformation, immortality, acquisition of tumorigenicity, and malignant progression. Analysis of the molecular alterations that occur at each stage indicated that morphological transformation results from both carcinogen-induced irreversible chromosomal/genetic mutations and reversible genetic events, including altered DNA methylation. Morphological transformation results from a block in the cellular differentiation of progenitor and determined stem-like cells in the SHE cell population via alternation in the expression of the H19 tumor suppressor gene and other genes. Immortality results from genetic mutations in growth factor responsiveness, including loss of growth suppression by TGF beta and autocrine growth factor production, and genomic stability, resulting in genomic instability and an increased mutation rate. Acquisition of tumorigenicity involves loss of tumor suppressor gene function, altered mitogenic signal transduction, mutation of oncogenes, acquisition of anchorage independent growth, and chromosomal aberrations. Malignant progression is associated with alterations in extracellular matrix growth characteristics, alterations in cytoskeleton structure, elevated fibrinolytic activity, secretion of proteases, and changes in extracellular matrix protein secretion. Together, these changes model the alterations observed during in vivo neoplastic transformation and possibly explain why the SHE assay, as a carcinogen screening tool, is able to identify carcinogens with a 80 to 85% accuracy.

Animals↗
PubMed2006

In vitro modeling of the clinical interactions between octreotide and 111In-pentetreotide: is there evidence of somatostatin receptor downregulation?

UNLABELLED: Some authors have suggested that chronic octreotide use enhances the efficiency of radiolabeled somatostatin receptor (sst) imaging. Conversely, desensitization of sst on tumor tissue (tachyphylaxis) may occur occasionally in patients on chronic octreotide therapy. Assuming that chronic exposure to octreotide induces tachyphylaxis, we hypothesized that chronic exposure of sst subtype 2 (sst2)-expressing cells to octreotide would downregulate binding of 111In-pentetreotide to sst and that this downregulation would be due to a reduction in the gene copy number for sst2. METHODS: The clinical scenarios of acute (24 h) and chronic (2 wk) octreotide use, followed by either nuclear imaging exposure (8.6 pmol/L) or therapeutic exposure (510 pmol/L) to (111)In-pentetreotide, were modeled in vitro. Receptor binding in IMR-32 human neuroblastoma cells (high sst2 expression) and PANC-1 human pancreatic cancer cells (no detectable sst2 expression) was evaluated. Gene copy numbers for sst subtypes 1-5 in IMR-32 cells were determined by quantitative polymerase chain reaction. RESULTS: Acute or chronic octreotide exposure at low or high doses did not significantly alter sst2 gene copy numbers or binding of either the diagnostic dose or the therapeutic dose of 111In-pentetreotide. CONCLUSION: In vitro exposure of cells to low or high doses of octreotide for 1-14 d does not result in the development of either tachyphylaxis or upregulation of sst as assessed by changes in gene expression or in high-affinity binding.

Cell Line, Tumor↗
PubMed2004

Identification of 14 quercetin phase II mono- and mixed conjugates and their formation by rat and human phase II in vitro model systems.

In this study, the HPLC, UV-vis, LC-MS, and 1H NMR characteristics of 14 different phase II mono- and mixed conjugates of quercetin were determined, providing a useful tool in the identification of quercetin phase II metabolite patterns in various biological systems. Using these data, the phase II metabolism of quercetin by different rat and human liver and intestine in vitro models, including cell lines, S9 samples, and hepatocytes, was investigated. A comparison of quercetin phase II metabolism between rat and human liver and intestinal cell lines, S9, and hepatocytes showed considerable variation in the nature and ratios of quercetin conjugate formation. It could be established that the intestine contributes significantly to the phase II metabolism of quercetin, especially to its sulfation, that organ-dependent phase II metabolism in rat and man differ significantly, and that human interindividual variation is higher for quercetin sulfation than for glucuronidation or methylation. Furthermore, quercetin conjugation by different in vitro models from corresponding origins may differ significantly. The identification of the various mono- and mixed quercetin phase II conjugates revealed significant differences in phase II conjugation by a variety of in vitro models and led to the conclusion that none of the in vitro models converted quercetin to a phase II metabolite mixture similar to the in vivo plasma metabolite pattern of quercetin. Altogether, the identification of a wide range of phase II metabolites of quercetin as presented in this study allows the determination of quercetin phase II biotransformation patterns and opens the way for a better-funded assessment of the biological activity of quercetin in a variety of biological systems.

Animals↗
PubMed1984

The activity of cefotiam on beta-lactamase-producing bacteria in an in-vitro model.

The activity of cefotiam was tested in an in-vitro model simulating human serum pharmacokinetics. Bacterial strains used for inoculation either produced a penicillinase or a cephalosporinase. The rate of hydrolysis and the MICs of cefotiam were determined in comparison with those of ampicillin and cephalothin. The influence of these beta-lactamases on the killing kinetics and the degradation of cefotiam in the in-vitro model were then measured. Although all beta-lactamases hydrolyzed cefotiam, only the chromosomal cephalosporinases, especially those from Enterobacter and Klebsiella, reduced the elimination of the bacteria by degradation of the drug. A rough correlation between the hydrolytic activity and MIC could be demonstrated for cefotiam but not for ampicillin, while a correlation between killing ability and MIC only existed for strains with cefotiam MICs higher than 16 mg/l.

Ampicillin↗
PubMed1999

CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs.

The present study characterized Chinese hamster ovary cells overexpressing a human intestinal peptide transporter, CHO/hPEPT1 cells, as an in vitro model for peptidomimetic drugs. The kinetic parameters of Gly-Sar uptake were determined in three different cell culture systems such as untransfected CHO cells (CHO-K1), transfected CHO cells (CHO/hPEPT1) and Caco-2 cells. Vmax in CHO/hPEPT1 cells was approximately 3-fold higher than those in Caco-2 cells and CHO-K1 cells, while Km values were similar in all cases. The uptake of beta-lactam antibiotics in CHO/hPEPT1 cells was three to twelve fold higher than that in CHO-K1 cells, indicating that CHO/hPEPT1 cells significantly enhanced the peptide transport activity. However, amino acid drugs also exhibited high cellular uptake in both CHO-K1 and CHO/hPEPT1 cells due to the high background level of amino acid transporters. Thus, cellular uptake study in CHO/hPEPT1 cells is not sensitive enough to distinguish the peptidyl drugs from amino acid drugs. The potential of CHO/hPEPT1 cells as an in vitro model for peptidomimetic drugs was also examined through the inhibition study on Gly-Sar uptake. Peptidomimetic drugs such as beta-lactam antibiotics and enalapril significantly inhibited Gly-Sar uptake whereas the nonpeptidyl compounds, L-dopa and alpha-methyldopa, did not compete with Gly-Sar for cellular uptake within the therapeutic concentrations. In conclusion, the present study demonstrates the further characterization of CHO/hPEPT1 cells as an uptake model as well as inhibition study and suggests their utility as an alternative in vitro model for drug candidates targeting the hPEPT1 transporter.

Animals↗
PubMedDate not supplied

[Drug-drug interactions: predictive in vitro models of in vivo interactions. Round table no. 6.XV].

Liver drug metabolism is a major source of drug interactions. Three major human in vitro models are employed to detect drug interactions in the preclinical phases of drug development: recombinant enzymes, human liver microsomes and primary human hepatocyte cell cultures. Results obtained from these models may vary during the different phases of drug development. Identification of the metabolic pathways, enzymes involved in drug metabolism (mainly cytochrome P450s), enzyme induction or inhibition allow us to detect the major pharmacokinetic drug interactions which can occur in man and to identify specific populations at risk for such interactions. In vitro models are essential to decide if and which future drug interaction studies should be performed in man. However, the clinical relevance of the potential pharmacokinetic drug interactions detected by these in vitro models remains to be determined and confirmed by human studies.

Animals↗
PubMed2001

Biomechanics of grade I degenerative lumbar spondylolisthesis. Part 1: in vitro model.

OBJECT: The authors sought to create and to evaluate an in vitro model of Grade I degenerative (closed-arch) spondylolisthesis. METHODS: The model of spondylolisthesis was created by two primary procedures: 1) resection of the disc; and 2) stripping of anterior and posterior longitudinal ligaments away from the vertebral bodies (VBs). In 13 vertebral levels obtained from three cadaveric lumbar spines, the tissues were resected sequentially in alternating order to determine the relative contribution of each resection to spinal instability. The entire specimens were loaded with nonconstraining torques and then individual levels were loaded with anteroposterior shear forces. The motion values were measured optoelectronically for each specimen at individual levels. CONCLUSIONS: The integrity of the disc was more important than attachment of the ligaments to the VB, but the resection of both structures was necessary to achieve substantial destabilization. The structures of the spine are highly resilient, and destabilization is difficult to achieve without performing extensive resection. Using the techniques described in this paper to alter normal spines, a level of spinal instability (Grade I; 25% slippage) that may represent spondylolisthesis can be modeled in vitro.

Aged↗
PubMed2006

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2.

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.

Algorithms↗
PubMedDate not supplied

Three-dimensional in vitro model of adipogenesis: comparison of culture conditions.

In vivo and in vitro studies have demonstrated both promise and current limitations in tissue engineering of fat. Herein, we report the establishment of a well-defined three-dimensional (3-D) in vitro model useful for systematic investigations of 3-D adipogenesis. Polyglycolic acid fiber meshes were dynamically seeded with 3T3-L1 preadipocytes; subsequently, cell-polymer constructs were hormonally induced and cultivation under three different conditions was evaluated. Regarding tissue coherence and intracellular lipid content, culture of cell-polymer constructs either dynamically in well plates or in stirred bioreactors yielded similar results, which were distinctly improved compared with static conditions in well plates. At the protein and mRNA levels, significantly increased expression of genes characteristic for a mature adipose phenotype was demonstrated for constructs dynamically cultured in well plates, as compared with static conditions. Furthermore, investigation of lipolysis under stimulating and inhibiting conditions demonstrated functionality of the dynamically differentiated constructs. Using dynamic culture conditions, the presented in vitro model system is suggested as a valuable tool serving both fat tissue engineering and basic research by facilitating investigations of tissue-inherent features not possible under conventional 2-D culture conditions.

3T3-L1 Cells↗
PubMed1995

Effects of high levels of fluoride on bone formation: an in vitro model system.

In order to develop an in vitro model for the study of the effects of different agents on biomineralization, a three-dimensional cell culture system was investigated at different levels of fluoride. Rat fetal osteoblasts were seeded onto collagen discs and maintained in a culture medium for 40 days. Results showed that, at 40 days, the cultured matrices had a Ca:P ratio, mineral content and Fourier transform infrared (FTIR) spectrum that were close to those seen for normal rat bone. Viable cells, observed by light microscopy, were present in the matrix at 40 days. The formation of a mineralized matrix in this experimental set-up provided a model for exploring in vitro the effects of high levels of fluoride on bone. The fluoride content of the mineral formed in the cultures showed a dose-dependent increase in fluoride content with time. Also, an increase in the crystallinity of the apatite in the presence of fluoride, was observed by FTIR. The Ca:P ratio and percentage mineral by weight showed no apparent differences among the groups. The three-dimensional model used for this study has the potential to be a powerful tool in the study of time-dependent effects of drugs and other factors on osteoblast cell functions and subsequently on matrix mineralization.

Animals↗
PubMedDate not supplied

Activity of PS-15 and its metabolite, WR99210, against Plasmodium falciparum in an in vivo-in vitro model.

An in vivo-in vitro model was used to assess the antimalarial activity of PS-15 and its metabolite, WR99210, against Plasmodium falciparum. WR99210, an antifolate triazine compound, was given as a single oral dose of 30 mg/kg to 8 Saimiri sciureus monkeys and, 3 months later, the parent compound, PS-15, was given similarly to the same monkeys. Serum samples were collected at various times after drug administration, serially diluted with control serum, and their antimalarial activity in vitro was determined against the multidrug-resistant K1 isolate of P. falciparum. Serum concentrations of PS-15 and WR99210 were estimated by high performance liquid chromatography. The maximum dilutions of serum that inhibited parasite growth were 20- to 86-fold higher 3 and 6 h after administration of PS-15 than following WR99210 administration. Substantial serum antimalarial activity was observed even at 48 h after medication with PS-15. Serum drug concentrations provided further evidence that PS-15 was absorbed far better from the gastrointestinal tract than WR99210. The substantial and sustained activity of PS-15 suggests that a single dose, or several smaller doses given once a day, should be effective in curing drug-resistant infections of P. falciparum.

Animals↗
PubMed1993

The isolated perfused bovine udder as an in vitro model of percutaneous drug absorption. Skin viability and percutaneous absorption of dexamethasone, benzoyl peroxide, and etofenamate.

Using udders from slaughtered cows as a new in vitro model of percutaneous drug absorption, the tissue viability and the percutaneous absorption of dexamethasone, benzoyl peroxide, and etofenamate were studied. The organ was perfused with gassed tyrode solution for up to 6 hr. As shown by measurement of glucose consumption, lactate production, lactate dehydrogenase activity, and pH in the perfusate, the tissue was viable over a 6-hr period. This was confirmed by a histological examination. Determination of the udder skin-fold thickness demonstrated that no edema developed within the perfusion period. A maximum skin penetration of dexamethasone was found after administration of dexamethasone dissolved in acetone with dimethyl sulfoxide, followed by ointment with salicylic acid, ointment without salicylic acid, and acetone solution. Experiments with benzoyl peroxide and etofenamate demonstrated that the perfused udder skin was capable of metabolizing drugs in vitro. In conclusion, the isolated perfused bovine udder is a new in vitro model, which maintains bovine udder skin with an isolated vasculature in a viable state. Using this in vitro model, we note it is possible to compare the dermal penetration, metabolism, and absorption of substances after topical administration of different drug formulations.

Animals↗
PubMed1988

Low Ca2+ stripping of differentiating cell layers in human epidermal cultures: an in vitro model of epidermal regeneration.

An in vitro model of the epidermal regeneration process is described. Incubation of multilayered, keratinizing cultures of human epidermal cells in Ca2+-free medium for 72 h results in a complete stripping of all suprabasal layers. When the Ca2+ stripped cultures are refed normal Ca2+ medium a reproducible series of morphologic and cell kinetic changes takes place. It is suggested that these changes represent a general pattern of regeneration after epidermal wounding. After an initial lag phase the regenerative response is principally effected by a recruitment to the proliferating pool of cells with a high rate of DNA synthesis. The cells seem to be programmed to rapid differentiation. Studies with cholera toxin suggest that this adenylate cyclase-stimulating agent is able to induce significant changes in the regenerative process causing a prolonged, but less intense, proliferative response leading to lateral growth rather than to rapid differentiation.

Calcium↗
PubMed1993

Master core apical movement during compaction: evaluation of two in vitro models.

This study evaluated two in vitro models (a split hard metal model and a split natural tooth model) for the assessment of master cone apical movement. Gutta-percha cones were fitted 0.5 and 1.0 mm short of the apical ends of the artificial canals. Photographs were taken before and after vertical or lateral compaction and measurements were made. The results demonstrated no significant difference between the two models.

Analysis of Variance↗