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PubMedDate not supplied

Murine endolymphatic sac development in tissue culture: an in vitro model for sac function.

Numerous studies have attempted to elucidate the function of the mammalian endolymphatic sac (ELS). All of these studies have been performed on in vivo specimens and are thus influenced by humoral and tissue factors extraneous to the sac. In contrast, an in vitro model would provide an opportunity to study ELS cells in a carefully controlled environment. This report presents our experience with tissue culturing the murine endolymphatic sac removed from 16 and 18 gestational day fetuses. Light (LM) and transmission electron microscopical (TEM) evaluations of the developing endolymphatic sac were performed over periods of one, four, and seven days in tissue culture. In order to confirm growth and maturation, three-dimensional reconstructions from serial sections of the cultured ELS were made and compared with published accounts of in vivo murine ELS development for equivalent periods of time. Both whole and dissected otocysts were grown in tissue culture and compared with one another. Two different tissue culture medias were investigated, each with and without the addition of collagenase, used to soften the dense fibrous capsule of the otocyst and thus facilitate dissection and histological preparation. The impact of collagenase and the tissue culture medias on endolymphatic sac growth were studied. Results demonstrated that murine ELS cells were able to differentiate and mature in tissue culture, as confirmed by LM, TEM, and three-dimensional reconstructions. After an initial delay, in vitro maturation of cells in tissue culture paralleled normal in vivo growth and in some specimens appeared to show accelerated maturation. This in vitro model should prove useful in efforts to define ELS function and in providing a technique for tissue culturing human ELS from normal and diseased ears.

Animals↗

PubMed2002

Role of thymidine phosphorylase in an in vitro model of human bladder cancer invasion.

PURPOSE: It has been previously demonstrated that the angiogenic factor thymidine phosphorylase is elevated significantly in invasive bladder cancer. We report that it is not merely an incidental finding. Thymidine phosphorylase has a functional role in bladder cancer invasion. MATERIALS AND METHODS: The superficial bladder cancer cell line RT112 was transfected by retroviral techniques to generate the RT112-TP clone that expressed significantly elevated levels of thymidine phosphorylase, comparable to those of invasive human bladder cancers. The empty vector control RT112-EV was generated for comparison. Growth of these transfectants was examined using a new in vitro model of bladder cancer invasion based on de-epithelialized rat bladder and by assessing growth as xenografts in nude mice. The effect of 5-deoxy-5-fluorouridine, a prodrug activated by TP to produce 5-fluorouracil, was also examined. RESULTS: RT112-TP high thymidine phosphorylase expressing cells invaded into the stroma of the in vitro model but wild-type RT112 and RT112-EV cells did not. This invasion was abolished by 5-deoxy-5-fluorouridine. Invasion correlated with thymidine phosphorylase expression on immunohistochemical testing. There was also a significantly greater xenograft growth rate for RT112-TP than for RT112-EV, confirming the malignant growth advantage conferred by thymidine phosphorylase. CONCLUSIONS: We demonstrated that thymidine phosphorylase may have a functional role in bladder cancer invasion and the apparent advantage of thymidine phosphorylase expression to tumor cells can be exploited by therapies that utilize prodrugs such as 5-deoxy-5-fluorouridine, which is activated by thymidine phosphorylase and inhibited invasion in our model.

Animals↗

PubMed2003

A quantitative in vitro model of smooth muscle cell migration through the arterial wall using the human amniotic membrane.

OBJECTIVE: The development of intimal hyperplasia involves smooth muscle cell (SMC) migration into the intima and proliferation. Matrix metalloproteinases and their tissue inhibitors play important roles in this process. In this study, we describe a novel in vitro model for studying SMC migration through the vessel wall. METHODS AND RESULTS: Human aortic SMCs (hASMCs) labeled with 125I-iododeoxyuridine or unlabeled were grown on the stromal aspect of the human amniotic membrane. Mechanical damage to endothelial cells grown on the basement membrane and addition of growth factors or platelets were characterized for their effect on SMC migration into the stroma both by histological methods and by measuring the radioactivity associated with the membrane after removal of noninvasive SMCs. To assess the reliability of the model, the cells were infected with a recombinant adenovirus encoding the tissue inhibitor of metalloproteinase-1 (TIMP-1). Addition of a platelet-derived growth factor gradient stimulated hASMC infiltration into the stroma. This effect was abolished with TIMP-1-transduced hASMC, confirming that TIMP-1 overexpression blocks SMC invasion of the stroma. CONCLUSIONS: This in vitro model of SMC migration in the vessel wall provides an inexpensive, quantitative, and reliable tool to study the molecular and cellular mechanisms of intimal hyperplasia.

Adenoviridae↗

PubMed1998

Cell fate specification in an in vitro model of neural development.

We have studied in an in vitro model of neural development the effect of neighboring cells on the fate of single fluorescently labeled precursor cells. In one line of experiments, PCC7-Mz1 embryonal carcinoma cells were transiently transfected with "green fluorescent protein" (GFP) and, following incubation with 0.1 microM all-trans retinoic acid (RA), the number and morphology of derivatives (neuronal or non-neuronal) was determined that form groups of GFP-expressing cells in a surrounding of unlabeled cells. Because single PCC7-Mz1 cells can produce single-lineage and mixed-lineage derivatives, they are individually pluripotent. In another line of experiments, we have analyzed the fate of GFP-expressing PCC7-MzN cells in different cellular environments. Whereas in the absence of other cells, PCC7-MzN cells exclusively differentiated to neuronal derivatives following RA induction (Lang, E., M. L. Mazauric-Stüker, A. Maelicke, J. Cell Biol. 109, 2481-2493 (1989)), they differentiated also to non-neuronal phenotypes (astrocytes and fibroblasts) when co-cultured with either PCC7-Mz1 stem cells or freshly RA-induced cells. The fate of PCC7-MzN cells could also be shifted in the absence of other cells when the cells were grown on laminin-coated surfaces. These results suggest that a putative fate-shifting activity (FSA) is released by PCC7-Mz1 and PCC7-MzN cells which requires, at least in the case of MzN cells, presentation by extracellular matrix-like structures in order to function in cell fate specification. Very few other cell types, in particular primary cultures of mouse forebrain cells of embryonic day 13, were capable of shifting the developmental potential of PCC7-MzN cells in a similar manner as PCC7-Mz1 cells do. We conclude that cell type specification in this model of neural development may occur by similar mechanisms as have been established in Drosophila neurogenesis. A default pathway (neuronal) is modulated by lateral signaling between neighboring cells so that cellular diversity can arise from initially homogeneous populations of progenitor cells.

3T3 Cells↗

PubMed2005

A novel 3-dimensional culture system as an in vitro model for studying oral cancer cell invasion.

Tissue microenvironment plays a critical role in tumour growth and invasion. This study established a novel 3-dimensional (3-D) cell invasion model for direct microscopic observation of oral cancer cell invasion into the underlying basement membrane and connective tissue stroma. A multilayer cell construct was developed using the OptiCell chamber, consisting of a lower layer of oral mucosa fibroblasts embedded in collagen gel and an overlaying upper layer of oral cancer cells. The two layers are separated by a basement membrane composed of reconstituted extracellular matrix. To verify the applicability of the cell invasion model, multilayer cell constructs of oral squamous cell carcinoma and oral mucosal fibroblasts were exposed to extrinsic urokinase-type plasminogen activator (uPA) or plasminogen activator inhibitor (PAI-1), which are known effectors of cell migration. In addition, the constructs were exposed to both normoxic and hypoxic culture conditions. Microscopic study showed that the presence of uPA enhanced cell invasion, while PAI-1 inhibited cell migration. Western blot and zymographic analysis demonstrated that hypoxia up-regulated uPA and matrix metalloproteinases (MMPs) expression and activity; conversely, PAI-1 level was down-regulated in response to hypoxic challenge as compared to normoxic condition. Our results indicated that the novel 3-D invasion model could serve as an excellent in vitro model to study cancer cell invasion and to test conditions or mediators of cellular migration.

Blotting, Western↗

PubMed1998

[Development of an in vitro model of culture using mouse fetal long bone].

An in vitro model of culture using mouse fetal long bone was firstly established using self-made turning-equipment. The bone formation, mineralization and resorption were observed simultaneously in cultured bone. It provided a useful model for investigating the relationship between bone, development and the effect of nutrients, toxicants and other factors.

Animals↗

PubMed1991

In vitro modeling of the magnetic resonance appearance of cerebral hemorrhage.

In vitro studies provide a framework for understanding the biophysical mechanisms in the evolution of the magnetic resonance (MR) appearance of cerebral hematomas. Varying concentrations of oxyhemoglobin, deoxyhemoglobin and methemoglobin in saline or in concentrated bovine serum albumin solutions were imaged at 1.5 T, allowing assessment of the relative relaxation effects of heme oxidation state, oxygenation, and protein concentration. The findings demonstrate the importance of susceptibility effects arising from local field heterogeneity in producing T2 shortening upon varying hemoglobin concentration for intracellular deoxyhemoglobin and methemoglobin. Correlation between the clinically observed MR appearance of acute and subacute hematomas with these in vitro models is presented.

Brain↗

PubMed2001

[Establishment of an in vitro model for rheumatoid arthritis as test system for therapeutical substances].

In our Tissue Engineering group a 3D in vitro model for rheumatoid Arthritis (in vitro pannus) was established with the aim to develop a standardized drug-screening test to analyze the effects of drugs and different biological substances. The advanced model consists of chondrocyte pellet cultures interacting with rheumatoid arthritis (RA) synovial cell cultures. To establish interactive 3D co-cultures defined rheumatoid arthritis synovial cell populations were centrifuged directly on chondrocyte pellet cultures. Histochemical stainings during time of co-culture revealed obvious invasion by RA synovial cell populations into the chondrocyte matrix. Gene expression analysis showed a downregulation of collagen type II expression in chondrocytes within 2 weeks after co-culture with RA synovial cells. Those interactive co-cultures allow the study of single cell populations as well as the cellular interactions in this system under in vitro conditions. Thus, the established co-culture model may be suitable for routine screening tests, which can be useful in supplementing animal experiments in basic research and drug testing.

Animal Testing Alternatives↗

PubMed1993

An in vitro model to study cellular photosensitizer uptake and photodynamic dose-response relationships of tumor cells.

Cellular fluorescence intensity (CFI) after incubation with varying concentrations of the photosensitizer Photofrin and the photodynamically induced dose-response relationships of hamster melanoma cells (A-MEL-3) were studied in a recently developed in vitro model. After administration of Photofrin to the extracellular serum-free medium, CFI was evaluated by flow cytometry together with constantly fluorescing latex particles used as a reference. After 5 min, 50% of maximal CFI was found, and after 60 min CFI was maximal. No further increase was obtained during the exposure to Photofrin over the incubation period of 4 h. During this plateau phase, CFI was significantly related to the concentration of Photofrin in the extracellular medium (r = 0.94; P < 0.001). Subsequent to increasing intervals of Photofrin exposure, cells were irradiated with laser light at 630 nm (40 mW/cm2, 4J). Cell viability as evaluated by trypan blue exclusion was significantly decreased with increasing concentrations of Photofrin in the medium, and significantly correlated with CFI during the plateau phase. After photodynamic treatment (PDT) cell fluorescence was reduced by about 15%. This was neither dose- nor time-dependent. On the basis of these findings we propose that CFI indicates photosensitizer uptake. This is also supported by the relation between CFI and phototoxicity. The latter also suggests that CFI might be useful to predict the PDT in vivo efficacy by this in vitro model. Besides measurements of photosensitizer uptake and cell photoxicity, the model demonstrates an excellent opportunity to study the molecular mechanisms of action associated with PDT.

Animals↗

PubMed2000

Validation of excised bovine nasal mucosa as in vitro model to study drug transport and metabolic pathways in nasal epithelium.

The present work aims at the validation of excised bovine nasal mucosa as an in vitro model to address transport and metabolism pathways relative to the nasal mucosal uptake of therapeutic peptides. Preservation of the viability of the excised tissue in the course of in vitro studies of up to 3 h was demonstrated by (i) positive viability staining, (ii) constant transepithelial electrical resistance (42 +/- 12 Omega cm(2)), (iii) constant rates of metabolic turnover, and (iv) linear permeation profiles of therapeutic peptides and (3)H-mannitol. Using 1-leucine-4-methoxy-2-naphthylamide as a model substrate, we observed no difference between bovine and human nasal aminopeptidase activity. By a series of therapeutic peptides, no direct correlation was found between their effective permeability coefficients (from 0. 1 x 10(-5) to 5 x 10(-5) cm s(-1)) and their respective molecular masses (from 417 to 3,432 Da), indicating that other factors dominate nasal permeability. For instance, the permeabilities of metabolically labile peptides were concentration dependent and saturable, as demonstrated for two short thymopoietin fragments, Arg-Lys-Asp (TP3) and Arg-Lys-Asp-Val (TP4). By permeation studies using gonadorelin and two gonadorelin derivatives, buserelin and Hoe 013, without and in the presence of the chemical enhancer bacitracin, we also verified the ability of the model to assess chemical enhancer effects and their reversibility. In conclusion, our work demonstrates the potential of the investigated in vitro model, excised bovine nasal mucosa, to explore mechanistic aspects of nasal transport and metabolism of therapeutic peptides.

Absorption↗

PubMed2006

An in vitro model system to study the damaging effects of prolonged mechanical loading of the epidermis.

Pressure ulcers are areas of soft tissue breakdown that result from sustained mechanical loading of the skin and underlying tissues. Today, little is known with respect to the aetiology of these ulcers. This study introduces an in vitro model system to study the effects of clinically relevant loading regimes on damage progression in the epidermis, the uppermost skin layer. Engineered epidermal equivalents (EpiDerm) were subjected to 6.7 and 13.3 kPa for either 2 or 20 h using a custom-built loading device. Tissue damage was assessed by (1) histological examination, (2) tissue viability evaluation, and (3) by the release of a pro-inflammatory mediator, interleukin-1alpha (IL-1alpha). Loading the EpiDerm samples for 2 h increased the IL-1alpha release, although no visible tissue damage was observed. However, in the 20 h loading experiments visible tissue damage and a small decrease in tissue viability were observed. Furthermore, in these experiments the IL-1alpha release increased with magnitude of loading. It is concluded that this in vitro model system can be applied to improve insight in the epidermal damage process due to prolonged mechanical loading and can serve as a sound basis for effective clinical identification and prevention of pressure ulcers.

Epidermis↗

PubMed1989

Lessons from in vitro models of small, irregular, multiple and tunnel-like stenoses relevant to clinical stenoses of valves and small vessels.

This review examines data on the accuracy of the simplified Bernoulli equation for quantitation of pressure drops across small, irregular, multiple and tunnel-like stenoses. This information is drawn from in vitro models of such cardiovascular stenoses and explores the limits of this simplification as they affect accuracy in special situations. Within the physiologic range, discrete small and irregular stenoses present no problems for the measurement of pressure drops using the simplified Bernoulli equation. Multiple side by side orifices of different dimension also give reasonable data using this approach. Tunnel-like stenoses of very small diameter and finite length produce underestimation of the true pressure drop through the stenosis when the simplified Bernouli equation is used. This underestimation is primarily due to neglect of the energy consumed by viscous friction in this situation. These considerations are especially pertinent to the problem of measuring pressure gradients across coronary vessels to assess their clinical significance as well as the adequacy of angioplasty and other intravascular interventional techniques. Because this area needs further exploration, some discussion of in vitro models as such as included.

Blood Flow Velocity↗

PubMed2003

Human mesenchymal stem cells as an in vitro model for human adipogenesis.

OBJECTIVE: To validate the human mesenchymal stem cells (hMSCs) as a new in vitro model for the study of human adipogenesis, to develop the optimal protocol for the differentiation of hMSCs into adipocytes, and to describe effect of mitogen-activated protein kinase on hMSC differentiation into adipocytes. RESEARCH METHODS AND PROCEDURES: hMSCs, obtained commercially, were differentiated by exposure to insulin, dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine three times for 3 days each. Various differentiation conditions were examined to optimize differentiation as measured by Oil Red O staining. The gene expression during adipogenic conversion was assessed by reverse-transcription polymerase chain reaction, real-time reverse-transcription polymerase chain reaction, and Western blotting. RESULTS: hMSCs differentiated into adipocytes to a different extent depending on the experimental conditions. We have found that differentiation medium based on medium 199 and containing 170 nM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM indomethacin, 1 microM dexamethasone, and 5% fetal bovine serum was optimal. However, the replacement of fetal bovine serum with rabbit serum (15%) led to further enhancement of differentiation. Inhibition of mitogen-activated protein kinase activation also facilitated adipogenic conversion of hMSCs. The pattern of genes expressed during hMSC differentiation into adipocytes (adipsin, peroxisome proliferator-activated receptor-gamma, CCAAT/enhancer-binding protein-beta, GLUT4, and leptin) was similar to that observed in other in vitro adipocyte models. DISCUSSION: hMSCs are renewable sources of noncommitted precursors that are able to differentiate into mature adipocytes under the proper hormonal and pharmacological stimuli. Thus, hMSCs represent a new model for the study of human adipogenesis.

1-Methyl-3-isobutylxanthine↗

PubMed2003

[In vitro model system of transepithelial migration of neutrophil by Helicobacter pylori].

BACKGROUND/AIMS: Migration of neutrophil across the intestinal epithelium is a structural hallmark of active intestinal diseases. One of the characteristic histologic features of Helicobacter pylori (H. pylori) infection is neutrophil infiltration of the gastric mucosa. This study was aimed to establish an in vitro model system of transepithelial migration of neutrophil by H. pylori. METHODS: Caco-2 cells were grown on polycarbonate membrane. Confluence and integrity were assessed by measuring the transepithelial electrical resistance (TER) and paracellular permeability of macromolecular marker. IL-8 mRNA expression was assessed after H. pylori infection by quantitative RT-PCR. Isolated neutrophils and H. pylori were applied to the basolateral and apical compartment in separate. Also neutrophil transmigration was assessed by light and electron microscopy. RESULTS: Paracellular leakage of 3[H]-mannitol was negligible over TER 166 Omega x cm2. IL-8 mRNA expression increased after H. pylori infection. Neutrophil transmigration was increased by adding of N-formyl-methionyl-leucyl-phenylalanine or H. pylori. CONCLUSIONS: This in vitro model system could be applied to the study of pathogenesis of H. pylori infection. A further study of quantification of transmigrated neutrophils is demanded to reach a confirmation.

Caco-2 Cells↗

PubMed1999

Development of new adherent mutant from human myeloma-derived cell line: in vitro model of anaplastic transformation of myeloma.

Anaplastic myeloma is a rare but distinct, biologically aggressive variant of myeloma which usually results from dedifferentiation or anaplastic transformation of the myeloma cells. The molecular mechanisms that determine the biologic behavior of anaplastic myeloma and effective treatment modalities have not been well known due to lack of in vitro models. In the present study, we have developed an anaplastically transformed mutant from a human myeloma-derived cell line. In the process of long-term culture of the myeloma-derived IM-9 cell line in low serum and nutrient conditions, an adherent mutant line was developed and named IM-9/AD. This mutant cell line displayed several characteristics resembling anaplastic myeloma such as: 1, large cells with large vesicular nucleus and prominent nucleolus, multinuclearity and high mitotic figures; 2, loss of leukocyte-associated antigens; and 3, higher tumorigenecity in scid mice than its parental cell line. This newly developed mutant cell line may serve as a readily available in vitro model to investigate the biology of anaplastic myeloma.

Animals↗

PubMed2002

Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier.

In keeping with the advance of biotechnology, cell culture becomes an important tool for investigating the transport and the metabolism phenomena. A cell line of human origin, the BeWo choriocarcinoma cell line, was used for the study of the transport and metabolism of opioid peptides across the in vitro model of the placental barrier. Opioid peptides, both naturally occurring and their synthetic analogs, are of interest to be developed as potent analgesics and were included in this study. The apparent permeability coefficients (Pe)s of the peptides containing 4-11 amino acid or analog residues were in the range of 0.23-14.6 x 10(-5) cm/s. The (Pe)s of these peptides were comparable to those of sucrose or dextrans, hydrophilic markers. The (Pe)s of low molecular weight (MW) peptides was not dependent on their MW or molecular size, whereas an inversely linear correlation between (Pe)s and molecular size was observed with the larger peptides. Molecular sieving of the BeWo monolayer restricted the transport of the peptides with MW> or =1033 Da or molecular size > or =6.6 A. Membrane partitioning ability and charge of the peptides were also investigated and found to be the minor factors regulating the extent of peptide permeation. Contrasting to the transport of Tyr-[D-pen-Gly-Phe-D-Pen] (DPDPE) peptide analog across the blood-brain barrier, the transport of DPDPE across the BeWo monolayers were not indicated to be via carrier-mediated transport. The major transport pathway of the opioid peptides across the BeWo monolayers was found to be via paracellular route. In metabolism studies, aminopeptidase was found to be a major enzyme type responsible for the degradation of naturally occurring peptides but not for the synthetic analogs. The finding obtained from the present study reveals the applicability of the BeWo cell line as an in vitro model for investigating placental transport and metabolism of opioid peptides.

Analgesics, Opioid↗

PubMed1998

MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy.

PURPOSE: Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. METHODS: Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. RESULTS: TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (> or = 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. CONCLUSIONS: Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.

Animals↗

PubMedDate not supplied

Diagnosis of urinary bladder rupture using ultrasound contrast cystography: in vitro model and two case-history reports.

Because urinary bladder rupture can be life threatening, a simple, safe technique for evaluating patients is desirable. Current diagnostic protocols involve radiographic imaging, but ultrasound-based contrast techniques have not been methodically evaluated in veterinary patients with urologic trauma. Ultrasound contrast cystography (contrast cystosonography) involves infusion of microbubbled saline solution through a urinary catheter. It was performed in an in vitro model and in 2 dogs with naturally occurring urinary bladder rupture. A positive result consisted of visualizing microbubbles sonographically in fluid surrounding the bladder immediately after infusion of contrast into the urinary catheter. A positive result was obtained both in the in vitro model and in the 2 dogs, with radiographic and surgical confirmation of naturally occurring intraperitoneal urinary bladder rupture in the dogs. Based on the results of this study, ultrasound contrast cystography appears to be more sensitive than two-dimensional (2D) abdominal sonography for detecting naturally occurring urinary bladder rupture in dogs.

Animals↗