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PubMed1998

In vitro-model for Toxoplasma gondii invasion into neuroepithelial cells.

In order to study the interactions of Toxoplasma gondii and neuroepithelial cells morphologically and biochemically we established an easy in vitro model, which simulates cellular contacts in congenital toxoplasmosis. Monolayer cultures of neuroepithelial cells from 13-14-day-old mouse embryos were prepared containing the typical ventricular cell types found in an embryonic brain, such as young neurons, macroglial and microglial cells. Ultrastructural investigations on cultures incubated for 1, 5 and 30 min or 1, 6, 12, 24 and 48 h with T. gondii indicated that all three cell types had been invaded by the parasites, multiplying in parasite vacuoles by means of endodyogeny. Microglial cells had already been penetrated by trophozoites within one minute and showed up to 3 or 5 parasite vacuoles per cell. Neurons and glial cells were invaded within 5 min and contained only one vacuole per host cell. All the parasite vacuoles were bounded by a membrane and bordered by the rough endoplasmic reticulum and mitochondria of the host cell after a few minutes. The vacuoles also contained some membranic tubuli. After 30 min some neuronal neurites were destroyed while the perikarya seemed to be unchanged. After 6 h the cytoplasm of the microglia lost more and more ribosomes and organelles. Neurons and glial cells showed no alterations. After 12h large areas of the vacuole membrane were folded up and lay curled up in the vacuoles. After 24 h incubation T. gondii had destroyed nearly all the microglial cells. The ultrastructure of neurons and glial cells now began to change in the same way as shown for microglial cells. The organelles and cellular membranes disintegrated and after 48 h incubation nearly all the cells in the neuroepithelial cell culture had fallen to pieces. For an identification of T. gondii in vitro by light microscopy or for the characterization of the cell surface we tried to label the parasites with 11 different FITC-stained lectins. None of the tested lectins bound to the parasites. We conclude that our in vitro-model for invasion of T. gondii in neuroepithelial cells opens an opportunity for studying the interaction of these cells or the pharmacological effects on this interaction under defined conditions.

Animals↗
PubMed2003

Follicular lymphoma cell lines, an in vitro model for antigenic selection and cytokine-mediated growth regulation of germinal centre B cells.

In the periphery, B cells differentiate in germinal centres (GCs) of secondary lymphoid organs. Isolated GC cells die quickly in vitro by apoptosis. Therefore, cell lines originating from follicular lymphomas, which are the malignant counterparts of GC B cells, would provide a stable in vitro model to study the immunobiology of GC B cells. We have established three novel human follicular lymphoma cell lines that were characterized with special reference to immunophenotypic features, response to B-cell receptor (BCR) triggering, response to cytokines and cytokine mRNA expression. One of the cell lines, HF-1A3, has a phenotype of a centrocyte. It expresses surface immunoglobulin G (sIgG) and dies by apoptosis following BCR cross-linking. Co-stimulation with interleukin-6 (IL-6), IL-15 or interferon-gamma (IFN-gamma) rescues HF-1A3 cells from BCR-induced apoptosis. The second cell line, HF-28, also represents phenotypically an IgG+ centrocyte. Ligation of its BCR leads to the cell-cycle arrest at G1 instead of apoptosis. HF-28 cells express both CD45RA and RO isoforms, which is unusual in B lymphocytes apart from plasma cells, thus suggesting a transition to plasma cell phenotype. The third cell line, HF-4.9, which phenotypically represents an sIgM+ centroblast, responds by proliferation to BCR cross-linking. These cell lines offer a unique in vitro model to study antigenic selection and cytokine-mediated growth regulation of human GC B cells.

Antigenic Variation↗
PubMed2001

Insights from a novel three-dimensional in vitro model of lyme arthritis: standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts.

OBJECTIVE: To develop a novel 3-dimensional (3-D) in vitro model of Lyme arthritis to use in the study of the interactions between Borrelia burgdorferi (Bb) and human synovial host cells with respect to phagocytosis and potential persistence of Bb as well as the induction of proinflammatory cytokines and chemokines. METHODS: Two distinct culture systems, consisting of synovial membrane explants or interactive synovial cells embedded in 3-D fibrin matrices, were chosen. Both systems were artificially infected with Bb, and the interactions between Bb and synovial tissue/cells were studied by histology, immunohistochemistry, and electron microscopy. Functional analyses included the induction/secretion of cytokines by Bb in the model system. RESULTS: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for >3 weeks. Bb invaded the synovial tissue and the artifical matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration. CONCLUSION: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis.

Borrelia burgdorferi Group↗
PubMed1978

The finite dose technique as a valid in vitro model for the study of percutaneous absorption in man.

An in vitro model of percutaneous absorption has been developed which permits close stimulation of conditions commonly associated with topical drug use in living man. Quantitative comparison of the absorption of selected compounds in the model and in living man was made to test the validity of the model. Excellent agreement has been found between the two sets of data, both with respect to the total amount absorbed and the kinetics of absorption.

Aspirin↗
PubMed1984

Variant generation and selection: an in vitro model of tumor progression.

Evidence for a new in vitro model of tumor progression was sought on the basis of the variant generation and selection hypothesis. The stability of a cloned murine tumor was examined during growth in standard tissue culture or in media containing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Analysis of subclones from the appropriate tumor populations revealed that growth of the L5178Y-F9 clone in 100 ng/ml TPA and 0.1% dimethyl sulphoxide (DMSO) for 2 days yielded a tumor which exhibited increased cellular heterogeneity for susceptibility to both syngeneic and allogeneic natural antibodies (NAb). Subsequent exposure of TPA- and DMSO-treated cells to two cycles of syngeneic NAb-mediated cytolysis resulted in tumor populations which expressed a reduced sensitivity to syngeneic NAb. Thus the elements of tumor variant generation and selection were demonstrated by means of this approach, and repeated cycles of the TPA treatment and NAb cytolysis produced tumor cells with a reduced susceptibility not only to NAb in vitro but also to anti-tumor natural resistance (NR) measured in a tumor elimination assay in vivo. These observations extend the support for the notion that tumor progression can proceed through variant generation and selection. Furthermore, the association of tumor variant generation with exposure to the combination of TPA and DMSO, both non-mutagens, offers a model for studying non-mutagenic mechanisms of tumor development.

Animals↗
PubMed1995

In vitro model of syndactyly replicates the morphologic features observed in vivo.

Syndactyly is a common congenital hand anomaly that may occur after exposure to teratogens. We have developed an in vitro model of syndactyly to investigate the molecular mechanisms underlying this malformation of digit development. Retinoic acid, which regulates pattern formation in vertebrate limb development and is associated with teratogenic malformations, was used in the development of this syndactyly model system. Pregnant Swiss-Webster mice were given retinoic acid by oral gavage on days 10 and 11 of embryonic development (E10 and E11, respectively). The mice were sacrificed on gestational days 13 and 17 (E13, E17) and immediately postnatally (PN). The fetuses were removed and the forelimbs dissected under the operating microscope. The E13 limbs were cultured for 4 days (E13+4) in an organ culture system using a serumless, chemically defined medium. The E17, PN, and E13+4 forelimbs were critically examined for malformations of digit separation and digit development. Retinoic acid-induced fetal mouse forelimb syndactyly was observed in all the groups; 81 percent of E17 limbs, 75 percent of PN limbs, and 77 percent of E13+4 limbs had syndactyly. The morphology of the digital malformations was similar in the E17, PN, and E13+4 limbs. This in vitro model permits further studies to characterize the molecular changes that occur during the development of a congenital hand anomaly.

Animals↗
PubMed2002

Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology.

Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCI2, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl2 demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology.

Cadmium Chloride↗
PubMed1991

Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium, on polyethyleneterephthalate microporous membranes, as an in vitro model.

Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells). Cells are cultivated in a serum-free medium, containing 1 microgram/ml insulin, 1 ng/ml epidermal growth factor, 10 micrograms/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen. Cells rapidly adhere, grow, and form confluent monolayers; after 15 days, scanning electron microscopy reveals numerous uniform microvilli. Domes, which develop on nonporous substrata, are absent on high porosity membranes. Culture medium from upper and lower compartments of microplate inserts and cell lysates were immunoprecipitated after labeling with [3H]glucosamine and leucine; analysis was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. [3H]transferrin is found mainly in the lower compartment and in cells; [3H]apolipoprotein B is released in both compartments, and fibronectin almost entirely recovered in the lower compartment; [3H]transferrin receptors and ferritin are only present in cell lysates. Binding experiments also show that transferrin receptors are accessible from the lower compartment. These results suggest that CaCo2 cells, cultivated in synthetic medium on membranes of appropriate porosity, could provide an in vitro model of the intestinal barrier, with the upper compartment of the culture insert corresponding to the apical pole facing the intestinal lumen and the lower one to the basal pole in contact with blood.

Adenocarcinoma↗
PubMed2004

Neuroblastoma survival and death: an in vitro model of hypoxia and metabolic stress.

Heterogeneous oxygen tension and access to metabolites in solid tumors may produce variability in response to adjuvant therapy. To better understand these microenvironmental features, we examined survival and proliferation of neuroblastoma (NB) cells in an in vitro model of hypoxia and metabolite deprivation. Human NB cells (SH-SY5Y) were subjected to a "self-generated" diffusion gradient of nutrient and oxygen deprivation in a modified in vitro "sandwich model." In this model, the extent of both hypoxia and metabolite deprivation were individually altered, and the effects of each were studied. Cellular proliferation was confirmed by proliferating cell nuclear antigen (PCNA) immunocytochemistry and morphology and hypoxia by vascular endothelial growth factor (VEGF) and pimonidazole immunocytochemistry. We examined apoptotic cell death using TUNEL analysis, assaying for plasma membrane transfer of phosphotidylserine and the presence of the anti-apoptotic protein Bcl-2 using immunocytochemistry. As predicted, cellular survival diminished with increasing duration and severity of hypoxia and metabolite deprivation; oxygen deprivation was determined to be the more important contributory factor to early survival and proliferation. PCNA immunocytochemistry confirmed decreasing fractions of proliferating cells as a function of distance from oxygen and metabolites. VEGF and Bcl-2 immunoreactivity increased with prolonged exposure and increased extent of oxygen/metabolite deprivation. TUNEL analysis and phosphotidylserine transfer demonstrated cellular death of hypoxic and metabolite-deprived NB cells in a manner consistent with a mitochondrial apoptotic pathway. This in vitro model demonstrates that increasing the severity of hypoxia and metabolite deprivation results in diminished proliferation and greater apoptotic death, observations analogous to that of clinical NB tumors.

Apoptosis↗
PubMed2001

In vitro models to study cellular differentiation and function in human prostate cancers.

In Vitro Models to Study Cellular Differentiation and Function in Human Prostate Cancers. To augment the currently available models of human prostate cancer in vitro, we have established extended life-span epithelial cultures from biopsies of well-differentiated prostate cancers. The genetic identity of the target cells was assessed by allelotyping, using microsatellites located on chromosome 8p, and microdissection of tissues and primary cell cultures. Cells with an extended life span (PxE6) were derived by recombinant retrovirus infection to introduce the human papilloma virus E6 gene (epithelial cells). Immunophenotyping of the resultant cell strains confirmed retention of differentiated cell functions, and the genotype of the E6-expressing epithelial cells was stable, while SV40-immortalized cultures were more unstable, leading to tetraploidy. All PxE6 cells eventually senesced, but an immortalized epithelial culture, P4E6, was derived from one of the epithelial cultures. The properties of this cell line, which remains close to diploid, are similar to those of early prostate cancer cells, and it retains expression of many prostate-associated antigens, such as prostate-specific antigen (PSA).

Allelic Imbalance↗
PubMed2001

Colonic fermentation as affected by antibiotics and acidic pH: Application of an in vitro model.

Antimicrobial substances such as vancomycin or metronidazole suppress normal gut flora, thereby preventing physiological fermentation of colonic substrates that may promote mucosal inflammation. This study was designed to establish an in vitro model of microbial metabolism in the colon under control and disturbed conditions (acidic pH) to investigate specific effects of vancomycin and metronidazole on the production of short chain fatty acids (SCFA), which play a pivotal role in maintaining homeostasis in the colon. The experiments were carried out with the colon simulation technique (Cositec) representing an in vitro model for the semi-continuous incubation of defined colon contents. Inocula and fermentable substrates were sampled from cecal contents of fistulated pigs. Disturbed microbial metabolism was generated by reduction of pH in the fermentation vessels from 6.7 to 5.8 and 5.1. In general, application of either vancomycin or metronidazole resulted in a significant decrease of SCFA production rates indicating substantial disturbance of the homeostasis of microbial metabolism. With low doses of vancomycin acetate and butyrate production rates were reduced and with high doses of the antibiotic propionate production was inhibited to a greater extent. Treatment with metronidazole inhibited butyrate production almost completely. Similarly, low pH caused a reduction in total SCFA production, which was mainly due to respective decrease of acetate synthesis. Metronidazole effects were not consistently changed at low pH. The Cositec system provides an excellent facility to test the effects of different antibiotics under defined conditions. In this study, both vancomycin and metronidazole affected microbial metabolism to a considerable extent. Both substances may thus be responsible for disturbances of colon function in vivo.

Acid-Base Equilibrium↗
PubMed2001

An in vitro model and case report that used gelatin sponge to restore amniotic fluid volume after spontaneous premature rupture of the membranes.

OBJECTIVE: The purpose of this project was to study an in vitro model for plugging membrane defects with gelatin sponge and to develop a method with which to use this material to treat premature rupture of the membranes. STUDY DESIGN: Fetal membranes were fixed over the opening of a flask that was filled with saline solution and gelatin sponge. Defects of various sizes were created, and the usefulness of differing sizes of gelatin sponge to obstruct the defects was observed. This technique was then applied to a case of previable, spontaneous premature rupture of the membranes. RESULTS: Fifteen amniotomies were performed in the in vitro model. The gelatin sponge obstructed all defects less than 7 mm in length, when pieces up to 1 x 1 cm in dimension (n = 8 amniotomies) were used. For larger defects or those defects with a complex shape (such as cruciate), gelatin sponge was not effective at arresting fluid loss (n = 4 amniotomies). An inspection of larger gelatin sponge pieces, after instillation through a 12-gauge angiocatheter, revealed 36% (15 of 42 pieces) of 1 x 1 - cm pieces remained intact. A case of spontaneous, previable premature rupture of the membranes was treated with this material. A favorable outcome was observed. CONCLUSION: Gelatin sponge is successful at arresting the egress of fluid through membrane defects when smaller defects are present. Complex or larger linear defects may not be treated by this method alone and necessitate adjuvant therapies. This therapeutic strategy can be applied to cases of previable, spontaneous premature rupture of the membranes.

Adult↗
PubMed1988

Plasmodium falciparum: the behavior of clinical isolates in an in vitro model of infected red blood cell sequestration.

An in vitro model of Plasmodium falciparum-infected red blood cell sequestration which uses C32 amelanotic melanoma cells as targets has been used to examine the binding capacity of infected red blood cells from subjects with naturally acquired P. falciparum infections of varying severity. The binding of infected red blood cells (IRBCs) to melanoma cells was specific to cells containing mature parasites. Variations in target cell density and in conditions of growth had significant effects on binding. Binding was pH dependent, being maximum at a pH of 6.9. Using standardized conditions the binding capacity of individual isolates of P. falciparum could be measured with a high degree of reproducibility. Binding capacity of IRBCs from 51 subjects between the ages of 6 months and 15 years varied between 12 and 1254 IRBCs per 100 melanoma cells when RBC suspensions at a 1% parasitemia and 4% hematocrit were used. Variation in binding was not related to the level of peripheral parasitemia of the isolate or to differences in adaptation to culture conditions. The binding capacity of parasitized cells from subjects with cerebral malaria did not differ from that of IRBCs from subjects with less serious clinical manifestations.

Adolescent↗
PubMed2001

Attenuation of catalase activity in the malignant phenotype plays a functional role in an in vitro model for tumor progression.

We have developed an in vitro model to study the molecular mechanisms of tumor progression. Using repeated treatments with ionizing radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we caused malignant progression of a papilloma producing mouse keratinocyte cell line, 308 cells. In a previous study we have shown that the malignant variants of 308 cells have elevated reactive oxygen species (ROS) levels, and have established a functional role for the pro-oxidant state in the progressed phenotype (Carcinogenesis 20 (1999) 2063). In this study, we have evaluated the status of intracellular defense mechanisms for ROS scavenging in the progressed phenotype to identify sources that contribute to their pro-oxidant state. Our results demonstrate that a reduction in several anti-oxidant defense mechanisms, including catalase and glutathione S-transferase mu, correlates with the emergence of the malignant phenotype. We provide evidence that attenuation of catalase activity may play a functional role in the malignant progression of mouse keratinocytes.

Animals↗
PubMedDate not supplied

Flutamide-hydroxypropy-beta-chiyclodextrin complex: formulation, physical characterization, and absorption studies using the Caco-2 in vitro model.

PURPOSE: The objective of this research was to formulate flutamide (FLT) in hydroxypropyl-beta-cyclodextrin (HPbetaCyD), and to investigate FLT transcellular permeation from the complex using the Caco-2 monolayer in vitro model. METHODS: Classical solubility data were used to derive thermodynamic parameters which, together with Differential Scanning Calorimetry (DSC), (1)H-NMR and (19)F-NMR, were used to characterize and derive stability constants for the FLT-HPbetaCyD complex. The Caco-2 cell line was used to examine the role of HPbetaCyD on the passage of FLT across cell monolayers in vitro. RESULTS: The solubility of FLT in water (1.46 mmol/L) increased almost 170 times (to 243.45 mmol/L) in the presence of 50% (w/v) HPbetaCyD. Solubility data for FLT in aqueous HPbetaCyD were used to derive thermodynamic parameters (DeltaG degrees at 298 K = -3.48, DeltaH degrees = 2.85, DeltaS degrees at 298 K = 21.24). The solubility of FLT in HPbetaCyD increased proportionally with an increase in temperature. The FLT-HPbetaCyD complex had an A(L)-type (DSC) isotherm, consistent with a linear increase in FLT solubility and unchanged stoichiometry. The DSC of free FLT and HPbetaCyD showed endothermic peaks at 110 degrees C and 300 degrees C, respectively. FLT-HPbetaCyD did not display a free-FLT endothermic response, but exhibited broadening of the endothermic peak in the HPbetaCyD region. (19)F- and (1)H-NMR chemical shifts of FLT moved upfield as a function of its increased solubility in the presence of HPbetaCyD. The FLT-HPbetaCyD stability constant, K(s) (1:1) was estimated to be 356 M(-1 )and 357 M(-1), from thermodynamic and (19)F NMR data, respectively. The apical-to-basal permeability coefficient (P(eff) = 4.75 x 10(-5) cm.s(-1)) for FLT across Caco-2 cell monolayers at 37; C increased as HPbetaCyD concentrations were reduced, indicative of transepithelial passage via passive diffusion of available free FLT in solution. Studies in the presence and absence of Ca(2+ )ruled out a significant paracellular transport component. CONCLUSIONS: FLT-HPbetaCyD is a relatively stable, 1:1 inclusion complex. Formation of this complex substantially increases the water solubility of FLT, but HPbetaCyD, except in high dilution, reduces transcellular passage of FLT in the Caco-2 cell in vitro model.

2-Hydroxypropyl-beta-cyclodextrin↗
PubMed2005

[The in vitro model of inhalation anesthesia using rodent ventilator].

OBJECTIVE: To set up a method of applying inhalation anesthesia in rodent using rodent ventilator and to study the dynamic procedure of the in vitro model. METHODS: The output port of the anesthesia machine was connected to the input port of the rodent ventilator, which was connected to a syringe simulating the lung. After supply of anesthetic gas, the gas samples from the input port of the ventilator and the syringe in the end-expiratory phase were collected at 10, 20, 30, 40, 50, 60, 90, 120, 180, 300, 600 and 900 seconds respectively and were determined using the gas chromatography(GC). The ratios of the anesthetic concentrations of the syringe to that of the input port were calculated (CE/CI). In elimination phase, the gas samples from the syringe were collected at 0,10, 20, 30, 40, 50, 60, 90, 120, 180, 300, 600 and 900 seconds respectively and were determined by GC. The ratios of the anesthetic concentrations of the gas at 10, 20, 30, 40, 50, 60, 90, 120, 180, 300, 600 and 900 seconds to that at 0 second were calculated(C'E/C0). RESULTS: CE/CI increased in the inhalation phase, there was an inverse relationship between CE/CI and time, the correlation coefficients were 0.90, 0.95 and 0.93 respectively (P < 0.01). The mathematical fitting equations were y = -0.19 + 0.19x(-1), y = -7.75 + 0.99x(-1), and y = -7.21 + 0.97x(-1) respectively. C'E/C0 decreased in the elimination phase,the correlation coefficients were 0.90, 0.94 and 0.95 respectively (P < 0.01). The mathematic fitting eqations were y = 5.65-0.02x(-1), y = 7.82-0.01x(-1),and y = 8.20-0.01x(-1), respectively. CONCLUSION: The in vitro model of rodent inhalation anesthesia using the rodent ventilator was set up. The establishment of this model has provided a basis for studies on inhalation anesthesia in rodents.

Anesthesia, Inhalation↗
PubMedDate not supplied

Development of a multilayered in vitro model for studying events associated with wound healing.

Simplified in vitro models such as cellular monolayer cultures have only limited usefulness in the study of cutaneous wound repair processes. This has stimulated the investigation of three-dimensional tissue equivalent systems such as the dermal and skin equivalent models. With the use of a wound system constructed of rat tail type I collagen and human dermal fibroblasts, experimental wounding was accompanied by problems with mechanical scoring of the plastic substratum which prevented cell migration. These problems were overcome with the use of a multilayered model in which a punch biopsy-wounded dermal equivalent (bilayered model) or skin equivalent (tri-layered model) was placed onto an acellular collagen lattice and fixed in place with polymerizing collagen. This model permitted observation of the process of cellular repopulation of the "wound space," into which both fibroblast and keratinocyte migration commenced within 1 day. The number of fibroblasts in this space increased dramatically over a period of 9 days, the cells appearing to migrate both over and through the acellular lower collagen layer. Keratinocyte reepithelialization of the "wound space" was completed after 5 days. With the model it was shown that platelet-derived growth factor--AB and epidermal growth factor had positive effects in increasing fibroblast number within the wound space. In conclusion, the model described here should facilitate the study of fibroblast and keratinocyte responses to a wound stimulus in vitro and be a plausible in vitro system for evaluating agents which may have a potential stimulatory or inhibitory effect on numerous cellular responses associated with wound healing.

Journal Article↗
PubMed2005

An in vitro model of traumatic brain injury utilising two-dimensional stretch of organotypic hippocampal slice cultures.

Traumatic brain injury (TBI) is caused by rapid deformation of the brain, resulting in a cascade of pathological events and ultimately neurodegeneration. Understanding how the biomechanics of brain deformation leads to tissue damage remains a considerable challenge. We have developed an in vitro model of TBI utilising organotypic hippocampal slice cultures on deformable silicone membranes, and an injury device, which generates tissue deformation through stretching the silicone substrate. Our injury device controls the biomechanical parameters of the stretch via feedback control, resulting in a reproducible and equi-biaxial deformation stimulus. Organotypic cultures remain well adhered to the membrane during deformation, so that tissue strain is 93 and 86% of the membrane strain in the x- and y-axis, respectively. Cell damage following injury is positively correlated with strain. In conclusion, we have developed a unique in vitro model to study the effects of mechanical stimuli within a complex cellular environment that mimics the in vivo environment. We believe this model could be a powerful tool to study the acute phases of TBI and the induced cell degeneration could provide a good platform for the development of potential therapeutic approaches and may be a useful in vitro alternative to animal models of TBI.

Animals↗