A novel in vitro model of human mesothelioma for studying tumor biology and apoptotic resistance

Like many tumors, malignant mesothelioma exhibits significant chemoresistance and resistance to apoptosis in vivo that is not seen in current in vitro models. To study the mechanisms of this multicellular resistance, biologically relevant in vitro models are necessary. Therefore, we characterized and tested human mesothelioma tissue grown in vitro as tumor fragment spheroids. After 5-10 d in culture, fragments from each of 15 human mesothelioma tumors rounded into spheroids. The tumor fragment spheroids maintained multiple characteristics of the original tumors for up to 3 mo including the presence of viable mesothelioma cells, macrophages, and a collagen-rich stroma. In 14-d-old spheroids, mesothelioma cells showed the same proliferation rate and expression of a death receptor, DR5, as in the original tumor. To determine responses to treatment, we treated tumor fragment spheroids grown from three separate tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL) plus cycloheximide, that induced near total apoptosis in three human mesothelioma cell lines (M28, REN, MS-1) grown as monolayers (94 +/- 6% apoptosis; mean +/- SEM). Compared with mesothelioma cells in monolayers, mesothelioma cells in the spheroids were resistant to TRAIL plus cycloheximide (32 +/- 4% apoptosis; mean +/- SEM). Apoptotic resistance of mesothelioma cells was significantly reduced by inhibiting either the PI3K/Akt pathway with LY294002 (47 +/- 6% apoptosis) or the mTOR pathway with rapamycin (50 +/- 17% apoptosis). We conclude that human mesothelioma can be maintained in vitro in a biologically relevant model that exhibits apoptotic resistance, thereby permitting study of its tumor biology and of novel approaches to therapy.     

A novel tissue engineered three-dimensional in vitro colorectal cancer model

The interactions of cancer cells within a solid mass with the surrounding reactive stroma are critical for growth and progression. The surrounding vasculature is recruited into the periphery of the growing tumour to supply cancer cells with nutrients and O₂. This study focuses on developing a novel three-dimensional (3-D) in vitro biomimetic colorectal cancer model using colorectal cancer cells and connective tissue cells. The 3-D model comprises a dense artificial cancer mass, created by partial plastic compression of collagen type I containing HT29 colorectal cancer cells, nested in a non-dense collagen type I gel populated by fibroblasts and/or endothelial cells. HT29 cells within the dense mass proliferate slower than when cultured in a two-dimensional system. These cells form tumour spheroids which invade the surrounding matrix, away from the hypoxic conditions in the core of the construct, measured using real time O₂ probes. This model is also characterized by the release of vascular endothelial growth factor (VEGF) by HT29 cells, mainly at the invading edge of the artificial cancer mass. This characterization is fundamental in establishing a reproducible, complex model that could be used to advance our understanding of cancer pathology and will facilitate therapeutic drug testing.     

A novel in vitro model of trophoblast-mediated decidual blood vessel remodeling

In vivo the extravillous trophoblasts (EVTs) penetrate the decidua and the first third of the myometrium to remodel the uterine spiral arteries and achieve the high-flow, low-resistance circulation characteristic of the intervillous space of the term placenta. Much of our understanding of these processes comes from histologic analysis of placental bed biopsies, a limited tissue source and one that can provide only a snapshot of a dynamic process. To better characterize these cellular interactions, we have developed an in vitro co-culture system in which first trimester villous explants are cultured at low oxygen tension in contact with 2-mm(2) sections of decidua parietalis from the same patient. Hematoxylin eosin counterstaining of paraffin sections shows that EVT columns form at the tips of the placental villi and adhere and penetrate the decidual surface. The decidual blood vessels in the path of the EVT show morphologic disruption. Immunohistochemical analysis of the co-cultures using both an endothelial specific anti-CD31 and an anti-smooth muscle actin antibody show a disruption of the integrity of the vessel lining together with a complete loss of organized smooth muscle actin surrounding the blood vessels. In contrast control decidua samples in the absence of placental villi exhibit blood vessels with a complete endothelial lining and an organized muscular sheath. Using both an anti-cytokeratin-7 and anti-Cdx-2 antibody specific to trophoblasts, we show that these changes coincide with invasion of the vessels by endovascular trophoblasts and penetration of the decidua by interstitial EVTs. No EVTs were found in the control decidua. Thus we conclude that this in vitro model mimics the physiologic change observed in vivo during trophoblast invasion into maternal decidual tissues, and as such it may provide useful information concerning the interactions between EVTs and decidual cells and vessels during early gestation.     

Cytotoxicity of human phagocytes studiedin vitro in a novel model based on Neutral Red absorbtion

The aim of these investigations was to establish a model for the study of neutrophil (NEU) and monocyte (MO) mediated cytotoxicity (TOX), and to study the protective actions of model protease inhibitor, peroxide scavengers and glucocorticoids in this model. Confluent human fibroblasts were used as target cells (T) and NEU and MO were used as effector cells (E), ratio E/T was 5–101. After triggering E with PMA (16–48 nM) for about 24 hours, remaining viable T were detected by incorporation of Neutral Red (NR). Oxidant-induced TOX was performed with H₂O₂ and t-BuOOH. In contrast to MO TOX, NEU TOX was inhibited by antiprotease and scavengers. On the other hand, MO TOX was inhibited by glucocorticosteroids. This indicates different TOX mechanisms by NEU and MO.     

Using human iPS cell-derived enterocytes as novel in vitro model for the evaluation of human intestinal mucosal damage

Objective and design

The primary component in gut mucus is mucin 2 (MUC2) secreted by goblet cells. Fluctuations in MUC2 expression are considered a useful indicator for evaluating mucosal damage and protective effect of various agents using animal studies. However, there are few in vitro studies evaluating mucosal damage using MUC2 as the indicator. Hence, we attempted to establish a novel in vitro model with MUC2 as the indicator for evaluating drug-induced mucosal damage and protective effect using enterocytes derived from human iPS cells.

Methods

Compounds were added into enterocytes derived from human iPS cells, and MUC2 mRNA and protein expression levels were evaluated. Further, the effect of compounds on membrane permeability was investigated.

Results

Nonsteroidal anti-inflammatory drugs were found to decrease MUC2 mRNA expression in enterocytes, whereas mucosal protective agents increased mRNA levels. Changes in MUC2 protein expression were consistent with those of mRNA. Additionally, our results indicated that indomethacin caused mucosal damage, affecting membrane permeability of the drug. Moreover, we observed protective effect of rebamipide against the indomethacin-induced permeability increase.

Conclusions

The developed model could facilitate evaluating drug-induced mucosal damage and protective effects of various agents and could impact drug development studies regarding pharmacological efficacy and safety.

     

Using human iPS cell-derived enterocytes as novel in vitro model for the evaluation of human intestinal mucosal damage

Objective and design

The primary component in gut mucus is mucin 2 (MUC2) secreted by goblet cells. Fluctuations in MUC2 expression are considered a useful indicator for evaluating mucosal damage and protective effect of various agents using animal studies. However, there are few in vitro studies evaluating mucosal damage using MUC2 as the indicator. Hence, we attempted to establish a novel in vitro model with MUC2 as the indicator for evaluating drug-induced mucosal damage and protective effect using enterocytes derived from human iPS cells.

Methods

Compounds were added into enterocytes derived from human iPS cells, and MUC2 mRNA and protein expression levels were evaluated. Further, the effect of compounds on membrane permeability was investigated.

Results

Nonsteroidal anti-inflammatory drugs were found to decrease MUC2 mRNA expression in enterocytes, whereas mucosal protective agents increased mRNA levels. Changes in MUC2 protein expression were consistent with those of mRNA. Additionally, our results indicated that indomethacin caused mucosal damage, affecting membrane permeability of the drug. Moreover, we observed protective effect of rebamipide against the indomethacin-induced permeability increase.

Conclusions

The developed model could facilitate evaluating drug-induced mucosal damage and protective effects of various agents and could impact drug development studies regarding pharmacological efficacy and safety.

     

A novel in vitro ELISA for estimation of glycoprotein content in human rabies vaccines

In vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r² = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.     

A novel murine model of disseminated trichosporonosis.

Serious infections caused by Trichosporon beigelii have been noted with increasing frequency in immuno-compromised patients. Progress in understanding the pathogenesis of this emerging infection has been limited by the lack of an animal model. We developed a CF1 mouse intravenous inoculation model of disseminated trichosporonosis to evaluate the pathogenicity of T. beigelii in transiently immunosuppressed mice. Four inocula (1 x 10(6), 1 x 10(7), 2 x 10(7), and 4 x 10(7) CFU per animal) of one clinical strain of T. beigelii 3001 were tested. Mice in groups of 10 were each injected with a single intravenous dose of one inoculum. Mortality correlated with inoculum size, as survival time was significantly shorter in mice injected with 4 x 10(7) or 2 x 10(7) CFU than in mice that received 1 x 10(7) or 1 x 10(6) CFU (P less than 0.01). Necrotizing abscesses with conidial and hyphal elements and neutrophil and macrophage infiltration were observed in all major organs examined. Resistance to infection was markedly lowered by immunosuppression with either cyclophosphamide or cortisone acetate, with a significantly shorter survival time and a greater fungal burden per organ in immunosuppressed animals than in normal animals (P less than 0.01). Nine additional strains were inoculated intravenously with around 5 x 10(6) CFU. Injection of each of these strains caused 100% mortality, in a pattern similar to that observed with strain 3001.     

人子宫内膜蜕膜化间质细胞与囊胚体外共培养建立人胚胎着床模型

目的建立有效的体外人胚胎着床模型,为体外研究人胚胎着床过程提供条件。方法将人子宫内膜蜕膜化的间质细胞与囊胚共同培养,光镜下观察囊胚在细胞上的定位、粘附及侵入过程;免疫荧光法测定共同培养系统中的角蛋白,以确定着床模型的建立。结果与蜕膜细胞共培养5—10h后,囊胚开始黏附在细胞层上,48h后侵入蜕膜细胞间;共培养48h后,胚胎及周围的内膜细胞表达角蛋白阳性。结论囊胚与蜕膜化细胞共同培养,可以成功建立体外胚胎着床模型,更好地模拟体内着床时状态,是较理想的体外研究模型。     

A novel model for biomechanical behavior of human brain in epidural hematoma injuries

To obtain an appropriate model for the simulation of the biomechanical behavior of brain tissue and the deformation of ventricles, in particular, we have developed a novel computerized plain strain finite element model. For optimum results, a multiple loading solutions approach using various tissue parameters for the simulation of epidural hematoma have been tested. For this purpose, CT-Scan of a patient with traumatic epidural hematoma has been modeled. By changing the tissue parameters (E and nu) and increasing intraventricular pressure gradient, the displacement of similar points in the modeled ventricle was compared with the true values obtained from patient's CT-Scan taken 3 months later after the resolution of hematoma. The magnitudes leading to least errors were determined. Best solutions were obtained with E=11-12 kPa and DeltaP=1.25-1.5 kPa (7.5-9.4 mmHg), which were consistent with the patient's clinical condition. Biomechanical modeling of unilateral displacement loadings, which are the conditions similar to surgical navigation systems, without considering ventricular geometry and their internal pressure resulted in unacceptable results.     

A novel inhibitor of glutamate release reduces excitotoxic injury in vitro.

Excessive release of glutamate has been implicated in the pathogenesis of excitotoxic neurologic disorders, such as stroke. BW 1003C87, an inhibitor of glutamate release and a putative Na+ channel antagonist, reduced veratridine-stimulated, tetrodotoxin- and dizocilpine-sensitive toxicity (measured by lactate dehydrogenase efflux) in neuron-enriched cortical cultures (IC50 = 5 microM). In contrast, BW 1003C87 (300 microM) had no effect on toxicity induced by direct application of 1 mM glutamate or 1 mM N-methyl-D-aspartate, or by depolarization with 50 mM KCl. Glutamate release inhibitors such as BW 1003C87 may provide a novel approach to protection from excitotoxicity.     

RAIN-Droplet: a novel 3D in vitro angiogenesis model

Angiogenesis is fundamentally required for the initialization, development and metastatic spread of cancer. A rapidly expanding number of new experimental, chemical modulators of endothelial cell function have been described for the therapeutic inhibition of angiogenesis in cancer. Despite this expansion, there has been very limited parallel growth of in vitro angiogenesis models or experimental tools. Here we present the Responsive Angiogenic Implanted Network (RAIN)-Droplet model and novel angiogenesis assay using an endothelial cell culture model of microvascular endothelial cells encapsulated in a spontaneously self-assembling, toroidal hydrogel droplet uniquely yielding discrete, pre-formed, angiogenic networks that may be embedded in 3D matrices. On embedding, radial growth of capillary-like sprouts and cell invasion was observed. The sprouts formed not only as outgrowths from endothelial cells on the surface of the droplets, but also, uniquely, from the pre-formed network structures within the droplet. We demonstrate proof of principle for the utility of the model showing significant inhibition of sprout formation (P<0.001) in the presence of bevacizumab, an anti-angiogenic antibody. Using the RAIN-Droplet assay, we also demonstrate a novel dose-dependent pro-angiogenic function for the characteristically anti-angiogenic multi-kinase inhibitor sorafenib. Exposure of endothelial cells in 3D culture to low, non-lethal doses (<1 μM) of sorafenib after initiation of sprouting resulted in the formation of significantly (P<0.05) more endothelial sprouts compared with controls over a 48-h period. Higher doses of sorafenib (5 μM) resulted in a significant (P<0.05) reduction of sprouting over the same time period. The RAIN-Droplet model is a highly versatile and simply constructed 3D focal sprouting approach well suited for the study of vascular morphogenesis and for preclinical testing of drugs. Furthermore, the RAIN-Droplet model has facilitated the discovery of a novel pro-angiogenic capacity for sorafenib, which may impact the clinical application and dosing regimen of that drug.     

Activin A increases invasiveness of endometrial cells in an in vitro model of human peritoneum

The aim of this study was to investigate whether activin A has an effect on the attachment and/or invasion of endometrial cells in a modeled peritoneum in vitro. Cultured endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs) were treated with activin A (6.25-50 ng/ml) and with activin A (25 ng/ml) with and without inhibin A or follistatin. Fluorescent labeled cells were added to confluent peritoneal mesothelial cells (PMCs) and to a monolayer of confluent PMCs grown in a Matrigel invasion assay. The rate of endometrial cell attachment and invasion through PMCs was assessed. The expression of cell adhesion proteins N- and E-cadherin was evaluated with real-time RT-PCR. Activin A (25 ng/ml) promoted invasion of the endometrial cells through the modeled peritoneum (>2-fold versus control) and this effect was partially reversed by inhibin A and follistatin. Activin A had no effect on the rate of attachment of the endometrial cells to the PMCs or in the rate of proliferation. In addition, activin A induced a decreased mRNA expression of E-cadherin in cultured EECs. In conclusion, activin A increases invasion of EECs and ESCs into modeled peritoneum. In EECs, this effect may be related to down-regulation of E-cadherin expression. Further studies are warranted to evaluate the role of activin-A in the genesis of the endometriotic lesion.     

A novel 3D in vitro culture model to study stromal-epithelial interactions in the mammary gland

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, the development of defined three-dimensional (3D) models is essential. In the present study, we have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo. Cocultures of a human mammary epithelial cell line MCF10A and human mammary fibroblasts obtained from reduction mammoplasties embedded in either a type I collagen or a mixed Matrigel-collagen matrix were carried out for up to 6 weeks. Histological and ultrastructural analysis confirmed the formation of ductal and alveolar structures. The importance of the stromal cells was apparent in both matrices; in the collagen gels the presence of reduction mammoplasty fibroblasts accelerated the initial formation of epithelial structures, and in the mixed Matrigel-collagen gels the presence of those fibroblasts was necessary for the formation of ductal structures. These models provide an excellent system to study tissue organization, epithelial morphogenesis, and breast carcinogenesis.     

A novel retinoic acid-resistant acute promyelocytic leukemia model in vitro and in vivo (review).

Differentiation-inducing therapy by all-trans retinoic acid (RA) is now a standard therapy in patients with acute promyelocytic leukemia (APL). Nearly all patients achieve complete remission by the treatment of all-trans RA, however, clinical remissions are usually of brief duration, and these patients often develop RA-resistant disease. The mechanisms of RA-resistance in APL cells are poorly understood and most clinical approaches have not been successful in overcoming RA-resistance. We have recently established a novel APL cell line (UF-1) with RA-resistant features. In addition, we have established human GM-CSF-producing transgenic (hGMTg) SCID mice system. UF-1 cells were inoculated either intraperitoneally or subcutaneously into hGMTg SCID mice and made the first RA-resistant murine APL model. These RA-resistant APL model systems in vitro and in vivo may be useful for investigating the molecular studies on the block of leukemic cell differentiation and as means to investigate the mechanisms of RA-resistance. Moreover, this murine model system will be important for developing novel therapeutic strategies in RA-resistant APL.     

Development of a novel three-dimensional in vitro model of oral Candida infection

Oropharyngeal candidiasis is emerging as a serious health problem in the elderly as well as other chronically immunosuppressed patient populations. Several approaches have been used to study the interactions between Candida and the oral mucosa in vitro. Recently, three-dimensional organotypic systems of the oral mucosa have been developed, which provide an organizational complexity that is between the culture of single cell types and organ cultures in vivo. In this report we describe the development of a novel three-dimensional system of the human oral mucosa based on an immortalized oral keratinocyte cell line. Unlike the commercially available cell line systems, this system also contains a connective tissue cell component, which ensures the quality and resemblance of the tissue model to the human oral masticatory mucosa and submucosa. Using a panel of Candida albicans strains with variable virulence in vivo, we showed that the extent of tissue damage, fungal invasion and host inflammatory response in this system was proportional to the well-documented in vivo virulence potential of these strains. Therefore, this investigation has added another useful tool in the study of host pathogen interactions in oral candidiasis.     

Computer-controlledin vitro model of the human left heart

The decision for surgical intervention in the treatment of stenosis and for regurgitation of the mitral valve demands an objective and quantitative evaluation of the severity of mitral valve disease. The availability of ultrasound techniques capable of analysing flow velocities across valves and to produce representative images of valve orifices has increased the interest in the hydraulics of cardiac valves. To isolate and study the determinants of transmitral flow, an in vitro model of the human left heart was built. From the model it is possible to differentiate the influence of the different determinants of left heart performance on transmitral flow: preload, compliance of the left atrium and ventricle, peripheral resistance (afterload) and heart rate. The mechanical part of the model consists of a reservoir connected to an elastic closed circuit (Latex pulmonary veins, left atrium, left ventricle and aortic arch) with replaceable mitral and aortic valves. The electronic part of the model drives and controls the hydraulic part, allowing the independent regulation and monitoring of left atrial and left ventricular pressures p, volumes V and 'pV-loops' throughout the cardiac cycle at different cardiac rhythms. Left atrial filling pressure and aortic resistance are variable in a controlled fashion. Echo-Doppler study of the mitral valve and the transmitral valve flow is possible both from an atrial and a ventricular window in the model. This technical note describes the model.     

An in vitro model for the study of human implantation

Citation Holmberg JC, Haddad S, Wünsche V, Yang Y, Aldo PB, Gnainsky Y, Granot I, Dekel N, Mor G. An In vitro model for the study of human implantation. Am J Reprod Immunol 2012; 67: 169–178 Problem Implantation remains the rate‐limiting step for the success of in vitro fertilization. Appropriate models to study the molecular aspects of human implantation are necessary in order to improve fertility. Methods First trimester trophoblast cells are differentiated into blastocyst‐like spheroids (BLS) by culturing them in low attachment plates. Immortalized human endometrial stromal cells and epithelial cells (ECC‐1) were stably transfected with GFP or tdTomato. Co‐culture experiments were monitored using Volocity imaging analysis system. Results This method demonstrates attachment and invasion of BLS, formed by trophoblast cells, into stromal cells, but not to uterine epithelial cells. Conclusion We have developed an in vitro model of uterine implantation. The manipulation of this system allows for dual color monitoring of the cells over time. Additionally, specific compounds can be added to the culture media to test how this may affect implantation and invasion. This model is a helpful tool in understanding the complexity of human implantation.