Vivo-like Morphology Research Articles

Physiological liver microtissue 384-well microplate system for preclinical hepatotoxicity assessment of therapeutic small molecule drugs.

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates.

Microfluidic-Based Reconstitution of Functional Lymphatic Microvasculature: Elucidating the Role of Lymphatics in Health and Disease.

The knowledge of the blood microvasculature and its functional role in health and disease has grown significantly attributable to decades of research and numerous advances in cell biology and tissue engineering; however, the lymphatics (the secondary vascular system) has not garnered similar attention, in part due to a lack of relevant in vitro models that mimic its pathophysiological functions. Here, a microfluidic-based approach is adopted to achieve precise control over the biological transport of growth factors and interstitial flow that drive the in vivo growth of lymphatic capillaries (lymphangiogenesis). The engineered on-chip lymphatics with in vivo-like morphology exhibit tissue-scale functionality with drainage rates of interstitial proteins and molecules comparable to in vivo standards. Computational and scaling analyses of the underlying transport phenomena elucidate the critical role of the three-dimensionalgeometry and lymphatic endothelium in recapitulating physiological drainage. Finally, the engineered on-chip lymphatics enabled studies of lymphatic-immune interactions that revealed inflammation-driven responses by the lymphatics to recruit immune cells via chemotactic signals similar to in vivo, pathological events. This on-chip lymphatics platform permits the interrogation of various lymphatic biological functions, as well as screening of lymphatic-based therapies such as interstitial absorption of protein therapeutics and lymphatic immunomodulation for cancer therapy.

Unravelling host-pathogen interactions by biofilm infected human wound models

Approximately 80 % of persistent wound infections are affected by the presence of bacterial biofilms, resulting in a severe clinical challenge associated with prolonged healing periods, increased morbidity, and high healthcare costs. Unfortunately, in vitro models for wound infection research almost exclusively focus on early infection stages with planktonic bacteria. In this study, we present a new approach to emulate biofilm-infected human wounds by three-dimensional human in vitro systems. For this purpose, a matured biofilm consisting of the clinical key wound pathogen Pseudomonas aeruginosa was pre-cultivated on electrospun scaffolds allowing for non-destructive transfer of the matured biofilm to human in vitro wound models. We infected tissue-engineered human in vitro skin models as well as ex vivo human skin explants with the biofilm and analyzed structural tissue characteristics, biofilm growth behavior, and biofilm-tissue interactions. The structural development of biofilms in close proximity to the tissue, resulting in high bacterial burden and in vivo-like morphology, confirmed a manifest wound infection on all tested wound models, validating their applicability for general investigations of biofilm growth and structure. The extent of bacterial colonization of the wound bed, as well as the subsequent changes in molecular composition of skin tissue, were inherently linked to the characteristics of the underlying wound models including their viability and origin. Notably, the immune response observed in viable ex vivo and in vitro models was consistent with previous in vivo reports. While ex vivo models offered greater complexity and closer similarity to the in vivo conditions, in vitro models consistently demonstrated higher reproducibility. As a consequence, when focusing on direct biofilm-skin interactions, the viability of the wound models as well as their advantages and limitations should be aligned to the particular research question of future studies. Altogether, the novel model allows for a systematic investigation of host-pathogen interactions of bacterial biofilms and human wound tissue, also paving the way for development and predictive testing of novel therapeutics to combat biofilm-infected wounds.

Luminal and Glandular Epithelial Cells from the Porcine Endometrium maintain Cell Type-Specific Marker Gene Expression in Air–Liquid Interface Culture

Two different types of epithelial cells constitute the inner surface of the endometrium. While luminal epithelial cells line the uterine cavity and build the embryo-maternal contact zone, glandular epithelial cells form tubular glands reaching deeply into the endometrial stroma. To facilitate investigations considering the functional and molecular differences between the two populations of epithelial cells and their contribution to reproductive processes, we aimed at establishing differentiated in vitro models of both the luminal and the glandular epithelium of the porcine endometrium using an air–liquid interface (ALI) approach. We first tested if porcine luminal endometrium epithelial cells (PEEC-L) reproducibly form differentiated epithelial monolayers under ALI conditions by monitoring the morphology and the trans-epithelial electrical resistance (TEER). Subsequently, luminal (PEEC-L) and glandular epithelial cells (PEEC-G) were consecutively isolated from the endometrium of the uterine horn. Both cell types were characterized by marker gene expression analysis immediately after isolation. Cells were separately grown at the ALI and assessed by means of histomorphometry, TEER, and marker gene expression after 3 weeks of culture. PEEC-L and PEEC-G formed polarized monolayers of differentiated epithelial cells with a moderate TEER and in vivo-like morphology at the ALI. They exhibited distinct patterns of functional and cell type-specific marker gene expression after isolation and largely maintained these patterns during the culture period. The here presented cell culture procedure for PEEC-L and -G offers new opportunities to study the impact of embryonic signals, endocrine effectors, and reproductive toxins on both porcine endometrial epithelial cell types under standardized in vitro conditions.Graphical Created with BioRender.com.

Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.

Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce’s medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce’s medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.

Microfluidic Live Imaging Technology to Perform Research Activities in 3D Models

Morphological dissimilarity and its evolution over time are one of the most unexpected variations found when comparing cell cultures in 2D and 3D. Monolayer cells appear to flatten in the lower part of the plate, adhering to and spreading in the horizontal plane while not extending vertically. Consequently, cells developed in two dimensions have a forced apex-basal polarity. Co-cultivation and crosstalking between multiple cell types, which control development and formation in the in vivo counterpart, are possible in 3D cultures. With or without a scaffold matrix, 3D model culture may exhibit more in vivo-like morphology and physiology. 3D cultures mimic relevant physiological cellular processes, transforming them into one-of-akind drug screening platforms. The structures and dynamics of regulatory networks, which are increasingly studied with live-imaging microscopy, must be considered to help and guarantee the functional maintenance of a 3D structure. However, commercially available technologies that can be used for current laboratory needs are minimal, despite the need to make it easier to acquire cellular kinetics with high spatial and temporal resolution, in order to improve visual efficiency and, as a result, experimentation performance. The CELLviewer is a newly developed multi-technology instrument that integrates and synchronizes the work of various scientific disciplines. The aim of this study is to test the device using two different models: a single Jurkat cell and an MCF-7 1 spheroid. The two models are loaded into the microfluidic cartridge for each experiment after they have been grown and captured in time-lapse for a total of 4 hours. The samples used are tracked under the operation of the optics after adaptive autofocus, while slipping inside the cartridge chamber and the 3D rotation was successfully obtained experimentally. The MitoGreen dye, a fluorescence marker selectively permeable to live cells, was then used to determine cell viability. To measure the model diameter, construct fluorescence intensity graphs along a straight line passing through the cell, and visualize the spatial fluorescence intensity distribution in 3D, Image J software was used.

Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection.

Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus–host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

In Vivo-Like Morphology of Intercalated Discs Achieved in a Neonatal Cardiomyocyte Culture Model.

In vitro cultures to be used in various analytical investigations of cardiomyocyte (CM) growth and function for enhancing insight into physiological and pathological mechanisms should closely express in vivo morphology. The aim of the studies is to explore how to use microfabrication and physical-cue-addition techniques to establish a neonatal rat CM culture model that expresses an end-to-end connected rod shape with in vivo-like intercalated discs (ICDs). Freshly isolated neonatal rat CMs were cultured on microgrooved polydimethylsiloxane substrate. Cell alignment and ICD orientation were evaluated using confocal fluorescence and transmission electron microscopy under various combinations of different culture conditions. Cyclic stretch and blebbistatin tests were conducted to explore mechanical and electrical effects. Laboratory-made MATLAB software was developed to quantify cell alignment and ICD orientation. Our results demonstrate that the mechanical effect associated with the electrical stimulation may contribute to step-like ICD formation viewed from the top. In addition, our study reveals that a suspended elastic substrate that was slack with scattered folds, not taut, enabled CM contraction of equal strength on both apical and basal cell surfaces, allowing the cultured CMs to express a three-dimensional rod shape with disc-like ICDs viewed cross-sectionally. Impact statement In this article, we describe how the tugging forces generated by cardiomyocytes (CMs) facilitate the formation of the morphology of the intercalated discs (ICDs) to achieve mechanoelectrical coupling between CMs. Correspondingly, we report experimental techniques we developed to enable the in vivo-like behavior of the tugging forces to support the development of in vivo-like morphology in ICDs. These techniques will enhance insight into physiological and pathological mechanisms related to the development of tissue-engineered cardiac constructs in various analytical investigations of CM growth and function.

Silk sericin-enhanced microstructured bacterial cellulose as tissue engineering scaffold towards prospective gut repair

As a first step towards the production of functional cell sheets applicable for the regeneration of gut muscle layer, microstructured bacterial cellulose (mBC) was assessed for its ability to support the growth of enteric nervous system (ENS) and gut smooth muscle cells (SMCs). To improve the cellular response, mBC was modified with silk sericin (SS) which has renowned abilities in supporting tissue regeneration. While SS did not impair the line structures imparted to BC by PDMS templates, similarly to the patterns, it affected its physical properties, ultimately leading to variations in the behavior of cells cultured onto these substrates. Enabled by the stripes on mBC, both SMCs and ENS cells were aligned in vitro, presenting the in vivo-like morphology essential for peristalsis and gut function. Interestingly, cell growth and differentiation remarkably enhanced upon SS addition to the samples, indicating the promise of the mBC-SS constructs as biomaterial not only for gut engineering, but also for tissues where cellular alignment is required for function, namely the heart, blood vessels, and similars.

Nanofibers Regulate Single Bone Marrow Stem Cell Osteogenesis via FAK/RhoA/YAP1 Pathway.

Understanding cell-material interactions is a prerequisite for the development of bio-inspired materials for tissue regeneration. While nanofibrous biomaterials have been widely used in tissue regeneration, the effects of nanofibrous architecture on stem cell behaviors are largely ambiguous because the previous biomaterial systems used for nanofiber-cell interactions could not exclude the interference of cell-cell interactions. In this study, we developed a unique micropatterning technology to confine one single stem cell in a microisland of the nanofibrous micropatterned matrix; therefore, eliminating any potential intercellular communications. The nanofibrous micropatterned matrix, which mimicked both the physical architecture and chemical composition of natural extracellular matrix, was fabricated by a combination of electrospinning, chemical crosslinking, and UV-initiated photolithography. Compared to the non-nanofibrous architecture, a bone marrow mesenchymal stem cell (BMSC) cultured on the nanofibrous microisland exhibited a more in vivo-like morphology, a smaller spreading area, less focal adhesion, and fewer stress fibers. The BMSC cultured on the nanofibrous microisland also had higher alkaline phosphatase activity, indicating nanofibrous architecture promoted BMSC differentiation. A mechanistic study reveals that nanofibers regulate single BMSC osteogenesis via the FAK/RhoA/YAP1 pathway. The nanofibrous micropatterned matrix developed in this study is an excellent platform to promote the fundamental understanding of cell-matrix interactions, ultimately provide valuable insights for the development of novel bio-inspired scaffolds for tissue regeneration.

Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports.

Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphology and functionality. However, due to the optical properties of the filter materials and the cells’ columnar shape, cell quality is hard to assess via light microscopy. In this study, we aim to evaluate transepithelial electrical resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 w to achieve diverse culture qualities, and TEER was measured before processing samples for histology. Culture quality was scored using morphological criteria (presence of cilia, confluence and cell polarity). We furthermore analyzed the correlation between cellular height (as a measure of apical–basal polarization) and TEER in fully differentiated routine cultures (biological variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm2. Only 5 % of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. We unveiled a highly significant (P < 0.0001) negative linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical–basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-015-1351-1) contains supplementary material, which is available to authorized users.

Topographical control of ocular cell types for tissue engineering

Visual impairment affects over 285 million people worldwide and has a major impact on an individual's quality of life. Tissue engineering has the potential to increase the quality of life for many of these patients by preventing vision loss or restoring vision using cell-based therapies. However, these strategies will require an understanding of the microenvironmental factors that influence cell behavior. The eye is a well-organized organ whose structural complexity is essential for proper function. Interactions between ocular cells and their highly ordered extracellular matrix are necessary for maintaining key tissue properties including corneal transparency and retinal lamination. Therefore, it is not surprising that culturing these cells in vitro on traditional flat substrates result in irregular morphology. Instead, topographically patterned biomaterials better mimic native extracellular matrix and have been shown to elicit in vivo-like morphology and gene expression which is essential for tissue engineering. Herein we review multiple methods for producing well-controlled topography and discuss optimal biomaterial scaffold design for cells of the cornea, retina, and lens.

Role of fibroblasts in the pathogenesis of atopic dermatitis

Atopic dermatitis (AD) is a common dermatosis that highly impairs a patient's quality of life. The recent discovery that epidermal barrier defects caused by an aberrant differentiation process of keratinocytes are comparably important to the well-characterized changes in immune response patterns attributed a crucial role to the keratinocytes. Fibroblasts are able to alter proliferation and differentiation of keratinocytes, but their role in AD is not yet fully understood. We sought to determine the role of fibroblasts in skin proliferation and differentiation in patients with AD. We used human 3-dimensional organotypic skin cultures consisting of atopic fibroblasts and healthy keratinocytes, as well as healthy fibroblasts and atopic keratinocytes, and compared them with their controls. The expression of differentiation markers in these organotypic cultures were analyzed by using immunohistology and quantitative RT-PCR. Furthermore, the fundamental role of fibroblast-secreted leukemia inhibitory factor was assessed by using small interfering RNA-mediated knockdown cultures. We observed that atopic fibroblasts influence the proliferation of keratinocytes and the terminal differentiation process, resulting in an in vivo-like morphology of AD. Subsequently, healthy fibroblasts were able to restore the structural deficits of the epidermis consisting of atopic keratinocytes. Partially, these effects were due to a reduced expression of the differentiation-associated cytokine leukemia inhibitory factor by atopic fibroblasts. These data demonstrate that fibroblasts and the modulation of fibroblast-derived factors might be new therapeutic targets for the alleviation of AD.

Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity

Introduction: The sandwich-cultured hepatocyte (SCH) model has become an invaluable in vitro tool for studying hepatic drug transport, metabolism, biliary excretion and toxicity. The relevant expression of many hepatocyte-specific functions together with the in vivo-like morphology favor SCHs over other preclinical models for evaluating hepatobiliary drug disposition and drug-induced hepatotoxicity.Areas covered: In this review, the authors highlight recommended procedures required for reproducibly culturing hepatocytes in sandwich configuration. It also provides an overview of the SCH model characteristics as a function of culture time. Lastly, the article presents a summary of the most prominent applications of the SCH model, including hepatic drug clearance prediction, drug–drug interaction potential and drug-induced hepatotoxicity.Expert opinion: When human (cryopreserved) hepatocytes are used to establish sandwich cultures, the model appears particularly valuable to quantitatively investigate clinically relevant mechanisms related to in vivo hepatobiliary drug disposition and hepatotoxicity. Nonetheless, the SCH model would largely benefit from better insight into the fundamental cell signaling mechanisms that are critical for long-term in vitro maintenance of the hepatocytic phenotype. Studies systematically exploring improved cell culture conditions (e.g., co-cultures or extracellular matrix modifications), as well as in vitro work identifying key transcription factors involved in hepatocyte differentiation are currently emerging.

Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

A serum-free, feeder cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1-wk-old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 μM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several wk. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiocyte monolayers. Serum protein secretion in conditioned media was analyzed by Western blot and indicated that albumin, transferrin, and haptoglobin levels were maintained in hepatocytes while albumin and haptoglobin declined over time in cholangiocytes. Quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte differentiation and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.

Endometrial cancer: experimental models useful for studies on molecular aspects of endometrial cancer and carcinogenesis.

There is definitely a need for the development of new drugs for the treatment and cure of endometrial cancer. In addition there are various new drugs or phyto-remedies under development which are intended for use in the treatment and prevention of breast cancer, for the treatment of menopausal symptoms and for hormone replacement therapy. The efficacy of novel drugs targeting steroid receptors in endometrial cancers has to be evaluated and the safety of other endocrine measures on endometrial cancers or on endometrial carcinogenesis has to be assessed. For these experimental purposes five main classes of experimental models are available: spontaneous endometrial tumorigenesis models in inbred animals (Donryu rats, DA/Han rats, BDII/Han rats), inoculation tumors from chunks of tumors (rat EnDA-tumor, human EnCa 101 tumor) or from inoculated tumor cell lines (rat RUCA-I cells, human Ishikawa and ECC-1 cells), developmental estrogenic exposure or chemical carcinogen exposure of CD-1 and ICR mice, transgenic approaches such as mice heterozygous regarding the tumor suppressor gene PTEN (pten(+/-)-mice) and endometrial tumor cell lines cultured under conditions promoting in vivo-like morphology and functions e.g. cell culture on reconstituted basement membrane. Although the number of models is comparatively small, most aspects related to functions of estrogenic or gestagenic substances are assessable, particularly if various experimental models are combined. Whereas models based on human endometrial adenocarcinoma cells are widely used, the properties and advantages of animal-derived models have mainly been ignored so far.

Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells.

In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used. The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exhibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.

Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration.

Extracellular matrix (ECM) geometry is an important modulator of cell polarity and function. For example, 3-dimensional matrices often more effectively induce differentiated cell function than traditional 2-dimensional substrates. The effect of ECM topology can be investigated in a controlled fashion using a technique whereby cells cultured on a single surface are overlaid with a second layer of ECM, thereby creating a "sandwich" configuration. Confluent monolayers of epithelial or endothelial cells overlaid in this fashion often reorganize into structures that are reminiscent of their native tissue. In the case of hepatocytes, the overlay causes a dramatic reorganization of the cytoskeleton, adoption of in vivo-like morphology and polarity, and expression of a wide array of liver-specific functions. In this short review, we use the sandwiched hepatocyte culture system to illustrate the effect of ECM geometry on cellular function. Pertinent studies are summarized in the context of defining the parallels, strengths, and limitations of this culture system as an in vitro model to study the physiology and morphogenesis of liver tissue. We also explore some of its potential uses as a model to study liver pharmacology and toxicology, and for the development of liver preservation techniques and liver-assist devices.