Primary culture of rabbit gallbladder epithelial cells in collagen gel matrix

Primary culture of gallbladder epithelial cells obtained from normal rabbits was attempted in collagen gel matrix for up to 6 weeks. Fluid medium containing 0.02% ethylenediaminetetraacetic acid and 0.25% trypsin was poured into the gallbladder lumen. The pellets obtained by centrifugation of recovered fluid contained many isolated epithelial cells and a few small cell clumps. These pellets were dispersed and embedded inside collagen gel matrix and cultured in William's medium E supplemented with fetal calf serum and epidermal growth factor. The three-dimensional outgrowth from individual cells and small cell clumps consisted predominantly of spherical cystic masses 2-4 days later. These cysts contained mucin and were covered by a single layer of cuboidal or low columnar epithelial cells. Electron microscopy revealed the epithelial arrangement of cells lining the cyst walls, and these cells were similar to gallbladder epithelial cells in vivo. These epithelial cells showed active mucin secretion into the cystic cavities. Cytokeratin was diffusely present in the cytoplasm. This isolation and culture system provides a reproducible and consistent method for sustained growth of normal gallbladder epithelial cells from normal tissue in primary culture and seems valuable for investigating pathologic conditions of the gallbladder.     

In vitro spermatogenesis by three-dimensional culture of rat testicular cells in collagen gel matrix

In an effort to improve in vitro spermatogenesis by potentiating the interactions between developing germ cells, somatic cells, and the extracellular matrix (ECM), the efficiency of the germ cell-somatic cell coculture in a three-dimensional (3D) collagen gel matrix was examined. Cells isolated from rat seminiferous tubules 18 days after birth were cultured for 22 days in a monolayer without ECM, collagen gel (CG), or collagen+Matrigel (CGM). After culture, the viabilities of the cultured cells in the monolayer, CG, and CGM culture were 42.8%, 70.7% and 76.1%, respectively. Occludin-positive cells in a cyst-like structure were found in the ECM gel matrix together with 3beta hydroxysteroid dehydrogenase-positive cells, suggesting the presence of functional Sertoli cells and Leydig cells, respectively. Flow cytometric analysis of DNA content revealed a significant increase in the haploid cell population in the CG and CGM compared to the monolayer culture. Transition protein 2 (TP2) and protamine 2-positive cells were found together with a significant increase in TP2 mRNA levels in cells cultured in CG and CGM over those in monolayer culture, suggesting the occurrence of the post-meiotic differentiation of spermatogenetic cells. Taken together, a 3D in vitro culture system for testicular cells using a collagen gel matrix could enhance viability, meiosis, and post-meiotic differentiation of germ cells to presumptive differentiating spermatids.     

Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.

The purpose of this study is to reconstruct an alveolus-like structure from alveolar type II epithelial cells in a culture condition. Isolated alveolar type II epithelial cells of the rat were cultured in a three-dimensional collagen gel matrix. Single type II cells formed cellular aggregates that had a lumen after cell division in this culture condition. Through proliferation of the component cells, these aggregates grew to assume a globular or branching structure, part of which in turn developed into a large, cystic alveolus-like structure. This structure consisted of flattened epithelial cells intermingled with cuboidal epithelial cells. In these structures, the surfactant production was confirmed by immunohistochemistry and electron microscopy. To our knowledge, this is the first report of a reconstruction of an alveolus-like structure in a three-dimensional collagen gel matrix culture. This culture system seems to provide an appropriate physiological environment in which to study the differentiation and disorders of pulmonary alveoli.     

Characterization of human extrahepatic biliary duct epithelial cells in culture

In order to study human bile duct cells in vitro, cystic ducts were obtained during cholecystectomy and treated with collagenase and mechanical abrasion to isolate biliary epithelial cells. The culture medium was supplemented with 50% of a bovine bile duct conditioned medium obtained by incubating minced bovine extrahepatic bile ducts for 24 hr in Dulbecco's modified Eagle's medium. Cells grew in monolayer and showed contact inhibition at confluency. The epithelial origin of primary cultures was verified by their growth pattern, ultrastructure, and indirect immunofluorescence for cytokeratin. The cultures showed specific immunofluorescence for lysozyme, collagen types I, III, and IV, fibronectin, and laminin, but were negative for collagen type V and factor VIII-associated antigen. Thus, these cultures provide an experimental model for the in vitro study of biliary atresia and other bile duct diseases.     

Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells

Recent advances made in the isolation, culture, and transplantation of defined populations of intrahepatic biliary epithelial cells and oval cells have permitted direct analysis of the functions, growth properties, and differentiation potential of these respective cell types in their untransformed or transformed states. This review provides a current and comprehensive examination of the various approaches that have been taken to isolate and culture intrahepatic biliary epithelial cells from normal and cholestatic liver and oval cells from preneoplastic liver. Emphasis is placed on comparing the phenotypic features and growth properties of these various biliary cell types in vitro as well as on describing their transplantation into ectopic tissue sites. In addition, the oval cell is evaluated in terms of its potential role as a 'facultative stem cell' during hepatocarcinogenesis.     

STAT3活化在脂多糖刺激大鼠胆管上皮细胞增殖中的作用

目的：探讨脂多糖(LPS)刺激大鼠肝内胆管上皮细胞（BEC）增殖过程中信号转导和转录激活因子3（STAT3）活化的意义。方法：大鼠随机分为对照组(control)、LPS组和雷帕霉素（RPM）处理组。分别于注射LPS后6、12、24、48和72 h采用动态比浊法鲎试验测定血浆LPS水平，酶联免疫吸附试验（ELISA）法检测肝组织匀浆IL-6水平，激光扫描共聚焦显微镜（LSM）检测BEC内磷酸化STAT3(p- STAT3)水平，免疫组织化学法检测BEC增殖。结果:大鼠注射LPS后6 h血浆LPS水平最高［（318±115） EU/L］，48 h接近control组水平［(29±11) EU/L］；IL-6 水平于注射LPS后12 h达峰值［(653.4±168.8) ng/g蛋白］，72 h接近control组水平［（249.4±50.7）ng/g蛋白］，LPS组BEC内p-STAT3的表达与IL-6水平呈显著正相关(r=0.944，P<0.05)；BEC增殖指数在12 h明显增加［（5.2±0.5）%］，24 h达到峰值［（12.8±3.0）%］，72 h接近control组水平［（4.2%±0.6%）］；给予RPM明显减轻LPS诱导的细胞STAT3活化和BEC增殖。结论:LPS刺激导致肝组织IL-6分泌增加，后者可能通过STAT3介导BEC增殖。     

An ultrastructural study of rat glomerular epithelial cells in culture

Cultured, rat glomerular podocytes were examined by electron microscopy and immunohistochemistry in order to determine their origin. The outgrowth of polygonal cells was noted first. Large arboroid cells were occasionally observed around the polygonal cells. Immunostaining of these large arboroid cells for the intermediate filament proteins showed an intensely positive reaction for vimentin, whereas cytokeratin was not detected. In the polygonal cells, however, both cytokeratin and vimentin were sometimes detected. Although scanning electron microscopy did not detect any specific irregularities in podocytes on the surface of the glomerulus, developing polygonal cells were observed. Both transmission and scanning electron microscopies revealed the polygonal cells to be similar to Bowman's capsule epithelial cellsin situ. The vascular poles of the cultured glomeruli were always in contact with the culture dish. A residual Bowman's capsule was also observed. These results suggest that polygonal cells originate from the epithelial cells of the residual Bowman's capsule, whereas large arboroid cells arise from podocytes. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

Effects of the mechanical properties of collagen gel on the in vitro formation of microvessel networks by endothelial cells

Vascularization by endothelial cells (ECs) is an essential element in tissue-engineering of organoids. Morphogenesis of these cells is regulated not only by the biochemical properties of the extracellular matrix (ECM) but also by its mechanical properties. Here, we investigated the effect of substrate mechanical properties on the formation of capillary-like networks by ECs; in particular, we examined the three-dimensional (3D) configurations of the resulting networks. Bovine pulmonary microvascular ECs (BPMECs) were cultured on a series of collagen gels of different stiffness but the same collagen concentration. Imaging techniques revealed that cells cultured in rigid and flexible gels formed 3D networks via different processes; cells formed dense, thin networks in the flexible gel, whereas thicker and deeper networks were formed in the rigid gel. Cross-sections of the networks revealed that those formed within the rigid gel had large lumens composed of multiple cells, whereas those formed within the flexible gel had small, intracellular vacuoles. The expression of vinculin, a focal adhesion protein, appeared to change with the mechanical properties of collagen gel. Our results indicate that the mechanical properties of adhesion substrates play an important role in regulating 3D network formation.     

Isolation and primary culture of rat renal cortical epithelial cells

Summary Methods are described for the isolation of rat renal cortical epithelial cells by a collagenase and hyaluronidase perfusion, followed by pressing fragments of renal cortex through an 80-mesh screen and further enzymatic dissociation in vitro. Primary monolayer cultures are derived from the resulting suspension of tubular fragments and cells. Fibroblast and endothelial cell overgrowth is suppressed by the use of medium lacking arginine and containingd-valine in place ofl-valine. Further separation of fibroblasts from epithelial cells is achieved by a technique that takes advantage of the differential rate of attachment of the two cell types. The presence of glomerular cells in the cultures is diminished by a complete medium change 48 h after plating the cells.     

Effects of transforming growth factor-beta on collagen synthesis by fetal rat lung epithelial cells

Studies were performed to characterize the effects of acute and chronic exposure to transforming growth factor-beta (TGF-beta) on collagen biosynthesis by fetal rat lung epithelial (FRLE) cells, a cell line established from the fetal rat lung alveolar epithelial cell. Neither condition of exposure to TGF-beta stimulated cell growth, but both conditions increased total protein synthesis. Quantitative evaluation by carboxymethyl-Trisacryl chromatography revealed that FRLE cells synthesized types I, III, IV, and V collagen under all circumstances. Acute and chronic exposure to TGF-beta increased total collagen production approximately 50% and 300%, respectively, with the increases in total collagen production exceeding those of total protein synthesis. In addition, these analyses indicated that the production of types I and III molecules was stimulated to a greater extent than was the synthesis of types IV and V molecules. Both experimental conditions increased the ratio of secreted to cell-associated molecules for types I and III molecules, decreased this ratio for type IV collagen, but minimally affected the culture distribution of type V collagen. Additionally, both conditions of exposure to TGF-beta were found to increase the proportion of the homotrimeric forms of types I and V molecules relative to their heterotrimeric counterparts. Thus, these studies establish that TGF-beta selectively and type-specifically alters collagen production without affecting growth in an epithelial cell line of fetal rat lung origin.     

Effects of transforming growth factor-beta on collagen synthesis by normal rat kidney epithelial cells.

The effects of transforming growth factor-beta (TGF-beta) on the growth of and collagen production by NRK52E cells, a clonal line established from normal rat kidney epithelial cells, have been characterized. NRK52E cells were grown in the absence or presence of TGF-beta for 4 days followed by incubation for 24 hours in serum-free medium containing [3H]proline. The secreted and cell-associated collagens produced by control and experimental cultures were isolated by limited pepsin digestion and differential salt fractionation. TGF-beta inhibited proliferation by about 50% but did not affect overall culture morphology. Both protein and collagen synthesis were increased in experimental cultures, but the increase in total collagen production exceeded that of total protein synthesis. Although NRK52E cells grown in the presence of TGF-beta continued to produce the same types of collagen (types I, III, IV, and V), their relative amounts were changed. In the experimental cultures, type I collagen production was increased eightfold, types III and V collagen levels were increased two-fold, but type IV production was only slightly enhanced. In addition to increasing total collagen production by about fivefold, TGF-beta increased the ratio of type I to type III about threefold but minimally affected the ratio of secreted to cell-associated molecules. These findings establish that TGF-beta specifically affects collagen production in NRK52E cells and that these alterations differ in many ways from the affects of epidermal growth factor. Because TGF-beta increased total collagen expression, these results provide additional evidence implicating this growth factor as a positive mediator of matrix accumulation in renal disease.     

Lateral boundary mechanosensing by adherent cells in a collagen gel system

Cell adhesion responses to in-depth physical properties such as substrate roughness and topography are well described but little is known about the influence of lateral physical cues such as tissue boundaries on the function of adherent cells. Accordingly, we developed a model system to examine remote cell sensing of lateral boundaries. The model employs floating thin collagen gels supported by rigid grids of varying widths. The dynamics, lengths, and numbers of cell extensions were regulated by grid opening size, which in turn determined the distance of cells from rigid physical boundaries. In smaller grids (200 μm and 500 μm wide), cell-induced deformation fields extended to, and were resisted by, the grid boundaries. However, in larger grids (1700 μm wide), the deformation field did not extend to the grid boundaries, which strongly affected the mean length and number of cell extensions (∼60% reduction). The generation of cell extensions in collagen gels required expression of the β1 integrin, focal adhesion kinase and actomyosin activity. We conclude that the presence of physical boundaries interrupts the process of cell-mediated collagen compaction and fiber alignment in the collagen matrix and enhances the formation of cell extensions. This new cell culture platform provides a geometry that more closely approximates the native basement membrane and will help to elucidate the roles of cell extensions and lateral mechanosensing on extracellular matrix remodeling by invasion and degradation.     

Inhibition of ERK promotes collagen gel compaction and fibrillogenesis to amplify the osteogenesis of human mesenchymal stem cells in three-dimensional collagen I culture

Tissue morphogenesis remains one of the least understood problems in cell and developmental biology. There is a disconnect between the mechanisms that apply to two-dimensional (2D) cultures and those seen in vivo. Three-dimensional (3D) culture presents a complex stimulus triggering cellular responses that are only partially understood. We compared 2D and 3D cultures of human mesenchymal stem cells in the presence of mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, to determine the role of extracellular signal-related kinase (ERK) in collagen-induced differentiation. 3D collagen I culture enhanced and accelerated the osteogenic differentiation of human mesenchymal stem cells (hMSC). Contrary to 2D results, the addition of PD98059 induced a significant amplification of osteogenic gene expression and matrix mineralization in 3D cultures. The inhibition of ERK altered cell-mediated compaction, proliferation, and resulted in the development of distinct tissue microstructure. Therefore, we suggest that the ability to reorganize collagen in 3D is an important step in ERK-mediated osteogenic differentiation. This work aims to propose a correlation between osteogenic differentiation and hMSC-directed collagen I remodeling. We present a potential mechanistic link (ERK) through which the three dimensionality of an engineered tissue acts to differentially induce and maintain cellular phenotype during tissue development.     

Vimentin expression of newly formed rat bile duct epithelial cells in secondary biliary fibrosis

Summary The intermediate filament profile and the growth fraction of hepatocytes and bile duct epithelial cells were studied in a rat model of biliary fibrosis secondary to common bile duct ligation and scission. Strong vimentin expression was observed in epithelial cells of newly formed bile ductules, while normal liver contained only few weakly positive bile duct epithelial cells. All epithelial cells reacted with a pan-cytokeratin antibody. A monoclonal antibody specific for human cytokeratin 7 selectively reacted with both normal and newly formed bile duct epithelial cells. The intermediate filament profile of hepatocytes was constant, showing no changes during proliferation or in periportal areas adjacent to excessive bile duct formations. The proliferation-associated antigen detected by the antibody Ki-67 was present in many hepatocytes, homogeneously distributed in the lobules, but was seen only in a small proportion of the epithelial cells of the newly formed bile ducts. We conclude that vimentin may serve as an indicator for cellular reorganization in the bile duct system, and that the epithelial cells of newly formed bile ductules in this particular model of secondary biliary fibrosis were most likely to be derived from an outgrowth of the biliary duct system and recruitment of preductular epithelial cells. No morphological or immunohistological evidence suggesting a derivation from hepatocytes by ductular metaplasia or from oval cells was obtained.     

羊膜上皮细胞胶原海绵复合体促大鼠预构皮瓣成活及其再血管化

目的　移植羊膜上皮细胞(AECs) 胶原海绵复合体促进预构皮瓣成活。 方法　第3代SD大鼠AECs、软骨细胞株分别植于胶原蛋白海绵支架,并提取AECs组上清液行促血管形成因子VEGF、TGFβ1、bFGF的ELISA检测。SD大鼠分正常对照组、空白对照组、软骨细胞组和AECs组。一期手术后第7、14 d取皮瓣组织行促血管形成因子的ELISA检测。二期手术后第7 d用计算机图像分析系统测定皮瓣存活率,取存活部位组织行HE染色、vWF免疫组织化学染色以分析微血管密度。 结果　AECs复合体上清液中的促血管形成因子分泌逐渐增多;组织内各因子含量以AECs组最高,AECs组皮瓣存活率明显高于空白对照组和软骨细胞组。AECs组皮肤微血管面积比、vWF因子阳性区面积比明显高于其他组。 结论　植入AECs胶原海绵复合体可促进预构皮瓣的存活,与其分泌的一些促血管形成因子促进预构皮瓣再血管化有关。     

Type VIII collagen. Synthesis by normal and malignant cells in culture

A novel protein belonging to the collagen family was originally purified from the culture medium of bovine aortic endothelial cells. This endothelial collagen, termed EC, was also found to comprise the major collagen type synthesized by a malignant astrocytoma-derived cell line. Examination of several cell strains derived from normal tissues revealed that EC was not synthesized by all endothelial cells; it was absent from human endothelial cells cultured from both large and small vessels but was present in bovine cells, including those from capillaries. Human foreskin fibroblasts also secreted this protein in small amounts relative to interstitial procollagens, but it was not detected in two human epithelial cell strains. EC was consistently observed in human cell lines derived from several carcinomas and comprised the major collagenous protein secreted by cells cultured from a Ewing's sarcoma. In contrast, malignant or transformed murine cells did not produce EC in vitro. In addition, the protein was not apparent after metabolic labeling of human cells from an epidermoid carcinoma, a fibrosarcoma, and two Wilms' tumors. EC-like proteins were isolated from cell culture medium by ion-exchange chromatography and were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cleavage with vertebrate collagenase, mast cell protease, and CNBr. In addition to the homologies displayed by comparative peptide mapping, these collagens exhibited other unusual properties that collectively were characteristic of EC from endothelial and astrocytoma-derived cells. These studies support the existence of a novel class of collagenous proteins that are secreted by a wide variety of cells derived from both normal and neoplastic tissues. This class of proteins, which manifests several unusual structural characteristics, has been designated type VIII collagen.