Pancreatic acinar cells rapidly lose their characteristic features when cultured in vitro. No successful cryopreservation methods have been reported. To solve the problem of storing pancreatic acinar material, we found that it could be preserved at nonfreezing, cold temperatures: above the freezing point of cell culture medium (−0.6°C) or at typical refrigeration temperatures (6.0–8.0°C) for up to 7 d. Under the conditions we defined, we determined that there was no significant dedifferentiation and no significant decrease in cell health. Good viability and enzyme content were realized after storage, as determined by growth in culture, histological evaluation, and enzyme content by ELISA (lipase and amylase).

The most commonly used means of assessing the invasiveness of cultured cells is the Boyden chamber assay, which requires that cells lyse Matrigel™, followed by migration through pores in a filter in response to a chemotactic gradient. This report describes a simple method, which greatly increases the speed and accuracy by which Boyden chamber assays can be analyzed, and permits the concurrent analysis of distinct cell subpopulations within specimens containing multiple-cell types.

Coronary vascular disease is one of the leading causes of mortality and morbidity in the United States. Therefore, a mechanistic understanding of coronary vessel morphogenesis would aid in the innovation of new therapies targeting vascular disorders. Moreover, a functionally equivalent in vitro model system allows for the delineation of the molecular mechanisms that regulate coronary vessel development. In this study, we present a novel in vitro model system. This three-dimensional (3-D) model system consists of a tubular scaffold, which is engineered from type-I collagen and has been optimized to support the growth of embryonic cardiac tissues. In this report, proepicardial (PE) cells, the developmental precursors of coronary vessels, have been isolated from several model species and cultured on this scaffold. In this model system, the PE cells were able to recapitulate several aspects of coronary vessel morphogenesis including epicardial formation, the epicardial to mesenchymal transformation, and de novo coronary vessel development or vasculogenesis. The differentiation of PE cells was characterized using a variety of specific protein markers. The potential uses of this novel coronary developmental model are discussed.

We have constructed an in vitro arterial wall model by coculturing bovine arterial endothelial cells (ECs) and smooth muscle cells (SMCs). When ECs were seeded directly over SMCs and cocultured in an ordinary culture medium, ECs grew sparsely and did not form a confluent monolayer. Addition of ascorbic acid to the culture medium at concentrations greater than 50 μg/ml increased the production of type IV collagen by the SMCs, and ECs formed a confluent monolayer covering the entire surface of SMCs. Histological studies showed that the thickness of the cell layer composed of ECs and SMCs increased with increasing duration of coculture. This arterial wall model, prepared by our method, may serve as a simple and good in vitro model to study the effects of factors such as biological chemicals and shear stress on cell proliferation and other physiological functions of arterial walls.

Osteoblasts are cells specialized in extracellular matrix production and mineralization. In collaboration with osteoclasts which are bone-resorbing cells, osteoblasts regulate bone homeostasis. The study of osteoblast differentiation from the earliest states of the differentiation can be performed using embryonic stem cells. Embryonic stem cells are pluripotent cells which have the capacity to give rise to all kinds of cells of the body. The main protocol to differentiate embryonic stem cells into osteoblast uses the generation of embryoid body which is a three-dimensional structure mimicking the developing embryo. Recently, it has been shown that human embryonic stem cells have the capacity to differentiate spontaneously into osteoblasts. In this manuscript, we showed that mouse embryonic stem cells have the capacity to differentiate spontaneously into osteoblasts, which can be visualized by the appearance of mineralization nodules and osteogenic markers.

Proton pump inhibitors (PPIs) could inhibit the secretion of gastric acid. Meanwhile, it could also decrease the secretion of other digestive glands besides gastric parietal cell. As we know, PPIs have been widely used to treat acute pancreatitis, and it is effective in clinical practice. However, research showed the side effect of PPIs on acute pancreatitis. The direct effect of PPI on pancreatic secretion is still unknown. Our experiment investigated the direct effect of PPIs on pancreatic exocrine by isolated pancreatic acini. In our study, isolated pancreatic acini were prepared as previously described by Williams, and cerulein was added to stimulate its secretion. The amylase release in the suspension was determined after the administration of different concentrations of omeprazole and Sandostatin; and its activity was also observed in different time phases. In our in vitro study, all results suggest that omeprazole has no direct repression on amylase release from isolated pancreatic acini.

The reconstruction of extensive tracheal defects is still an unsolved challenge for thoracic surgery. Tissue engineering is a promising possibility to solve this problem through the generation of an autologous tracheal replacement from patients' own tissue. Therefore, this study investigated the potential of three different coculture systems, combining human respiratory epithelial cells and human chondrocytes. The coculture systems were analyzed by histological staining with alcian blue, immunohisto-chemical staining with the antibodies, 34betaE12 and CD44v6, and scanning electron microscopy. The first composite culture consisted of human respiratory epithelial cells seeded on human high-density chondrocyte pellets. For the second system, we used native articular cartilage chips as base for the respiratory epithelial cells. The third system consisted of a collagen membrane, seeded with respiratory epithelial cells and human chondrocytes onto different sides of the membrane, which achieved the most promising results. In combination with an air–liquid interface system and fibroblast-conditioned medium, an extended epithelial multilayer with differentiated epithelial cells could be generated. Our results suggest that at least three factors are necessary for the development towards a tracheal replacement: (1) a basal lamina equivalent, consisting of collagen fibers for cell–cell interaction and cell polarization, (2) extracellular factors of mesenchymal fibroblasts, and (3) the presence of an air–liquid interface system for proliferation and differentiation of the epithelial cells.

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways.