Culture of porcine fetal pancreatic neurons.

OBJECTIVES: Pancreatic neurons have not been cultured commonly. Cultured neurons can be continuously observed, and their external environment is easy to be controlled. We report here a simple method for separating and cultivating neuronal cells from pancreas. METHODS: Pancreata of fetal swine were digested with collagenase. Clusters were collected with a sieve and digested with trypsin. Digested clusters were collected and cultured in Dulbecco modified Eagle medium containing serum and basic fibroblast growth factor. Cultured cells were investigated morphologically. RESULTS: Cultured cells formed spiderweblike colonies. These cells were distinguished into Schwann cells and 2 types of neurons. The neurons were positive on immunocytochemical staining with antigrowth-associated protein-43 and cytochemical staining for cholinesterase. One type of neuron was located in the central cluster and had very long processes extending radially. The other type of neuron was sparsely scattered, had long processes, and was connected to other neurons. The neurotransmitter of these neurons was concluded to be acetylcholine. CONCLUSIONS: Using our method, neuronal cells were readily cultured from pancreatic tissue. These cells will be useful in elucidating the physiology and pharmacology of pancreatic neurons.

Erythropoietin-induced proliferation of gastric mucosal cells.

AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS: Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine (BrdU). RESULTS: Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of anti- erythropoietin antibody (P<0.01). CONCLUSION: These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.

Preservation of the characteristics of the cultured human type II alveolar epithelial cells.

The human type II alveolar epithelial cells lost their specific characteristics during cultivation. We examined the ultrastructural and biochemical nature of the human type II cells cultured by two culture systems. To make a physiological alveoli model, the epithelial cells were seeded onto the cell culture insert and allowed contact with the air directly. The cells exposed to the air expressed polarity and immature lamellar bodies in their cytoplasm. Separately, the alveolar epithelial cells were cultured as spheroids to construct the three-dimensional condition. These cells expressed mature morphological characteristics as epithelial cells and lamellar bodies. The expression of the surfactant apoprotein-A (SP-A) and -C (SP-C) mRNA was compared in the cells cultured as a monolayer, the air exposed and the spheroids. SP-A mRNA was detected in all the cultured epithelial cells, but SP-C mRNA, a specific protein for the type II cells, was expressed only in the cells forming spheroids. The expression of uPA, one of the fibrinolytic enzymes, its receptor (uPAR) and its inhibitor-1 (PAI-1) were also examined. The epithelial cells exposed to the air and formed spheroids expressed a larger amount of uPA mRNA than the monolayer, although the amount of uPAR mRNA were comparable in these cells. The amount of PAI-1 mRNA significantly increased when the epithelial cells were exposed to the air. These results indicate that the type II alveolar epithelial cells induced and preserved their specific characteristics by taking the physiological three-dimensional structure, and these characteristics were partially restored by exposure to the air. Those findings suggest that the alveolar epithelial cells should be cultivated in three-dimensional form with contact to the air to regenerate an appropriate alveolar tissue.