Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic fibroblast growth factor (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-20 ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serumfree medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained >5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.During embryonic development neurons and glia are generated from the neuroepithelial cells of the neural tube. Although the factors that control the growth and differentiation of these cells are largely unknown, the survival and growth of central nervous system neurons appear to be regulated by trophic factors. Nerve growth factor, the prototypical neurotrophic factor, supports the survival of cholinergic neurons of septum, striatum, and nucleus basalis (1-3). Basic fibroblast growth factor (bFGF) has a much broader range of effects and supports the survival of a variety of neurons from different regions of the brain (4-8); its most significant effect, however, is on fetal rat hippocampal neurons (4). Both neuronal survival and neurite elongation are increased in the presence of bFGF (4). Moreover, at concentrations >5 ng/ml, bFGF has short-term proliferative and morphodifferentiation effects on neuronal precursor cells from mesencephalon, telencephalon, cerebral hemispheres, and spinal cord (9-11). Proliferation and differentiation of primary neurons from both fetal and adult striatum in response to a combination of nerve growth factor and bFGF, only epidermal growth factor, or bFGF have been reported (12-14). Furthermore, long-term mixed cultures of neurons and glia have been established from fetal and neonatal rat and mouse brains (15, 16). However, in many cases these cell lines were transformed in vitro and caused tumors when injected into syngeneic animals (15, 16).Unavail...

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The effects on morphology and diagnostic interpretation of delayed processing of refrigerated effusion samples have not been well documented. The potential for cellular degeneration has led many laboratories to reflexively fix samples rather than submit fresh/refrigerated samples for cytologic examination. We sought to determine if effusion specimens are suitable for morphologic, immunocytochemical, and DNA-based molecular studies after prolonged periods of refrigerated storage time. Ten fresh effusion specimens were refrigerated at 4 degrees C; aliquots were processed at specific points in time (days 0, 3, 5, 7, 10, 14). Specimens evaluated included four pleural (3 benign, 1 breast adenocarcinoma) and six peritoneal (2 ovarian adenocarcinomas, 1 malignant melanoma, 2 mesotheliomas, 1 atypical mesothelial) effusions. The morphology of the cytologic preparations from the 10 effusions was preserved and interpretable after 14 days of storage at 4 degrees C. The immunocytochemical profile of the samples (AE1/AE3, EMA, calretinin, and LCA) was consistent from day 0 to day 14. Amplifiable DNA was present in all samples tested on day 14. We conclude that cytopathologic interpretation of effusion samples remains reliable with refrigeration at 4 degrees C even if processing is delayed.