Cultured embryonic chick heart cells have been utilized as a model system for characterization of various membrane transport mechanisms. One advantage of this system is that the cells may be grown with differing geometries to minimize diffusion limitations and to optimize the growth configuration for particular techniques, such as ion-selective microelectrode measurements, fluorescent dye indicators, patch clamp, etc. A spontaneously contracting strand of cells embedded in a collagen matrix has recently been developed for measurements of cytoplasmic free ions by nuclear magnetic resonance (NMR) spectroscopy. These same strands, which provide the large numbers of cells needed for NMR, can be subdivided into small fragments ideal for cryopreservation prior to electron probe X-ray microanalysis (EPXMA). The aims in this study were to characterize the ultrastructure of cardiac cells within the strand, to demonstrate the quality of preservation obtainable by quick freezing methods, and to quantitatively map with EPXMA the distribution of physiologically relevant elements in thin, freeze-dried cryosections of the cells.Cells were isolated by serial trypsinization of 11-day old embryonic chick hearts. Strands of cells approximately 100 cm in length and 0.2 mm in diameter were obtained by extrusion of a cell-collagen mixture through polyethylene tubing into media within a culture dish. Three to five millimeter segments of 1-day old strands which contracted spontaneously were preserved by rapid immersion in liquid nitrogen-cooled liquid propane at 〜-185°C and stored in liquid nitrogen prior to being (a) cryosectioned for subsequent EPXMA or (b) freeze-substituted for conventional transmission electron microscopy (CTEM). Segments of strands were also chemically preserved in 2.5% glutaraldehyde in 0.1 M sodium cacodylate and processed as above for comparative CTEM. Cryosections of the frozen strands were cut at <-140°C with a dry glass knife and placed directly onto pre-cooled, carbon-coated, 200 mesh, fine bar nickel grids with a precooled implement. The grids were transferred to a liquid nitrogen cooled copper well for freeze drying at 10‒3 Torr over 24 to 48 hours. Prior to EPXMA, the grids were coated with 〜1OOÅ carbon.
Physiologically viable and synchronously beating heart cells can be grown in varying geometric configurations for parallel biochemical, electrophysiological, ultrastructural and x-ray microanalytical studies. Our objective in these experiments was to develop a method by which heart cells grown to confluency either as a sheet, spherical aggregate or polystrand could be rapidly frozen prior to cryosectioning and electron probe x-ray microanalysis.A single-sided propane jet freezer, which has been shown to achieve excellent freezing of confluent cell sheets prior to freeze fracture, was constructed according to Pscheid et al.; a specimen holder was also designed and constructed which facilitated rapid transfer (<10 sec) of the various cultured cells from media to the jet freezer (FIG. 1a,b).
Ultra-rapid freezing followed by cryoultramicrotomy is essential for the preservation of diffusible elements in situ within cells prior to scanning transmission electron microscopy and quantitative energy dispersive x-ray microanalysis. For cells or tissue fragments in suspension and for monolayer cell cultures, propane jet freezing provides cooling rates greater than 30,000°C/sec with regions up to 40μm in thickness free of significant ice crystal formation. While this method of freezing has frequently been applied prior to freeze fracture or freeze substitution, it has not been widely utilized prior to cryoultramicrotomy and subsequent x-ray microanalytical studies. This report describes methods devised in our laboratory for cryosectioning of propane jet frozen kidney proximal tubule suspensions and cultured embryonic chick heart cells, in particular a new technique for mounting frozen suspension specimens for sectioning. The techniques utilize the same specimen supports and sample holders as those used for freeze fracture and freeze substitution and should be generally applicable to any cell suspension or culture preparation.
Quantitative biological electron probe x-ray microanalysis (EPXMA) ideally makes use of two approaches in examination of samples: static raster probing and quantitative x-ray imaging. Efficient use of expensive microanalytical equipment requires intelligent decisions on the appropriate strategy for optimizing data collection. A low resolution, short dwell time map (for example, 64 × 64 pixels by 2 seconds) may survey one to several cells at low magnification and provide adequate statistics for most structures and elements. Elements, notably Ca, present in low concentrations within small regions may require the acquisition equivalent of long static raster probes, increased numbers of images from low resolution maps, higher resolution maps, longer dwell time maps, or a combination of these options.We examined raster probe and mapping results in cultured heart cells. In these cells EXPMA has been demonstrated to be an appropriate tool to observe the results of perturbation of the Na-Ca gradient, brought about by incubation of cells in low Na or Na-free media.
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