Biodegradable microspheres. V: Stimulation of macrophages with microparticles made of various polysaccharides.

The interaction between four different microparticulate drug carriers and macrophages was investigated in vitro. The microparticles, consisting of crosslinked starch (1,4-alpha-D-glucan with 1,6-alpha-branches), dextran (1,6-alpha-D-glucan with 1,3-alpha-branches), lichenan (1,3-beta-D-glucan), or mannan (1,6-alpha-D-mannan with 1,2-alpha- and 1,3-alpha-branches), were investigated for their macrophage stimulatory properties. Macrophage stimulation was assayed by the uptake of [14C]glucosamine and stimulatory indices were calculated. Microparticles made of crosslinked lichenan were most stimulatory, followed by the biologically inert mannan and dextran microparticles. Biodegradable starch microparticles were less stimulatory to the macrophages than the other microparticles. All microparticles were phagocytosed to the same extent and stimulated the macrophages to release oxygen radicals. Lichenan, mannan, and dextran microparticles induced morphological changes in the macrophages when given in nontoxic doses. No morphological changes were observed when the macrophages were exposed to starch microparticles or soluble polysaccharides.

The fixation, dehydration, drying and coating of cultured cells of SEM.

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues. Studies on cultivated cells indicate that the glutaraldehyde component of a glutaraldehyde-based fixative does not contribute to the effective osmotic pressure of the fixative and thus the osmolarity of the buffer, and other components, must be equalized to that of the medium in which the cells grow. Even small deviations from this ideal effective osmotic pressure will result in osmotically induced artefacts. Disturbances of pH and temperature of the cultures prior to and during fixation will result in changes in the appearance of many cellular structures such as microspikes and ruffles. We find that osmium fixation is advisable in most instances for best possible membrance preservation and that even long periods of glutaraldehyde fixation do not compensate for osmium fixation. Dehydration always results in shrinkage. Freeze drying (FD) and critical point drying (CPD) also give rise to shrinkage, the former to a lesser degree than the latter. A gold-palladium alloy gives a less granular coating that does gold alone. When cultured cells are studied, a metal thickness of between 5 and 15 nm is usually sufficient to give rise to an adequate secondary electron production and to avoid charging even at accelerating voltages of 30-40 kV. Without treatment with OsO4 a thicker metal coating is required.

The fine structure of "dividers" and "non-dividers" in phase II human glial cell cultures.

A method is described which allows a comparison in the transmission electron microscope (TEM) of cells with different remaining proliferative capacity from one and the same culture. The method takes advantage of a mini-cloning technique employing hapatotactic palladium islands in combination with micro-dissection and preparation for TEM of islands carrying various numbers of cells after 10 days in culture, when all miniclones have become density dependent growth inhibited. By means of this technique non-dividers were compared with miniclones of dividers composed of five to eight cells originating from single cells. Moreover, large, immotile cells without peripheral ruffling activity, known to be non-dividers, were compared with small, ruffling cells, known to be dividers, in the reflection-interference mode in sparse cultures of living cells, and in the TEM mode as whole cell preparations after critical point drying of cells cultured on formvar-coated, gold EM-grids. Non-dividers proved to contain a moderate number of residual bodies, well developed Golgi areas, and often branched or circular mitochondria; they were thinly spread over the substratum with many focal points of contact, and large areas of close apposition between cell and substratum.

Visualization of the peripheral weave of microfilaments in glia cells.

A peripheral weave of microfilaments is visualized in human glia cells. In this weave small numbers of microfilaments converge to structures in the cell edge. Similar assemblies of microfilaments seem to be attached to structures on the surface of microspikes. Together with filaments splaying from the paracrystalline arrangement in microspikes, these units make up the peripheral weave. The filaments of the weave come in close contact with each other and with filaments of internal actin fibres.

Red blood cell preservation in protein-poor media. 2. Studies of changes in red cell shape during storage.

Red cells stored for up to 35 days have been studied by scanning electron microscopy. Formation of spicules occurred under all storage conditions in CPD and CPD-adenine whole blood, CPD-adenine red cell concentrate, and saline-adenine-glucose red cell concentrate. The number of cells with normal or near normal shape was larger if the blood was stored as a moderately concentrated suspension in saline-adenine-glucose medium or in autologous plasma as compared to the storage as whole blood. The occurrence of echinocytes during storage at +4 degrees C was not correlated to red cell ATP. It was shown that a drastic reduction of ATP leads to an increased formation of both spherocytes and echinocytes. The change of red cell shape during storage at +4 degrees C thus can be due to two processes, one unrelated to ATP and another related to ATP. Why storage as red cell concentrate is superior is not fully understood.

Growth stimulation of aged cells in culture.

Human glial cultures of any passage consist of two populations of cells: those with mitotic ability, and the non-dividers. The fraction of non-dividers increases with age of the culture, and dominates in late passages. When cells from midphase II cultures (passage 28) were sparsely seeded in dishes containing agarose partially covered by small, isolated, palladium squares (haptotactic islands) they settled on the palladium squares but not on the agarose. 58% of the cells divided, and formed mini-clones which became density growth inhibited within 10 days in medium with 5% serum. The non-dividers comprised 42%. They showed a characteristic indolent motility pattern. When these cultures were exposed to 15% fetal calf serum and 2 ng/ml mEGF (mouse epidermal growth factor) for another 5 days, nine per cent of the solitary cells had divided, and DNA measurements showed another 20% to have entered the S phase. About 40% of the initial single cells presented morphological alterations after the stimulation which are known to be early signs of entrance into the cell cycle after blockage in G1/G0. The present results in combination with earlier findings suggest that "old" cells approaching the non-dividing state become increasingly insensitive to stimulation by growth-promoting factors.

Plasma membrane motility of cultured human glia cells in phase II and III.

A human diploid glia line (U-787 CG) was cultured on haptotactic islands using a miniclone method recently described. The surface morphology and motility pattern of the cells were studied using timelapse cinemicrophotography and scanning electron microscopy. Proliferating cells showed locomotion and ruffling activity with intermittent associated macropinocytosis. Non-dividers, viz, cells which had not divided 10 days after seeding on the haptotactic islands, were immobile, larger than the dividers, rather flat with few microvilli, and showed only very occasional ruffles almost completely without associated macropinocytosis. Our present findings show that phase III cells growing singly on haptotactic islands in serum-containing medium behave like phase II cells starved of growth factors or density growth inhibited. The results corroborate the theory that stationary cells have depressed ruffling activity and associated macropinocytosis as compared with proliferating cells.