A total arrest of cell membrane movement occurred after incubation for 2 h of cultured fibroblasts with the polycation DEAE-dextran. The immobilized cells resumed normal membrane undulations after exposure for 5 min to the polyanion dextran-sulphate. The influence of DEAE-dextran on the electrophoretic mobility, which was seen after 10 min of incubation was also erased by a subsequent incubation with dextran-sulphate. No influence on growth of the culture was seen.
Polyions are, depending on electric charge, powerful tools for enhancement and suppression of several in vitro reactions such as the following; infection with many viruses (Toyoshima & Vogt, 1969), antibody complement-dependent cytotoxicity (Ebbesen, 1972), and certain cellular immune reactions (Martz & Benaceraf, 1973; Yu, Yan & Pearson, 1975). In this paper we describe an attempt to use a polycation and a polyanion for controlling in vitro movement of fibroblasts.
MATERIALS AND METHODS
The polycation used was DEAE-dextran, mol.wt 2 × 106and the polyanion dextran-sulphate, mol. wt 5 × 105 (Pharmacia, Uppsala, Sweden); with 50 μg/ml Eagle’s Minimum Essential Medium (MEM) these compounds do not alter the original pH value of 7.2.
For cinemicrography, 2 × 105 secondary BALB/c mouse embryo fibroblasts were seeded in a 4-cm* Rose chamber (Paul, 1965) containing 2 ml of MEM with Hanks’ balanced salt solution pH 7-2 containing Ca2+ 1.3 mM, Mg2+ 0.8 mM and 10% calf serum. After 24 b at 30 °C, polyion was added and time-lapse cinemicrography under phase contrast (Güttler & Larsen, 1970) was carried out.
Electrophoresis was done with a Carl Zeiss cytopherometer (Forrester & Salaman, 1967). With about 106 cells in a 23-cm2plastic bottle the subconfluent culture was drained of medium and washed twice with 5 ml of MEM, followed by the addition of 2 ml of 0.25% trypsin in phosphate-buffered saline, pH 7’2. After incubation for 5 min at 37 °C, the cells were harvested in 10 ml MEM, spun down (500 g for 10 min at 4 °C), resuspended in MEM and spun down again. After the last centrifugation, the cells were resuspended (106cells/ml) in MEM containing 25 μg/ml of the compound to be tested. After 10 min and 2 h respectively, at 37 °C, the cells were washed twice in MEM and resuspended in aqua dist. containing per 1., 40 g Sorbitol, 8 g NaCl, 0.2 g KC1, 1.15 g Na2HPO4, and 0.2 g KH,P04, pH 7.2–7.4.
The influence of polyion on growth of the fibroblasts was measured by seeding 105 cells/4 cm2 and counting the cells in parallel cultures. On day 1 after seeding, 12 cultures were counted and each type of treatment used in the cinemicrographic experiment was administered to 12 cultures which were then counted on day 2. The cultures were washed 3 times before trypsinization and counting was done with and without trypan blue. MEM with Hanks’ buffer and 10% calf serum was used as medium throughout.
Time-lapse cinemicrography revealed that cells cultured in plain medium (MEM) moved randomly about throughout the observation period (Fig. 1). The addition of 50 μg of the polycation DEAE-dextran per ml of MEM resulted after 2 h in a dramatic reduction in cell movements, followed 10 min later by total immobilization (Fig. 5), whereas incubation with dextran-sulphate had neither enhancing nor inhibitory effect on cell mobility. In one of the three experiments, the immobilization of the cell membrane by DEAE-dextran was preceded by a short burst of spasms of the cell. Simultaneous with arrest of membrane undulation, we noticed an immobilization of the intracytoplasmic movement of small organelles or inclusions. Removal of the DEAE-dextran-containing medium followed by incubation for 5 min with MEM containing 50 μg of the polycation dextran-sulphate per ml of MEM and thereafter refilling of the chamber with plain MEM, restored full cell mobility after 15 min (Fig. 9).
The electrophoretic mobility of fibroblasts brought into suspension by trypsinization was altered according to the net charge of the polyion with which the cells were preincubated (Table 1). Preincubation for 10 min gave the same result as preincubation for 120 min.
Counting before polyion treatment and 24 h later revealed a doubling in number of viable cells with no differences (P < 0.1) between the differently treated cultures.
Immobilization of the fibroblasts during DEAE-dextran treatment is not due to permanent damage, since the arrest could be reversed and since growth of the cultures (Moroson, 1971) was unaffected.
Nullification of the polycation DEAE-dextran effects on cell movement and electrophoretic mobility by the polyanion dextran-sulphate suggests that the positively charged residues of DEAE-dextran are essential to its effects. Local paralysis of the cell membrane is also known to occur opposite the anode of cells in an electric potential gradient where an even concentration of electrolytes is ensured (Weiss & Scott, 1963). The positively charged polylysin inhibits pseudopod formation in amoeba and this inhibition is reversed by the negatively charged polyglutamate (Wolpert & Gingell, 1968). The alteration in electrophoretic mobility is induced by DEAE-dextran much faster than membrane immobilization, which shows that immobilization might be a secondary effect of change in cell surface charge, or the 2 DEAE-dextran effects are unrelated.
A gluing of cells to substratum is a likely effect of DEAE-dextran (Nordling, 1967), as we find it more difficult to detach fibroblasts from DEAE-Sephadex beads (Pharmacia) than to loosen fibroblasts growing on negatively charged CM-Sephadex beads (unpublished); however, it hardly explains the final immobilization. First, in one case violent membrane undulations were observed just prior to the arrest of membrane movements. Second, intracellular streaming also appeared to come to a standstill. A direct effect on intracellular contractile protein (Young & Perdue, 1972) and/or a redistribution of membrane proteins (Edidin & Weiss, 1972) seem more likely mechanisms. We have previously found that cells treated with fluorescent DEAE-dextran initially show a diffuse staining which modifies during the next two hours into a granular fluorescence (Hesse, Ebbesen & Kristensen, 1978).
The authors wish to express their gratitude to Dr R. Rask-Nielsen and Professor H. Holter for their suggestions and help during the preparation of this manuscript.
This investigation was supported by grants from Daell Fonden, The Danish Cancer Society, P. Carl Petersens Fond, Anders Hasselbalchs fond til leukæmiens bekæmpelse, Kong Christian den X’s foundation, and The Danish Medical Research Council.