Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers.

Abstract

Improved media have reduced the amount of serum protein required for clonal growth of normal human and chicken fibroblast-like cells. In the presence of limiting amounts of serum protein, attachment of colonies to tissue culture plastic surfaces is weak. Treatment of the culture surface with polylysine or other basic polymers causes the cells to adhere much more tightly. Growth is also improved on the surfaces treated with basic polymers, and further reductions in the concentration of serum are possible. At sufficiently low protein concentrations, growth of some types of cells is totally dependent on the use of a treated surface. Several different types of normal human and chicken fibroblast-like cells show improved growth on polylysine-coated surfaces, but no improvement was obtained in growth of a line of SV-40 transformed WI-38 cells. Acidic and neutral polymers are generally inactive. Collagen and gelatin improve growth slightly, but the effect is much less than that obtained with basic polymers. Both natural and synthetic polymers with an excess of basic groups are active, including histone, polyarginine, polyhistidine, polylysine, polyornithine, and protamine. The only critical requirement appears to be a polymer that carries a positive charge at a physiological pH.The amount of serum protein required for clonal growth of normal human and chicken cells has been reduced significantly by recent improvements in medium formulation (9, 16). ~ During experiments seeking to determine the role of the serum protein that was still required, we observed that colonies grown in the presence of limiting amounts of serum protein were poorly attached to the culture surface. Such colonies frequently came loose as sheets of cells during the mild mechanical In press. disturbances involved in removing the medium and fixing and staining the cultures.Polylysine-coated surfaces have been used to attach live myoblasts, amoeba, and sea urchin eggs firmly to surfaces used in microscope observation (15). This is believed to be accomplished through adsorption of polylysine molecules to the surfaces, followed by interaction between cationic sites on the attached molecules and anionic sites on the surface of the cells.We have tested polylysine and a variety of other synthetic and natural polymers for their effect on the attachment of vertebrate cells grown in the presence of minimal amounts of serum protein to culture surfaces. The attachment is much more stable on culture surfaces that have been coated

Growth-dependent synthesis of c-myc-encoded proteins: early stimulation by serum factors in synchronized mouse 3T3 cells.

“…In the present study, the expression of c-myc was monitored by measuring the relative rates of synthesis of the protein products of the gene in tightly synchronized subconfluent cells. This was achieved by combining the use of polylysine coating of the culture dishes with the use of medium that is similar to one d G 1 which allows the serum-free growth of simian virus 40-transformed 3T3 cells (31,40,42). In agreement with the documented improvement of growth in low serum (31), polylysine coating extended the time required to achieve growth arrest.…”

A low molecular weight extract of bovine serum albumin stimulates rabbit blastocyst cell division and expansion in vitro

“…Substitution of the standard culture medium by MCDB 104, an extremely complex tissue culture medium (McKeehan & Ham, 1976) with an extensive range of cofactors, did not give the same stimulatory effect as the low molecular weight extract (M. T. Kane, unpublished data). This indicates that the BSA factor is probably not one of the usual forms of a vitamin or other growth factor.…”

Immobilization of 293 cells using porous support particles for adenovirus vector production

“…These results clearly show that the treatment of reticulated PVF resin surface with polyethyleneimine is effective in promoting the immobilization of 293-F cells within BSPs. In mammalian cell culture, coating the plastic culture surface with a positively charged polymer such as poly-lysine and polyethyleneimine has been employed to facilitate the attachment and spreading of many types of anchorage-dependent cells (McKeehan and Ham 1976;Rüegg and Hefti 1984;Vancha et al 2004). Reportedly a macroporous cellulose carrier treated with polyethyleneimine and stabilized by cross-linking has been successfully used for high-density culture of hybridoma cells (Ong et al 1994) and recombinant L-929 cells (Terashima et al 1994).…”