No abstract

No abstract

Endothelial cell growth factor(s) from several previously untested human tumor cell lines (i.e., SK-HEP-1, MG63, A375, TE671-C1, RD) were detected using a low cell inoculum growth assay. The final cell density in the 2-cm2 wells was determined by a highly sensitive DNA content measurement performed directly in the tissue culture plates. The sensitivity of the assay to human tumor cell growth factors depended critically on the low cell inocula, 2,000 to 5,000 cells/well. Most of the bovine endothelial cells used were cloned from primary cultures; all the cell lines obtained from various fetal and nonfetal sources responded to the growth factor(s) (up to a 16x stimulation) as well as to endothelial cell growth supplement. Dose response curves showing the cell specific response of bovine endothelial cells were obtained. The growth stimulatory activity and the in vivo chick embryo chorioallantoic membrane assay responses correlated sufficiently to imply that the assay is detecting tumor angiogenesis factor or some closely related activity. This in vitro assay should prove useful in the identification and purification of tumor-derived factors and in the elucidation of the role of these factors in the events comprising angiogenesis.

A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10(9) cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O2 and CO2 were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10- to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4- ad 40-liter reactor also has been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10 x 10(6) cells/ml with a total yield of 8.7 x 10(11) cells from 360 liters of medium.

No abstract