| Abstract: | In order to obtain human granulocytic colony-stimulating factor (G-CSF) in large quantities, a large-scale culture system of human G-CSF-producing cells has been established. The cell used for this system was T3M-1, which grew in a monolayered sheet in F-10 synthetic medium supplemented with 10% fetal bovine serum. T3M-1 cells grew in rolling bottles at the velocity of 0.5 r.p.m. with about 22 hr. of population doubling time. When the culture reached confluency, it was incubated in a serum-free medium supplemented with 1% bovine serum albumin. The conditioned medium was harvested every week, concentrated by Amicon PM-10 membrane, and loaded on a Sephadex G-75 column. The molecular weight of G-CSF was estimated at about 30,000. This G-CSF was stable over a pH range of 1.0 to 11.0 at 4°C for 21 hr. The CSF activity was destroyed by either trypsin or chymotrypsin, but resisted to RNase and DNase. A slight decrease in the activity was produced by treatment with neuramidase. G-CSF stimulated granulocytic colony formation of human and mouse marrow cells. By using the roller bottle culture system, we could obtain more than 100 liters of cultured medium in a month, which was able to form about 150,000,000 colonies of human bone marrow cells. The recovery of the human G-CSF activity from gel-filtration column was very high (91.7%), and a large increase of specific activity was obtainable (13.3-fold). This culture system is therefore expected to aid in the large-scale preparation of human G-CSF, thereby facilitating further studies on this granulopoietic factor. |
