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Large‐scale preparation and characterization of human colony‐stimulating factor

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.