Cellular Activities in Tissue Cultures of Leukemic Human Bone Marrow.

Bone marrow specimens were obtained by aspiration from the sternum. Two to 3 ml. of aspirate were injected into a test tube containing 9 ml. of tissue culture medium and 1 ml. of heparin ( Liquaemin heparin, Organon ) . Bone marrow particles were collected with a capillary pipette from the sediment and from the fatty surface layer. The bone marrow particles were transferred into a second and then into a third ( and sometimes into a fourth) tube containing 5 ml. of medium each. The particles were then distributed on the coverslips in Leighton-tubes, or in Sykes-Moore chambers.1 Medium was added and the cultures were incubated at 37 C. The medium was first changed 24 hours later, and then at approximately 5 to 7 day intervals. Five to 12 Leighton-tubes were set up from one bone marrow specimen, each tube carrying 3 to 8 bone marrow particles. Coverslips were periodically removed, rapidly dried and stained with Wright stain. The tissue culture medium consisted of Hanks balanced salt solution containing amino acids and vitamins (Eagle basal medium),2 excess glutamine ( 10 ml. 200 mM per 1000 ml. medium), and 0.5 per cent lactalbumine hydrolysate; 20 per cent calf serum was added. The medium contained 100 units or penicillin and 0.01 mg. streptomycin per ml. The pH preferred for Leighton-tube cultures was 7.4; for chamber cultures, 7.0. Observations on living cultures maii tained in Sykes-Moore chambers were made by high power magnification phase contrast microscopy. The bone marrow particles were grown between the coverslip and a perforated cellophane membrane. Certain cultures were grown also in T flasks. Morphological observations with approximately 200X magnification were possible. Bacteriological sterility was regularly checked and a few contamined