STUDIES OF THE CULTURE OF LEUKEMIC CELLS IN VITRO*

Abstract

With the exception of a mouse leukemic cell line isolated by DeBruyn and described in 1955,l it has not been possible to propagate leukemic cells continuously in the absence of a feeder layer of nonleukemic cells. In 1938 Bichel2 reported that leukemic cells from mice would not reproduce in the culture media used at that time, but required a feeder layer of chick fibroblasts.These studies were confirmed and extended by DeBruyn et at.; who grew leukemic lymphoblasts on a supporting layer of mouse fibroblasts.In this paper I shall describe the successful culture of several strains of L-5178, a lymphocytic mouse leukemia. The medium developed, which contains peptone and very high levels of folic acid, permits the continuous reproduction, in vitro and in the absence of feeder layers, of the leukemic cells.? In support of the statement that leukemic cells representative of L-5178 reproduce in the medium devised, several lines of evidence have been obtained. Titration studies in susceptible strains of mice with cells grown in culture have demonstrated that there is no loss of virulence on long-continued propagation in vitro. The cells in culture retain their morphologic characteristics unaltered, continue to grow in the supernatant fluid of the medium, and retain their round-cell character and staining properties. Obtained from the ascitic cavity of mice during the logarithmic phase of their growth, cultures resume logarithmic rates of reproduction with an intervening lag period of no more than 5 hours. In addition, cytological studies have demonstrated that the diploid character of the cells is retained after 60 cell generations in c~l t u r e .~ Finally, single cells, isolated from populations grown in culture, have given rise to genetically homogeneous populations of cells with the same general properties as those exhibited by the parent strain^.^ The medium used is presented in TABLES 1, 2, and 3. The defined components are essentially those of a medium described by Scherrer;6 however, toxic as well as nonessential components have been omitted. Two alterations, essential for the growth of the leukemic cells, were necessary: one, a high level of folic acid, to satisfy an unusually high requirement for this vitamin,' and, two, the presence of a peptone, since reproduction of the leukemic cells is acutely dependent on one or more substances in such materials. Work concerning the isolation and characterization of the growth factors of peptone is in pr~gress.~ Other additions to the modified medium of Scherrer are glutamine, ascorbic acid, and glutathione. I t is unlikely that each of the required metabolites is present as yet at an optimal level, for emphasis has been

The Presence of Tumour Specific Membrane Antigen in the Serum of Rats with Chemically Induced Sarcomata

“…2 x 105 cells were seeded in 3 cm Falcon petri dishes and cultured in 3 ml of tissue culture medium (Fisher, 1958) and a reaction was defined as positive when the Fl was equal to or greater than 2-5, again based on extensive studies using control sera of different kinds. Absorption of immune serum with crossslinked tumour-bearing serum.…”

Biological Effects of Corynebacterium Parvum. III. amplification of resistance and impairment of Active Immunity to Murine Tumours

“…Tumourrs.The following tumours were obtained from the Chester Beatty Research Institute and maintained in ascitic form in the mice specified: R-I (radiation induced CBA leukaemia; Hewitt, 1962) in CBA-p; Hepatoma 129 (induced by CC13 in a C3H mouse, Andervont and Dunn, 1955) in CBA-p and BALB/c; Adj. PC6A (adjuvant induced BALB/c plasmacytoma, Potter and Robertson, 1960) in BALB/c and L5178 (DBA/2 leukaemia, Fischer, 1958) Hepatoma 129 grows rapidly from inocula of less than 10 cells and causes death with gross ascites and no evidence of metastases. Our form is not strain specific; originally a C3H tumour, it is routinely passaged in CBA against a minor histocompatibility barrier and a secondary strain is passaged in BALB/c mice against a strong histocompatibility barrier.…”

Effects of Valine Deprivation on the Biosynthesis of Ribosomal and Messenger RNA in a Mammalian Cell Line (L 5178 Y)

“…The L5178Y cell line is grown in suspension without stirring in Fisher's medium supplemented with 5O/, dialysed calf serum [13]. Doubling time is 12-14 h. To induce starvation, cells are centrifuged a t 37" washed once with Fisher's medium lacking valine, then resuspended in this medium a t a concentration of 1 to 1.5 x lo8 cells/ml.…”