Replacement of serum by insulin, transferrin, albumin, phosphatidyl choline, cholesterol, and some trace elements in cultures of mouse myeloid leukemia cells sensitive to inducers of differentiation

Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 00144827/79/140421-08$02.00/0

Cell Research 124 (1979) 421428

Experimental

REPLACEMENT

OF SERUM

PHOSPHATIDYL ELEMENTS CELLS

BY INSULIN,

CHOLINE, IN CULTURES

SENSITIVE

TRANSFERRIN,

CHOLESTEROL, OF MOUSE

TO INDUCERS

Y. HONMA, T. KASUKABE,

ALBUMIN,

AND SOME TRACE MYELOID

LEUKEMIA

OF DIFFERENTIATION

J. OKABE and M. HOZUMI

Departmentof Chemotherapy, Ina-machi,

Saitama Cancer Center Research Institute, Kitaadachi-gun, Saitama 362, Japan

SUMMARY In serum-supplemented culture conditions, mouse myeloid leukemia cells (Ml) could be induced by various inducers including dexamethasone to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzymes, and change into forms that were morphologically similar to macrophages and granulocytes. Ml cells grew in the absence of serum in synthetic medium supplemented with insulin, transferrin, and several trace elements. The cells were maintained for more than 6 months in this medium. Induction of differentiation by dexamethasone was obtained with the cells that had been grown in serum-free medium for several days. However, the ability to be induced to differentiate in this medium was then lost with further growth of the cells in this medium. These cells could be induced to differentiate by dexamethasone with the aid of albumin, phosphatidyl choline, and cholesterol, but not by the inducer alone. The cells were leukemogenic in syngeneic mice. Macrophage-like cells that developed spontaneously from Ml cells were continuously maintained in this environment without morphological alteration or loss of their differentiated properties such as formation of Fc receptors, phagocytosis, and lysozyme synthesis.

Mouse myeloid leukemia Ml cells were induced by various inducers to differentiate into forms that are functionally and morphologically similar to macrophages and granulocytes [ 1, 21. Differentiated Ml cells produced a proteineous factor(s) stimulating their own differentiation [ 31. Differentiation-resistant Ml cells produced a factor(s) that inhibited induction of differentiation of sensitive Ml cells, while differentiation-sensitive Ml cells did not produce [4]. The differentiation-stimulating factor(s) and the inhibitory factor(s) were released from the cells to the conditioned medium of the cultures. These findings suggest that differentiation of Ml cells is regulated by some factors released from the other Ml cells.

When Ml cells were incubated without serum in conventional culture medium, all the cells died several days later. The use of serum entails serious disadvantages of introducing an unknown and uncontrolled set of substances which influence growth and differentiation of Ml cells. This not only raises problem of fluctuation of the cellular responses, but also makes it difficult to analyze biological activities released by the cells into the culture medium. Therefore, there is a need for tissue culture medium in which serum is replaced by purified serum components. Recently, role of serum in supporting growth of some mammalian cells in culture has been substantially clarified. Most of the serum required for cell growth Exp Cdl Res 124 (1979)

422

Honma et al. Experimental

media

Serum-free medium was prepared according to Hutchings & Sato [5], except antibiotics. Serum-free Ham’s F12 medium (SFF12) containing sodium bicarbonate at 1.2 g/l, ‘300 &ml kanaiycin sulfate, 15 mM HEPES (N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid), and trace elements (0.5 mM MnCIZ .4 HZO; 0.5 nM (NH,),Mo,O,, .4 H,O; 0.25 nM NiSO,. 6 H,O; 15 nM H,SeO,; 250 nM Na,SiO, .9 H,O: 0.25 nM SnCl,: 2.5 nM Na,VO,. 4 H,O: 50 nM C&6,) were used ih all experiments. Bo&e serum albumin was dissolved in phosphate-buffered saline (PBS), 100 mg/ml. Cholesterol (25 mg/ml) and phosphatidyl choline (100 mglml) were dissolved in ethanol. Dexamethasone solution was prepared in ethanol at a concentration of lo+ M. Lipopolysaccharide was suspended in sterile PBS, 500 pg/ml.

Fie. 1. Abscissa: davs in culture: ordinate: cell no. (cl05 cells/ml). e Growth curve of mveloid leukemia Ml (SF-S) cells in serum-free medium. -Cells were inocul&ed at 4X 104 Assay of the properties of cells/ml in SFF12 medium supplemented with 0, 10% differentiated cells calf serum; 0, insulin (5 &ml) and transfen-in Lysozyme activity was determined by a modification (5 dml). of the lvsoolate method of Osserman & Lawlor [15] with lyiopiates containing 1% agar, l/l5 M sodium phosphate buffer (pH 6.6), 0.05 M NaCl and 0.5 mg/ml in culture can be replaced by a combination heat-killed Micrococcus lysodycticus [ 161.After 24 h at the diameters of the clear zone were measured. of some serum components including hor- 27”C, Fc receptors, and phagocytic and locomotive activities mones and transferrin [5-121. were assayed by the procedures reported previously In this paper we show that in Ml cell cul- [17, 181. The percentages of cells that were morphologically similar to granulocytes and macrophages tures established in a serum-free medium, were determined in May-Griinwald-Giemsa stained serum is replaced by insulin, transferrin, al- smears.

bumin, phosphatidyl choline, cholesterol, and some trace elements. Moreover, the serum-free culture conditions allow growth of macrophage-like cells that developed spontaneously from Ml cells. MATERIALS

AND METHODS

Cells Cells used in this experiment were myeloid leukemia Ml cells that were established from an SL strain mouse with mveloid leukemia rll and macroDhane-like Mm-l cells thit spontaneously developed f&m kl cells [ 131. These cells were kindlv SUDDliedbv Professor Y. Ichikawa and Dr M. Maeda,‘i
Materials Bovine crystalline insulin, human transferrin, bovine serum albumin (crystallized and lyophilized), cholesterol, L-a-phosphatidyl choline (Type III-S, from soybean), and dexamethasone were obtained from Sigma Chemical Co., St Louis, MO. Trace elements were obtained from Wako Pure Chemical Co., Tokyo. Ham’s F12 medium was supplied by Nissui Seiyaku, Tokyo. Lipopolysaccharide (Boivin type) from Salmonella typhimurium was obtained from Difco Laboratories, Detroit, USA.

RESULTS In SFF12 medium, myeloid leukemia Ml cells degenerated and died. However, the growth rate of the cells in SFF12 medium supplemented with insulin (5 pg/ml) and transferrin (5 &ml) was similar to that in SFF12 medium supplemented with 10% calf serum (SFF12S medium). On the other hand, saturation density of the cells in the serum-free hormone-supplemented medium

Differentiation

of leukemia cells in serum-free medium

423

Fig. 2. Phase-contrast photomicrographs of cells cultured in SFFl2H medium. (a) SF-S cells cultured for 4

months; (b) Mm-l-SF cells cultured for 3 months in SFFl2H medium. Bar, 50 pm.

(SFF12H medium) was lower than that in the serum-supplemented medium. Fig. 1 shows a typical growth curve of the cells. Morphologically, the cells in SFF12H medium were similar to those in SFF12S medium. The cells had a large round nucleus and a small, strongly basophilic cytoplasm (figs 2, 3). The cells had been cultured for more than 6 months in the serum-free me-

dium. Omission of either insulin or transferrin resulted in degeneration of the cells. This indicates that maintenance of the cell culture may require at least insulin, transferrin and several trace elements. Sensitive Ml cells cultured in SFF12S medium could be induced differentiation by various inducers including dexamethasone and bacterial lipopolysaccharide, while un-

Fig. 3. Cell smears of SF-S cells (a) and Mm-l-SF cells (b), stained with May-Griinwald-Giemsa solution. x1250. Exp Cell Res 124 (1979)

424

Honma et al.

Table 1. Induction Ml cells cultured medium

Cell We SF-S

SF-R

of lysozyme activity of in serum-free (SFFlZH) Lysozyme act. KJlw protein)

Treatment None Dex (10-r M) Dex (10-r M), serum (10%) Dex (1O-7M), BSA, PC and Chl Dex (10-r M), BSA Dex (10-r M), PC and Chl BSA, PC and Chl None Dex (lO-B M), BSA, PC and Chl

1.8 7.1 40.8 38.4 16.8 9.9 2.4 0 0

Lysozyme activity was determined 4 days after seeding with the materials indicated. One unit of activity was defined as the amount equivalent to 1 pg of egg white lysozyme under these conditions. Dex, dexamethasone; BSA, bovine serum albumin (3 mg/ml); PC and Chl, phosphatidyl choline (100 pg/ ml) plus cholesterol (25 &ml). Average data from four separate experiments are shown.

treated Ml cells did not. However, the Ml cells cultured in SFFl2H medium for a long time were unable to differentiate by any inducers (tables 1,2). Then, the following experiments were Table 2. Induction

of morphological

carried out to examine the response to the inducer of cells cultured in SFF12H medium. Sensitive Ml cells cultured in SFF12S medium were washed with PBS and cultured in SFF12H medium. The differentiation-associated properties of the cells were assayed 4 days later. When the inducer was added, most of the cells were induced to differentiate, judging by the induction of phagocytosis, lysozyme activity, adherence to the dish, and morphological changes similar to macrophages and granulocytes. On the other hand, resistant Ml cells were not induced to differentiate by the inducer when cultured in SFF12H medium. The inducibility of differentiation in sensitive Ml cells cultured in SFF12H medium (SF-S cells) was labile and decreased during culture of the cells for a long time. Although loss of the inducibility of differentiation varied with experiments, the ability was lost within 9-20 days. All inducers we tested could hardly induce differentiation of SF-S cells cultured for more than 100 days. However, the cells could be induced differentiation by dexamethasone or lipopolysaccharide when the cells were cultured in serum-supplemented SFF12H medium (table 1). Similar results were obtained

changes of SF-S cells by inducer with aid of some

materials Morphological changes (%)

Treatment Untreated Dexamethasone (10-r M) Dexamethasone, albumin, cholesterol and phosphatidyl choline Lipopolysaccharide. (0.1 pg/ml) Lipopolysaccharide, albumin, cholesterol, and phosphatidyl choline

Myeloblastic cells

Cells in intermediate stages

99.1 78.9

0.9 18.4

0 2.7

33.3 80.4

43.0 18.1

23.7 1.5

27.9

47.3

24.8

Mature macrophages or granulocytes

Morphological changes were determined 5 days after seeding with the materials indicated. Average data from three separate experiments are shown. Exp Cell Res 124 (1979)

Differentiation

of leukemia cells in serum-free medium

Table 3. Differentiation-stimulating factor(s) of conditioned medium of cells cultured in SFFIZH medium

Treatment

Cont. (%, v/v)

None Treated

25 :i

Table 4. Inhibitory factor(s) for induction differentiation in conditioned medium SF-S and SF-R cells

Phagocytic act. (% of phagocytic cells)

Lysozyme act. Wlmg protein)

Cell we

Cont. (%, v/v)

Expt I

SF-S

1.4 21.1 33.3

1.1 33.2 58.9

SF-R

10 30 10

5.1 14.6 81.5

425 of of

Inhibitory activity (%)

Cells were inoculated at 5x 106 cells in 10 ml of SFFlZH medium containing albumin, phosphatidyl choline, and cholesterol with or without 10-r M dexamethasone. The culture medium was collected 3 days later, and dialysed against PBS. In the control culture, dexamethasone was added before dialysis. The effect of the conditioned medium on the inductions of phagocytosis and lysozyme activity in Ml cells was examined [3]. Average data from three separate experiments are shown.

when a set of bovine serum albumin (3 mg/ ml), phosphatidyl choline (100 Fglml), and cholesterol (25 pglml) was used instead of serum (tables 1,2), although addition of the materials to SFF12H medium did not affect the cell growth. The inducibility of differentiation in this culture remained for more than 6 months. These results indicate that SF-S cells can be induced to differentiate by the inducer with the aid of albumin, phosphatidyl choline, and cholesterol, but not by the inducer alone. Resistant Ml cells cultured in SFF12H medium for a long time (SF-R cells) were not induced differentiation by the inducer even when SFF12H medium was supplemented with albumin, phosphatidyl choline, and cholesterol. The differentiated Ml cells induced by glucocorticoids produced the differentiation-stimulating factor(s) of Ml cells, and the factor(s) was released to the conditioned medium [3]. When SF-S cells were treated by dexamethasone with albumin, phosphatidyl choline, and cholesterol, the cells were induced to produce the factor(s) and

Expt II 1.2 49.5

Exot I. The cells (5~ lOVm1) were incubated in SF’Fl2H medium for 2 days. The conditioned medium was harvested and the activitv which inhibited the induction of phagocytosis in sensitive Ml cells by treatment with 10-r M dexamethasone for 2 days was measured [4]. Phagocytic activity of the control cells was 47.5 %. Expt II. The activity which inhibited the induction of lvsozvme activitv in sensitive Ml cells bv treatment of BOO--7 ‘M dexamethasone for 4 days was measured. Lvsozvme activitv of the control cells was 44.3 U/mg protein. The activity was calculated as the inhibition percentage. Average data from four separate experiments are shown.

released it to the conditioned medium (table 3). Untreated resistant Ml cells produced inhibitory factor(s) for the induction of their own differentiation, while the factor(s) was hardly detected in culture of sensitive Ml cells. We postulated that the factor(s) may be associated with resistibility to differentiation [4]. The conditioned medium of SF-R cells was harvested and the inhibitory activity for induction of phagocytosis in sensitive cells by the inducer was determined. Table 4 shows that the inhibitory activity in conditioned medium of SF-R cells is much stronger than that in conditioned medium of SF-S cells. We confirmed the inhibitory effect of the conditioned medium on the other differentiation-associated properties, morphological changes and lysozyme activity. These results showed that SF-R cells produced the inhibitory factor(s), but it was hardly detected in the culture of SF-S cells. Previous reports indicate that the surExp Cell Res 124 (1979)

426

Honma et al.

Table 5. Leukemogenicity

of Ml or Mm-l cells in syngeneic SL strain mice

Cell type

Culture medium

Sensitive Ml SF-S Resistant MI SF-R Mm-l Mm-l-SF

Stock culture medium SFFlZH Stock culture medium SFF12H Stock culture medium SFF12H

Leukemia developed/no. of recipient

8/S 8/8 o/10 o/10

Survival days (mean & SD.) 40.8Az4.3 42.3k3.1 22.6k2.9 25.4k3.5

Sensitive and resistant Ml cells were cultured in stock medium (Eagle’s minimum essential medium with a 2-fold concentration of amino acids and vitamins with 10% calf serum). Cells were washed three times with PBS and inoculated intraperitoneally into SL strain mice (10scells/mouse). Mm-l-SF cells, Mm-l cells cultured in SFF12H medium for longer than 100days.

vival of mice inoculated with sensitive Ml cells is longer than that with resistant Ml cells [ 191. Therefore, the leukemogenicity of the cells and survival times of mice inoculated with SF-S or SF-R cells were investigated (table 5). All mice inoculated with 105of SF-S or SF-R cells died of leukemia. The survival times of mice inoculated with SF-R cells were similar to those of mice inoculated with resistant Ml cells. The survival times of mice with SF-S cells were longer than those of mice with SF-R cells and were similar to those of mice inoculated with sensitive Ml cells. These re-

02466 Fig. 4. Abscissa:

time in culture (days); ordinate: cell no. (X 105cells/ml). Growth curve of macrophage-like (Mm-l-SF) cells. Cells were cultured in SFF12 medium supplemented with: 0, 10% calf serum; 0, insulin and transfertin.

Exp Cd Res 124 (I 979)

sults suggest that SF-S cells and SF-R cells are related to the sensitive and the resistant Ml cells, respectively. Macrophage-like (Mm-l) cells, which developed spontaneously from a clonal line of Ml cells, were cultured in SFF12H medium. Morphologically, the cells in SFF12H medium were similar to those in SFF12S medium (figs 2, 3). The cells adhered to and spread over the dish surface. Their cytoplasm had various forms-fungiform, triangular, rectangular, or round. The cells were stained with May-Grtinwald-Giemsa solution The predominant type of cells was those with eccentrically located nuclei and enlarged cytoplasm. Fig. 4 shows a typical growth curve of the cells in SFF12H medium. Some characteristics of the cells in SFF12H medium are shown in table 6. The cells remained for more than 6 months in this culture condition. There was no significant difference in the functional and biochemical characteristics between cells in SFF12H medium and those in SFF12S medium. The Mm-l cells in SFF12H medium were intraperitoneally inoculated into syngeneic SL mice (table 5). During an observation period of 4 months, no tumor or leukemia developed, although all the mice inoculated

Differentiation

Table 6. Some properties of Mm-l cells cultured in serum-free medium Culture condition (medium)

Fc receutors (% of cells with rosette) Phagocitosis (% of phagocytic cells) Lysozyme activity (U/mg protein) Adherence to dish (% of adherent cells) Morphology

SFF12S

SFFl2H

80-85

80-85

>98

>98

149

126

>80 Macrogy-

>80 Macrof;;fse-

Average data from four separate experiments are shown.

with SF-S or SF-R cells developed leukemia and died within 2 months. Therefore, Mm-l cells cultured in SFF12H medium, as well as those in serum-supplemented medium [ 131, suppressed leukemogenicity in the mice. These results indicate that Mm-l cells in the serum-free medium are closely related to those in the serum-supplemented medium. DISCUSSION In some mammalian cell lines, serum-free synthetic medium, supplemented with some hormones and transfer-r-in supports clonal growth, long-term cultivation, and a growth rate equal to that of serum-supplemented medium [5-121. We have found that a set of insulin, transfer-r-in, and several trace elements can support clonal growth and longterm cultivation of mouse myeloid leukemia Ml cells for more than 6 months. Other hormones and serum components which were tested and failed to promote growth of Ml cells in SFF12H medium were fetuin, albumin, epidermal growth factor,

of leukemia cells in serum-free medium

427

fibroblast growth factor, chorionic gonadotoropin, prolactin, follicle-stimulating hormone, luteinizing hormone, thyroxine, progesterone, estrogen, testosterone and hydrocortisone. Most of the serum required for clonal growth of erythrocytes and granulocyte-macrophage precursor cells and B lymphocytes in culture can be replaced by a combination of four normal serum components; albumin, transferrin, lecithin (phosphatidyl choline) and selenium [7, 81. Myeloid leukemia cells cultured in serum-free medium showed requirement for insulin, transferrin and selenium. No requirement of lecithin and albumin for cell growth was demonstrated in these conditions of culture, although these components were required for induction of cell differentiation in cells cultured in serum-free medium for a long time. Induction of differentiation was obtained with sensitive cells that had been grown in the serum-free medium for several days. However, SF-S cells cultured in SFF12H medium for a long time were not induced differentiation by glucocorticoids or lipopolysaccharides in this medium. On the other hand, the cells could be induced differentiation when a set of albumin, phosphatidyl choline, and cholesterol was added to the serum-free medium. A requirement for phosphatidyl choline has been shown before in the formation of a granulocytemacrophage colony by normal mouse bone marrow cells [7]. No cholesterol requirement was shown in these culture conditions. However, cholesterol was added routinely in our experiments, because phosphatidyl choline dispersions prepared without cholesterol tend to deplete cell membrane of cholesterol [20]. A requirement for albumin by hematopoietic cells has been shown before [20]. Albumin binds mainly by hydrophobic interaction with a wide Exp Cell Res 124 (I 979)

428

Honma et al.

variety of molecules [21]. One function of albumin in culture may bind with the inducer of differentiation, such as dexamethasone and lipopolysaccharide. In addition, our results as well as those of others [8, 211 suggest that albumin plays a role in making lipid available to the cells, a function also provided in vivo by serum lipoproteins. The present results show that the resistant Ml cells cultured in the serum-free medium, as well as those in the serumsupplemented medium, produced inhibitory factor(s) of their own differentiation and released them into the culture medium (table 4). Moreover, differentiated Ml cells cultured in serum-free medium produced the factor(s) stimulating their own differentiation (table 3). The use of serum entails the serious disadvantage of introducing an unknown and uncontrolled set of substances which influence growth and differentiation of myeloid leukemia cells. In fact, induction of differentiation of Ml cells was dependent, to some extent, on the lot of serum used in culture medium. Therefore, the serum-free culture may provide an excellent system for analysis and understanding of the biological significance of the factors involved in growth and differentiation of the cells.

Exp Cell Res 124 (1979)

REFERENCES 1. Ichikawa, Y, J cell physio174 (1962) 223. 2. Hozumi, M, Honma, Y, Okabe, J, Tomida, M, Kasukabe, T, Takenaga, K & Sugiyama, K, Oncogenic viruses and host cell genes (ed Y Ikawa & T Odaka) p. 341. Academic Press, New York (1979). 3. Honma, Y, Kasukabe, T & Hozumi, M, Exp cell res 111(1978) 261. 4. Okabe, J, Hayashi, M, Honma, Y & Hozumi, M, Int j cancer 22 (1978) 570. 5. Hutchines. S E & Sato. G H. Proc natl acad sci US 75 (1978)901. 6. Hayashi, I &.Sato, G H, Nature 259 (1976) 132. 7. Guilbert, L J & Iscove, N N, Nature 263 (1976) 594. 8. Iscove, N N & Melchers, F, J exp med 147 (1978) 923. 9. Yarnane, I, Murakami, 0 & Kato, M, Proc sot exp biol med 149 (1975) 439. 10. Allegra, J C & Lippman, ME, Cancer res 38 (1978) 3823. 11. Rizzino, A & Sato, G H, Proc natl acad sci US 75 (1978) 1844. 12. Bottenstein, J E & Sato, G H, Proc natl acad sci US 76 (1979) 514. 13. Maeda, M & Ichikawa, Y, Gann 64 (1973) 265. 14. Honma, Y, Kasukabe, T, Okabe., J & Hozumi, M, Cancer res 39 (1979) 3167. 1.5. Osserman, E F & Lawlor, D P, J exp med 124 (1966) 921. 16. Kasukabe, T, Honma, Y & Hozumi, M, Gann 68 (1977) 765. 17. Lotem, J & Sachs, L, Int j cancer 15 (1975) 731. 18. Honma, Y, Kasukabe, T & Hozumi, M, Gann 68 (1977) 405. 19. -J natl cancer inst 61 (1978) 837. 20. Cooper, R A, Arner, E C, Wiley, J S & Shattil, S J, J clin invest 55 (1975) 115. 21. Arai, S, Yamane, I, Tanno, Y & Takishima, T, Proc sot exp biol med 154 (1977) 444. Received April 23, 1979 Revised version received July 2, 1979 Accepted July 11, 1979