Biochemie der Ernährung
Prof. Dr. Stefan Lorkowski
Monocytic and macrophage-like cell lines
Isolating and culturing primary monocytes and macrophages for functional analyses is technically demanding and expensive. Several attempts have been made therefore to generate immortalized monocyte/macrophage-like cell lines that can be cultured easily in the amounts required for functional studies. In the following published monocyte/macrophage-like cell lines are briefly described.
ANA-1 (Mus musculus)
The ANA-1 cell line were established from bone marrow cells of C57BL/6 (H-2b) mice infected with J2 recombinant retrovirus for immortalization (Cox et al. 1989). This cell line exhibits dimorphic growth patterns with both adherent and non-adherent cells. Their morphology is characterized by numerous vacuoles and eccentric nuclei. ANA-1 cells are positive for FcyR (Ly-17), Mac-1, Ly-5 and heat stable antigen (HSA) as well as Ly5.1 and Ly6B.2 (Cox et al. 1989). These cells also express lysozyme continuously, show a very high constitutive phagocytic activity and are tumoricidally active upon LPS treatment, thus belonging to the myelomonocytic lineage. ANA-1 cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotics (Cox et al. 1989).
AML-193 (Homo sapiens)
The AML-193 cell line has been established from a 13-year-old female with acute monocytic leukemia (Lange et al. 1987). Interleukin (IL)-3 and granulocyte/macrophage colony stimulating factor (GM-CSF) act synergistically to stimulate growth of these cells. Granulocyte colony stimulating factor (G-CSF) also supports short term and long term growth of AML-193 cells and acts synergistically with GM-CSF in inducing proliferation of the cells. Cultures can be maintained by addition or replacement of fresh medium (DMEM with 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml GM-CSF and 5% fetal bovine serum). The cultures should be started at a density of 3 x 105 cells/ml and maintained between 3 x 105 and 1 x 106 cells/ml. The cells require GM-CSF for long term growth. Conditioned medium from U-87MG cells can be used at a concentration of 10% to replace GM-CSF in the culture medium (Santoli et al. 1987).
C7 (Mus musculus)
The C7 cell line has been established from bone marrow of male adult 129/Sv p53-/- mice (Miyamoto et al. 1998). The cells share the characteristics of their cell surface molecules and phagocytotic activity with macrophages. The differentiation potential of C7 cells to osteoclasts is maintained in the presence of M-CSF and abolished by treatment with lipopolysaccharide (LPS) that is known to activate macrophages. The C7 cell line can be cultured in alpha-MEM containing 10% fetal bovine serum and 1 ng/ml human or murine M-CSF. After reaching confluence cells can be divided 1:2 to 1:4 every four to six days using trypsin/EDTA after washing twice with PBS. C7 cells have a doubling time of about 80 to 100 hours.
DH82 (Canis familiaris)
The DH82 cell line was established from neoplastic progenitor cells of canine malignant histiocytosis of a ten year old male Golden Retriever (Wellman et al. 1988). The cells have a macrophage-like morphology and are able to phagocytose latex particles. They are positive for Fc-γ receptors and negative for Fc-µ and C3b receptors. However, DH82 cells do not produce IL-1. They can be cultured with Eagle's Minimum Essential Medium (EMEM) supplemented with 2 mM glutamine, 1% non essential amino acids and 15% fetal bovine serum. Subcultures are prepared by treating cells with 0.25% trypsin/0.03% EDTA solution at 37°C. A subcultivation ratio of 1:4 to 1:8 is recommended.
DMBM-2 (Mus musculus)
The DMBM-2 cell line has been established from bone marrow of a female C3H/HeJ mouse (Monner et al. 1997). The macrophages grow in aggregates, mostly loosely adherent, and some of them have long processes. The cells can be cultured in Roswell Park Memorial Institute (RPMI)-1640 containing 20% horse serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 1.25 µg/ml vitamin B12, 75 µM α-thioglycerin, 2 mM L-glutamine and 2.5% 10x L-929 supernatant. Cells can be split 1:10 every four to six days by detaching the cells by flushing adherent cells with medium through a 25G needle. Cells grow best in round Corning plates and have a doubling time about 16 to 22 hours.
GG2EE (Mus musculus)
GG2EE cells were isolated from C3H/HeJ mice. Immortalization was achieved by infection with J2 recombinant retrovirus (Blasi et al. 1987). Cells grow in suspension but may also adhere loosely to culture vessels (Cox et al. 1989). This cell line belongs to the macrophage lineage and is positive for MAC-1, MAC-2 and Fc receptor (Blasi et al. 1987). In addition these cells express Ly-5.1 antigens as well as M169 and F4/80 antigens, but they do not show any tumoricidal activity upon stimulation with LPS (Blasi et al. 1987). GG2EE cells are cultivated in DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotics (Cox et al. 1989).
HD11 (Gallus gallus)
HD11 cells were derived drom chicken bone marrow cells after transformation with MC29 virus (avian leukemia virus) (Beug et al. 1979). The morphology of these cells is amoeboid, often with polar shape and a diameter of 15-20 µm. The cells are slightly adherent in culture and display numerous lamellipodia. The nucleus is located eccentrically and the cytoplasm contains granules and vacuoles (Beug et al. 1979). HD11 cells are macrophage-like in morphology and functionality and show phagocytic activity. These cells are positive for Fc receptor and Mac antigen (Beug et al. 1979). HD11 cells are maintained in DMEM supplemented with 8 % FCS, 2 % chicken serum and 10 mM HEPES (Beug et al. 1979).
HL-60 (Homo sapiens)
HL-60 is a promyelocytic cell line derived by Collins et al. in 1977. Peripheral blood leukocytes were obtained by leukopheresis from a 36-year-old Caucasian female with acute promyelocytic leukemia. HL-60 cells differentiate spontaneously but differentiation can be also stimulated by butyrate, hypoxanthine, phorbol-12-myristate-13-acetate, 12-O-tetradecanoylphorbol-13-acetate, dimethylsulfoxide (DMSO), actinomycin D and retinoic acid (Collins et al. 1978). The cells exhibit phagocytic activity and responsiveness to chemotactic stimuli (Gallagher et al. 1979). The HL-60 cell line is positive for myc oncogene expression. Cultures can be maintained by addition of fresh medium or replacement of medium (RPMI-1640 containing 10% fetal calf serum) every two to three days depending on cell density. Alternatively, cultures can be established by centrifugation with subsequent resuspension in fresh medium at a cell density of 5 x 106 cells/ml. Cultures are maintained at cell concentrations between 5 x 105 and 2 x 106 cells/ml.
IC-21 (Mus musculus)
The IC-21 cell line was obtained by transformation of normal C57BL/6 mouse peritoneal macrophages with SV40 (Mauel et al. 1971). This line shares many properties with normal mouse macrophages and displays macrophage specific antigens. IC-21 cells have phagocytic and cytolytic properties, can lyse tumor targets in vitro and appear to be more differentiated than cells of the P388D1 macrophage line (Walker et al. 1975 and 1980). The cells produce large quantities of acid so RPMI-1640 containing 10% fetal calf serum should be changed at least three times per week. The cell monolayer should be rinsed with 10 to 15 ml of calcium and magnesium free PBS and an additional 10 to 15 ml of the same solution. The culture should be stood for 5 to 10 minutes at room temperature. The flask is then struck to dislodge the cells and 5 to 7 ml of the cell suspension are added to a flask containing less than 10 ml of growth medium. Once the cells have attached (after one to two days) additional medium should be added. Subculturing is necessary when cells became confluent. A subcultivation ratio of 1:2 to 1:4 is recommended.
INF-3A (Mus musculus)
INF-3A cells were derived from bone marrow of C3H/HeN (H-2k) mice (Cox et al. 1989). Immortalization was performed by infection with J2 recombinant retrovirus. Cells grow in suspension with some cells loosely adherent. INF-3A cells are characterized by a large nuclear to cytoplasmic ratio and few cytosolic vacuoles. This cell line is positive for FcyR (Ly-17), Mac-1, Ly-5 and heat-stable antigen (HAS) (Cox et al. 1989). INF-3A cells also expresses lysozyme and show phagocytic activity, which places this cell line into the myelomonocytic lineage (Cox et al. 1989). Upon stimulation with LPS these cells have shown tumoricidal activity. INF-3A cells are maintained in DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotics (Cox et al. 1989).
J774A.1 (Mus musculus)
J774 tumor arose in a female BALB/c/NIH mouse with a reticulum cell sarkoma during a plasmacytoma induction program in 1968 (Hirst et al. 1971). The initial tumor of which the cells designated as J774.A1 had been isolated was ascitic. J774A.1 macrophages are active in antibody dependent phagocytosis (Ralph et al. 1975). Their growth is inhibited by dextran sulfate, p-phenylendiamin and LPS. J774.A1 cells synthesize large amounts of lysozyme and exhibit minor cytolysis but predominantly antibody-dependent phagocytosis. Interleukin 1β is synthesized continuously by this cell line (Ralph et al. 1977b). J774.A1 cells have a doubling time of 17 hours and can be cultured under the same conditions as RAW 264.7 macrophages.
J2-BM (Mus musculus)
The J2-BM cell line was obtained from femurs of C3H/HeJ mice, whose B-cell and macrophage response to LPS is genetically knocked out (Blasi et al. 1985). For immortalization cells were infected with J2 recombinant retrovirus and the cells were found to express v-myc and v-raf. J2-BM cells are a non-adherent, monocytic cell line characterized by the expression of lysozyme and nonspecific esterase (Blasi et al. 1985). They are positive for MAC-1 and Fc receptors as well as mouse heat-stable antigen, asialo-GM1 glycolipid and F4/80 antigen. Propagation of J2-BM cells requires the presence of dextran beads (Blasi et al. 1985).
Kasumi-3 (Homo sapiens)
The Kasumi-3 cell line was established from blast cells of a 57-years-old male myeloperoxidase-negative acute leukemia patient from Japan (Asou et al. 1996). Kasumi-3 cells express CD7, CD4, CD13, CD33, CD34, HLA-DR and c-Kit on their cell surface, a finding that is compatible with that of acute myelocytic leukemia, subtype M0 (AML-M0). Kasumi-3 cells have t(3;7)(q27:q22), del(5)(q15), del(9)(q32), and add(12)(p11) chromosomal abnormalities. The breakpoint of 3q27 is located near the EVI1 gene and high levels of expression of the EVI1 gene were observed. Kasumi-3 cells treated with 12-O-tetradecanoylphorbol-13-acetate show maturation of the monocytic lineage. Treatment with either IL-2, IL-3, IL-4, GM-CSF or stem cell factor induces proliferation, a characteristic of undifferentiated leukemia. Kasumi-3 cultures can be maintained by the addition of fresh medium or replacement of the medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension at a cell density of 3 x 105 cells/ml. Depending on cell density (between 3 x 105 and 3 x 106 cells/ml) the medium should be renewed every two to three days.
M1 (Mus musculus)
The M1 cell line was established in 1969 in vitro from spontaneous myeloid leukemia of SL strain mice (Ichikawa et al. 1969). The cells can be induced to become macrophage-like by a variety of stimulating agents including dexamethasone, LPS and WEHI-3 conditioned medium (Ralph et al. 1983). M1 cultures can be maintained by addition or replacement of fresh RPMI-1640 medium containing 10% fetal calf serum. Cultures are started at a cell density of 1 x 105 cells/ml and maintained between 1 x 105 cells/ml and 1 x 106 cells/ml. Medium has to be renewed every two to three days.