- Email: [email protected]
Establishment of growth factor-dependent MOPC 104E cell line in vitro
CELLULAR
IMMUNOLOGY
m,24-31
(1985)
Establishment of Growth Factor-Dependent MOPC 104E Cell Line in Vitro’ KEDAR SHRESTHA,*** RAYMOND N. HIFL%MOTO,*+~AND VITHAL K. GHANTA*,~ *Department of Microbiology, and tcomprehensive Cancer Center, University of Alabama in Birmingham, Birmingham, Alabama 35294 Received March 30, 1984; accepted August 5, 1984 The MOPC 104Ecell line has been adapted to grow in vitro using a combination of feeder layer and growth factor(s). The growth of this myeloma cell line is dependent on the presence of growth factor(s). Growth-promoting activity generated from T-cell-mitogen-stimulated, Corynebacterium pawurn-stimulated spleen cell culture supematant, and peritoneal adherent cell culture supematants gives dose-dependent proliferation. Generation of growth factors in the serum-free bovine serum albumin-substituted media and a rapid assay system based on [)H]thymidine uptake for the quantitation of growth promoting activity are described. 8 1985 Academic Rss, Inc.
INTRODUCTION Although there has been some successwith the establishment of long-term culture of normal human and mouse B cells (1, 2), maintenance of B-cell clonal expansion is poorly understood. Recent studies with normal B-cell lines of mouse and human origin and short-term proliferative response of stimulated B cells have identified the requirement for a T-cell-derived mediator involved in the clonal expansion of activated B cells (3-6). Studies of Howard et al. (1) indicate that there might be a multitude of soluble mediators required for B-cell clonal expansion. Myeloma, although neoplastic, still retains some of the properties of normal differentiated B cells (7). MOPC 104E, a monoclonal IgM-secreting murine myeloma, responds to lymphokine/monokine preparations by clonal expansion. In this paper, we describe the establishment of the MOPC 104E cell line in culture that shows dependency on the growth-promoting activity of monokines and lymphokines. This line of myeloma should provide a uniform tool for the understanding of molecular events leading to the clonal expansion of B cells at the later stages of their development. METHODS Mice. Female BALB/c mice were obtained from the Simonsen Laboratories and West Seneca Laboratories (Gilroy, Calif., and West Seneca, N.Y., respectively) and ’ This study was supported by NIH Grants AG02327, CA27765, and a grant from the American Cancer Society, PDT-236. * To whom correspondence should be addressed. 24 OOQ8-8749185 $3.00 Cqyright 8 1985 by Academic R-es, Inc. All rights of reproduction in any form trw’ved.
MOPC
104E CELL
LINE
25
maintained in plastic cageson rodent pellets and water ad libitum. Mice were used when they were about 2 to 4 months of age. Medium. RPM1 1640 was supplemented with 2 mM glutamine, 100 U/ml penicillin, and 100 &ml streptomycin. Tumor cells. MOPC 104E was maintained in BALB/c mice in ascites form. Ascites were collected from tumor bearing mice. Tumor cells were washed with media containing 10% fetal calf serum (FCS)3media. Contaminating red blood cells were removed by 0.83% NH&l treatment. After having been washed twice, cells were suspended in 10% FCS media at a cell density of lo7 cells/ml. Preparation of spleen cell culture supernatants. BALB/c mice were given 350 pg Corynebacterium parvum ip. Seven to eight days later spleens were removed and a single-cell suspension was made. Red blood cells were removed by treatment with 0.83% NH&l, washed 3X with media, and suspended at 10’ cells/ml in 10% FCS media or media substituted with 1.5 mg/ml bovine serum albumin (BSA) and 5 X low5 M 2-mercaptoethanol. Concanavalin A (ConA) or phytohemagglutinin (PHA) was added at 10 or 5 &ml, respectively. Following 24 hr of incubation at 37°C cells were removed by centrifugation, and supematants were collected and stored at -20°C. Generation of growth factors from Corynebacterium parvum-activated macrophages. BALB/c mice were given 350 pg C. parvum ip, and, 7 to 8 days later, peritoneal exudate cells were collected under ether anesthesia with 5 ml of 10% FCS media. Cells were washed once and suspended in 10% FCS media at a density of 3-5 X lo6 cells/ml. One milliliter of cell suspension was plated in a 35-mm tissue culture plate and incubated for 15-30 min at 37°C. Nonadherent cells were removed by washing the plates 2X with warm RPM1 1640. Cultures were fed with 4 ml BSA medium and incubated for an additional 24 hr at 37°C. Supematants were collected, centrifuged, and stored at -20°C. Initiation of in vitro culture. MOPC 104E cells in 20 ml at lo7 cells/ml in 10% FCS media were plated in 75-cm2 tissue culture flasks. After 4 hr of incubation at 37°C the majority of the nonadherent cells were removed by aspiration of the media. Twenty-five milliliters of 10% FCS media was added and the flasks were incubated at 37°C. A small number of MOPC 104E cells, along with the adherent cells remaining in the flask, were fed with fresh 10% FCS media every week. After 6 to 8 weeks, nonadherent cells were removed and grown at a density of 5 X lo4 to 1 X IO5 cells/ml in the presence of C. parvum and T-cell-mitogen-stimulated spleen cell culture supematant. The culture media was replaced every 48 to 72 hr. The growth factor requirement was judged by their inability to grow in the presence of 10% FCS media alone. Growth factor-dependent proliferation of MOPC 104E. Growth factor preparations from C. parvum-stimulated spleen cells restimulated with ConA or PHA were concentrated using YMlO Diaflo membrane (Amicon Corp., Lexington, Mass.) and passed through a Sephadex G- 10 column. Cells were suspended at a density of 5 X lo4 cells/ml in 10% FCS media or growth factor preparation at an equivalent of 20 to 25% of crude preparation. Five milliliters of cell suspension were incubated 3 Abbreviations used: FCS, fetal calf serum; BSA, bovine serum albumin; ConA, concanavalin A; PHA, phytohemagglutinin; SRBC-D, sheep red blood cells conjugated with Dextran B-1355; cpm, mean counts per minute.
26
SHRESTHA,
HIRAMOTO,
AND
GHANTA
in 25-cm2 tissue culture flasks. Cells were counted in a hemocytometer in the presence of 0.15% trypan blue. Assay of growth factor-dependent proliferation as detected by 13athymidine uptake. Growth factor activities generated from cell preparations from normal and C. panturn-immune mice were analyzed as described below. One-tenth milliliter of MOPC 104E cells (5 X lo4 cells/ml) was mixed with 0.1 ml of various doubling dilutions of growth factor preparations in the wells of 96-well flat-bottom microtiter plates. After 48 hr, each well was pulsed with 1 &i [3H]thymidine (sp act 6.7 Ci/mmol, Research Products Int., Elk Grove, Ill.) for 4 hr. At the end of the incubation, the cells were harvested onto glass-fiber filters and the radioactivity was counted in a Beckman LS 8000 liquid scintillation counter. Data are expressed as mean counts per minute (cpm) with standard deviations. In vivo growth as measured by IgM production. After 4 months of factordependent proliferation in vitro, cultured MOPC 104E cells were washed with saline (0.9% NaCl solution) and suspended in saline at lo7 cells/ml. One tenth ml of cell suspension was given to BALB/c mice iv. Tumor growth was monitored by using previously established procedures of tumor IgM quantitation in the sera of animals (8, 9). Monitoring MOPC 104E IgM. The monoclonal IgM produced by the MOPC 104E myeloma has unique characteristics of reacting with bacterial Dextran B-l 355. Sheep red blood cells conjugated with Dextran B-1355 (SRBC-D) were used in the radial hemolysis in gel assay. For the quantitation of MOPC 104E IgM, sera from tumor-bearing mice were transferred into the wells of a SRBC-D agarose plate and were allowed to diffise at 5°C for 22 hr followed by lysis with complement at 37°C. The area of lysis of unknown samples was compared with that of standard samples to obtain the micrograms of MOPC 104E &M/mouse (10). RESULTS In Vitro Adaptation of MOPC 104E Myeloma MOPC 104E was adapted to grow in vitro in the presence of adherent cells present in the ascites. An adherent feeder layer was generated by incubating MOPC 104E ascites at high cell density ( lo7 cells/ml; 20 m1/75-cm* flask). After 4 hr of incubation, nonadherent cells were discarded. Flasks containing adherent cells, plus residual MOPC 104E, were fed with 25 ml of 10% FCS media. In the presence of feeder layer, MOPC 104E grew steadily but slowly. Cultures were fed once a week with fresh media containing 10% FCS. MOPC 104E cells were removed every 2 to 3 weeks. In vitro adaptation in the presence of feeder layer was necessary for the cell survival. Within this adaptation period, growth factors were not sufficient for the establishment of the cell line. After 6 to 8 weeks of adaptation, MOPC 104E showed proliferative response in the presence of growth factors. Growth-Factor Dependency of Cultured MOPC 104E MOPC 104E cells were maintained in the presence of ConA-stimulated C. parvum immune spleen cells at lo-fold dilution of growth factors in 10% FCS media. Alternatively, concentrated preparations of the factors similarly prepared in BSA media were used after proper dilution. Cultures were given fresh growth factor
MOPC 104E CELL LINE
27
every 72 hr. Figure 1 shows the growth of MOPC 104E in the presence of growth factor generated in BSA media and in 10% FCS media control. The results show that the MOPC 104E, after adaptation in vitro, require growth factors for continuous proliferation. When 5 X lo4 MOPC 104E cells/ml were cultured in the presence of growth factor, the approximate doubling time was - 12 hr and reached a cell density of -4-5 X lo5 cells/ml by 60 hr. Cells cultured in the presence 10% FCS media alone did not survive. Conditions for the Production of the Growth Factor in Serum-Free Media Normal or C. parvum-immune spleen cells were stimulated with ConA or PHA or incubated in the absence of mitogen in serum-free BSA-substituted medium. Normal spleen cells stimulated with ConA or unstimulated were unable to generate growth factors, whereas, when stimulated with PHA, normal spleen cells were able to generate significant levels of growth factor activity (Fig. 2). Spleen cells from C, parvum-immune animals, on the other hand, were able to generate considerable amounts of growth factors without stimulation with T-cell mitogen. Addition of T-cell mitogen to the spleen cells from C. parvum-stimulated animals resulted in an increase in the growth-promoting activity of the supernatant. Proliferation Is Dose Dependent When serial doubling dilutions of the growth factor preparations (concentrated and desalted ConA-BSA or PHA-BSA supernatant) were used to quantitate proliferative response, a dose-dependent uptake of [3H]thymidine was observed (Fig. 3). Dose-responsewas converted into percentagemaximum responseand quantitated using probit analysis as described for quantitation of T-cell growth factor (11). Concentrated standard preparations were diluted 1O-fold and doubling dilutions
6-
0
24 lncubatlan
I 46 72 Time (hr)
FIG. 1. Dependency of MOPC 104E cultured line on growth factors. Growth of MOPC 104E cells suspended in media containing 25% ConA-stimulated culture supematant (0) and growth of MOPC 104E suspended in media control (0). Quadruplicate cultures were examined at 36 and 60 hr after initiation of the cultures.
28
SHRESTHA, HIRAMOTO,
AND GHANTA
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FIG. 2. Growth factor activity in different preparations of spleen cell culture supematant prepared in BSA-substituted media; normal spleen cells (Cl); C. parrum-immune spleen cells (A); C. parrum-immune spleen cells stimulated with 10 &ml ConA (A); normal spleen cells stimulated with 5 pg/ml PHA (0); C. parrum-immune spleen cells stimulated with 5 &ml PHA (0); and normal spleen cells stimulated with 10 &ml of ConA (m).
were assayed (Fig. 4). Similarly, doubling dilutions of 24- and 36-hr PHA-BSA crude preparations were also assayed. The doubling dilution (log 2) of control growth factor at which the curve crossesthe 50% maximum activity (probit value of 5) corresponds to 1 unit of growth factor. Thus, the growth factor activity of test samples were obtained by dividing dilution (x-axis) of test samples at probit value of 5 by dilution of the control growth factor corresponding to 1 unit. The 24-hr preparation contained 1.65 units/ml and 36-hr preparations had 2.1 units/ml activity. The 36-hr preparation contained more growth factor than the 24-hr preparation. 60-
60-
40P P *
30-
B I
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FIG. 3. Response of MOPC 104E to the concentrated and desalted preparations of growth factors generated from C. purvum-immune spleen cells by stimulation with 10 &ml ConA (A) and 5 &ml PHA (0) in BSA-substituted media.
29
MOPC 104E CELL LINE
I
2
3
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7
LOG2 DILUTION OF FACTOR SAMPLE
FIG. 4. Probit analysis of proliferative activity present in C. purvum-immune spleen cells stimulated with 5 &ml PHA. Concentrated standard (7-8X) diluted IO-fold (0); 24-hr (A) and 36-hr (0) culture supematant.
Growth Factor Activity in Adherent Peritoneal Exudate Cell Culture Supernatant Factors generated in the culture supernatant of adherent peritoneal exudate cells were also able to support the growth of MOPC 104E cells. The proliferative response to the peritoneal adherent cell culture supematant is shown in Fig. 5. The growth promoting activity was completely retained by Diaflo XM 100 membrane, indicating that the factor is unable to pass through the membrane with a retention capacity of 100,000 Da. The proliferative response was dose dependent. In Vivo Growth of Cultured Line We have previously shown that the growth of MOPC 104E in vivo can be monitored by quantitating tumor IgM in the sera of tumor-bearing mice. Thus, the in vivo growth of the in vitro line was monitored by determining tumor IgM in the sera of the tumor-bearing mice (Fig. 6). Logarithmic growth of the MOPC 104E
DILUTION
FIG. 5. Growth factor activity generated by C. purvum-immune adherent peritoneal cells. Crude (A), Diaflo XM 100 membrane-retained (0), and fihrate from DiaIIo XM 100 membrane retained in YMlO Diaflo membrane (0). Both XM 100 and YM 10 concentrate were brought back to original volume prior to the assay.
30
SHRESTHA, HIRAMOTO,
AND GHANTA
was observed as determined by the MOPC 104E IgM. The results indicate that the factor-dependent line is capable of growing in vivu and the clonal expansion of MOPC 104E is supported by the host. DISCUSSION Myelomas are difficult to maintain in continuous culture. However, once established, they assume autonomous growth and show poor immunoresponsiveness (12-18). We have established a MOPC 104E cell line in continuous culture that shows complete dependency on the growth factors generated by lymphoreticular cells. This line was developed using a combination of feeder layer and cytokine(s) generated from C. pawurn-immune spleen cells stimulated with T-cell mitogens. Once established, this line showed growth factor-dependent proliferative response to the growth stimuli generated from a variety of sources, such as PHA-stimulated spleen cells and C. parvum-activated peritoneal macrophages, but not normal spleen cells or normal spleen cells stimulated with ConA in serum-free media. The growth is rapid, with a doubling time of - 12 hr, and the absence of factors results in cell death. Namba and Hanaoka (19) have previously described the development of a MOPC 104E cell line that gave proliferative response to a macrophage-derived factor of 50K Da. However, the factors generated from the adherent cells in our hands did not pass through the XM 100 untrafiltration membrane, indicating them to be of high molecular mass; or, conversely, they could be aggregatein nature or associated with BSA and, thus, unable to pass through the ultrafiltration membrane. Factor production from C. panturn-stimulated spleen cells was increased by the addition of T-cell mitogen, which indicates active involvement of T-cells. There may be, however, two possible reasons for this. First, properly activated (PHA or C. parvum) T cells might be directly involved in the production of the factors, or,
’ IO
20
30
40
, 50
DAYS
FIG. 6. Growth of MOPC 104E in BALB/c mice.. Five mice were given 1 X 106 MOFC 104E-D iv, and tumor IgM was quantitated in the sera every 4 to 5 days. The values are mean k SD.
MOPC 104E CELL LINE
31
T cells produce macrophage-stimulatory factors for the production of macrophagederived growth factors. Factor-dependent proliferation can be quantitated using [3H]thymidine uptake assay, and thus can be used for rapid quantitation of B-cell growth-promoting activity. In recent years B-cell growth-promoting activity has been identified and quantitated in [3H]thymidine uptake assay using short-term proliferative response of preactivated normal B cells (3-6), or by a BCLl cell line whose growth in vitro is enhanced by the presence of the factors (20). Howard et al. (1) have shown that the growth of a continuous culture of normal murine B-cells can be supported by growth factors from a variety of sources, including T cells, macrophages, or T-cellmitogen-stimulated spleen cell culture supematants. Thus, the growth-promoting activity seen in the various culture supematants for MOPC 104E growth is not surprising; it only indicates that the B-cell clonal expansion can be induced by a variety of factors. However, the nature of the factor(s) and its mode of action might be different, but still result in clonal expansion of a responsive population. This MOPC 104E line grows in vivo and secretes immunoglobulin. When lo6 cells were given iv, three out of five mice died within 45 to 50 days with a large tumor burden, indicating that the cultured myeloma cell has retained its tumorigenicity. In conclusion, we have established a MOPC 104E cell line that is dependent on growth factors for its proliferation. We believe this line could be a very useful tool for the study of biological and biochemical properties of factors involved in the clonal expansion of immune B cells. ACKNOWLEDGMENT We thank Dr. Phillipa Marrack, National Jewish Hospital and Research Center, Denver, Colorado, for critical reading of the manuscript.
REFERENCES 1. Howard, M., Kessler, S., Chused, T., and Paul, W. E., Proc. N&l. Acad. Sci. USA 78, 5788, 1981. 2. Sredni, B., Sieckmann, D. G., Kumagai, S., House, S., Green, I., and Paul, W. E., J. Exp. Med. 154, 1500, 1981. 3. Muraguchi, A., Kasahara, T., Oppenheim, J. J., and Fauci, A. S., J. Immunol. 129, 2488, 1982. 4. Garchon, H., Levi-Strauss, M., Camaud, C., and Bach, J., J. Immunol. 130, 777-780, 1983. 5. Muraguchi, A., and Fauci, A. S., .I. Immunol. 129, 1104, 1982. 6. Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Torkatsu, K., and Paul, W. E., J. Exp. Med. 155, 914, 1982. 7. Lynch, R. G., Rohrer, J. W., Gdermatt, B., Giebel, H. M., Autry, J. R., and Hoover, R. G., Zmmunol. Rev. 48,4S, 1979. 8. Ghanta, V. K., and Hiramoto, R. N., J. Natl. Cancer Inst. 52, 1199, 1974. 9. Hiramoto, R. N., Ghan& V. K., McGhee, J. R., and Hamlin, N. M., J. Zmmunol. 111, 1546, 1973. 10. Ghan@ V. K., Hamlin, N. M., and Hiramoto, R. N., J. Immunol. 109, 810, 1972. 11. Gillis, S., Fenn, M. M., Ou, Winny, and Smith, K. A., J. Immunol. 120, 2027, 1978. 12. Hamburger, A., and Salmon, S. E., J. Clin. Invest. 60, 846, 1977. 13. Horibata, A., and Harris, A. W., Exp. Cell. Res. 60, 61, 1970. 14. Laskov, R., and Schati, M. D., J. Exp. Med. 131, 515, 1970. 15. Loachim, H. L., Osserman, E. F., and Agar, H. A., Fed. Proc. 25 (Abstr. 419), 1301, 1966. 16. Cohn, M., Sympos. Quant. Biol.. 32, 211, 1967. 17. Periman, P., .I. Natl. Cancer Inst. 46, 403, 1971. 18. Pettergill, S. G., and Sorensen, G. D., Exp. Cell. Res. 47, 608, 1967. 19. Namba, Y., and Hanaoka, M., J. Immunol. 109, 1193, 1972. 20. Thoman, M. L., and Weigle, W. O., J. Immunol. 130, 233, 1983.
IMMUNOLOGY
m,24-31
(1985)
Establishment of Growth Factor-Dependent MOPC 104E Cell Line in Vitro’ KEDAR SHRESTHA,*** RAYMOND N. HIFL%MOTO,*+~AND VITHAL K. GHANTA*,~ *Department of Microbiology, and tcomprehensive Cancer Center, University of Alabama in Birmingham, Birmingham, Alabama 35294 Received March 30, 1984; accepted August 5, 1984 The MOPC 104Ecell line has been adapted to grow in vitro using a combination of feeder layer and growth factor(s). The growth of this myeloma cell line is dependent on the presence of growth factor(s). Growth-promoting activity generated from T-cell-mitogen-stimulated, Corynebacterium pawurn-stimulated spleen cell culture supematant, and peritoneal adherent cell culture supematants gives dose-dependent proliferation. Generation of growth factors in the serum-free bovine serum albumin-substituted media and a rapid assay system based on [)H]thymidine uptake for the quantitation of growth promoting activity are described. 8 1985 Academic Rss, Inc.
INTRODUCTION Although there has been some successwith the establishment of long-term culture of normal human and mouse B cells (1, 2), maintenance of B-cell clonal expansion is poorly understood. Recent studies with normal B-cell lines of mouse and human origin and short-term proliferative response of stimulated B cells have identified the requirement for a T-cell-derived mediator involved in the clonal expansion of activated B cells (3-6). Studies of Howard et al. (1) indicate that there might be a multitude of soluble mediators required for B-cell clonal expansion. Myeloma, although neoplastic, still retains some of the properties of normal differentiated B cells (7). MOPC 104E, a monoclonal IgM-secreting murine myeloma, responds to lymphokine/monokine preparations by clonal expansion. In this paper, we describe the establishment of the MOPC 104E cell line in culture that shows dependency on the growth-promoting activity of monokines and lymphokines. This line of myeloma should provide a uniform tool for the understanding of molecular events leading to the clonal expansion of B cells at the later stages of their development. METHODS Mice. Female BALB/c mice were obtained from the Simonsen Laboratories and West Seneca Laboratories (Gilroy, Calif., and West Seneca, N.Y., respectively) and ’ This study was supported by NIH Grants AG02327, CA27765, and a grant from the American Cancer Society, PDT-236. * To whom correspondence should be addressed. 24 OOQ8-8749185 $3.00 Cqyright 8 1985 by Academic R-es, Inc. All rights of reproduction in any form trw’ved.
MOPC
104E CELL
LINE
25
maintained in plastic cageson rodent pellets and water ad libitum. Mice were used when they were about 2 to 4 months of age. Medium. RPM1 1640 was supplemented with 2 mM glutamine, 100 U/ml penicillin, and 100 &ml streptomycin. Tumor cells. MOPC 104E was maintained in BALB/c mice in ascites form. Ascites were collected from tumor bearing mice. Tumor cells were washed with media containing 10% fetal calf serum (FCS)3media. Contaminating red blood cells were removed by 0.83% NH&l treatment. After having been washed twice, cells were suspended in 10% FCS media at a cell density of lo7 cells/ml. Preparation of spleen cell culture supernatants. BALB/c mice were given 350 pg Corynebacterium parvum ip. Seven to eight days later spleens were removed and a single-cell suspension was made. Red blood cells were removed by treatment with 0.83% NH&l, washed 3X with media, and suspended at 10’ cells/ml in 10% FCS media or media substituted with 1.5 mg/ml bovine serum albumin (BSA) and 5 X low5 M 2-mercaptoethanol. Concanavalin A (ConA) or phytohemagglutinin (PHA) was added at 10 or 5 &ml, respectively. Following 24 hr of incubation at 37°C cells were removed by centrifugation, and supematants were collected and stored at -20°C. Generation of growth factors from Corynebacterium parvum-activated macrophages. BALB/c mice were given 350 pg C. parvum ip, and, 7 to 8 days later, peritoneal exudate cells were collected under ether anesthesia with 5 ml of 10% FCS media. Cells were washed once and suspended in 10% FCS media at a density of 3-5 X lo6 cells/ml. One milliliter of cell suspension was plated in a 35-mm tissue culture plate and incubated for 15-30 min at 37°C. Nonadherent cells were removed by washing the plates 2X with warm RPM1 1640. Cultures were fed with 4 ml BSA medium and incubated for an additional 24 hr at 37°C. Supematants were collected, centrifuged, and stored at -20°C. Initiation of in vitro culture. MOPC 104E cells in 20 ml at lo7 cells/ml in 10% FCS media were plated in 75-cm2 tissue culture flasks. After 4 hr of incubation at 37°C the majority of the nonadherent cells were removed by aspiration of the media. Twenty-five milliliters of 10% FCS media was added and the flasks were incubated at 37°C. A small number of MOPC 104E cells, along with the adherent cells remaining in the flask, were fed with fresh 10% FCS media every week. After 6 to 8 weeks, nonadherent cells were removed and grown at a density of 5 X lo4 to 1 X IO5 cells/ml in the presence of C. parvum and T-cell-mitogen-stimulated spleen cell culture supematant. The culture media was replaced every 48 to 72 hr. The growth factor requirement was judged by their inability to grow in the presence of 10% FCS media alone. Growth factor-dependent proliferation of MOPC 104E. Growth factor preparations from C. parvum-stimulated spleen cells restimulated with ConA or PHA were concentrated using YMlO Diaflo membrane (Amicon Corp., Lexington, Mass.) and passed through a Sephadex G- 10 column. Cells were suspended at a density of 5 X lo4 cells/ml in 10% FCS media or growth factor preparation at an equivalent of 20 to 25% of crude preparation. Five milliliters of cell suspension were incubated 3 Abbreviations used: FCS, fetal calf serum; BSA, bovine serum albumin; ConA, concanavalin A; PHA, phytohemagglutinin; SRBC-D, sheep red blood cells conjugated with Dextran B-1355; cpm, mean counts per minute.
26
SHRESTHA,
HIRAMOTO,
AND
GHANTA
in 25-cm2 tissue culture flasks. Cells were counted in a hemocytometer in the presence of 0.15% trypan blue. Assay of growth factor-dependent proliferation as detected by 13athymidine uptake. Growth factor activities generated from cell preparations from normal and C. panturn-immune mice were analyzed as described below. One-tenth milliliter of MOPC 104E cells (5 X lo4 cells/ml) was mixed with 0.1 ml of various doubling dilutions of growth factor preparations in the wells of 96-well flat-bottom microtiter plates. After 48 hr, each well was pulsed with 1 &i [3H]thymidine (sp act 6.7 Ci/mmol, Research Products Int., Elk Grove, Ill.) for 4 hr. At the end of the incubation, the cells were harvested onto glass-fiber filters and the radioactivity was counted in a Beckman LS 8000 liquid scintillation counter. Data are expressed as mean counts per minute (cpm) with standard deviations. In vivo growth as measured by IgM production. After 4 months of factordependent proliferation in vitro, cultured MOPC 104E cells were washed with saline (0.9% NaCl solution) and suspended in saline at lo7 cells/ml. One tenth ml of cell suspension was given to BALB/c mice iv. Tumor growth was monitored by using previously established procedures of tumor IgM quantitation in the sera of animals (8, 9). Monitoring MOPC 104E IgM. The monoclonal IgM produced by the MOPC 104E myeloma has unique characteristics of reacting with bacterial Dextran B-l 355. Sheep red blood cells conjugated with Dextran B-1355 (SRBC-D) were used in the radial hemolysis in gel assay. For the quantitation of MOPC 104E IgM, sera from tumor-bearing mice were transferred into the wells of a SRBC-D agarose plate and were allowed to diffise at 5°C for 22 hr followed by lysis with complement at 37°C. The area of lysis of unknown samples was compared with that of standard samples to obtain the micrograms of MOPC 104E &M/mouse (10). RESULTS In Vitro Adaptation of MOPC 104E Myeloma MOPC 104E was adapted to grow in vitro in the presence of adherent cells present in the ascites. An adherent feeder layer was generated by incubating MOPC 104E ascites at high cell density ( lo7 cells/ml; 20 m1/75-cm* flask). After 4 hr of incubation, nonadherent cells were discarded. Flasks containing adherent cells, plus residual MOPC 104E, were fed with 25 ml of 10% FCS media. In the presence of feeder layer, MOPC 104E grew steadily but slowly. Cultures were fed once a week with fresh media containing 10% FCS. MOPC 104E cells were removed every 2 to 3 weeks. In vitro adaptation in the presence of feeder layer was necessary for the cell survival. Within this adaptation period, growth factors were not sufficient for the establishment of the cell line. After 6 to 8 weeks of adaptation, MOPC 104E showed proliferative response in the presence of growth factors. Growth-Factor Dependency of Cultured MOPC 104E MOPC 104E cells were maintained in the presence of ConA-stimulated C. parvum immune spleen cells at lo-fold dilution of growth factors in 10% FCS media. Alternatively, concentrated preparations of the factors similarly prepared in BSA media were used after proper dilution. Cultures were given fresh growth factor
MOPC 104E CELL LINE
27
every 72 hr. Figure 1 shows the growth of MOPC 104E in the presence of growth factor generated in BSA media and in 10% FCS media control. The results show that the MOPC 104E, after adaptation in vitro, require growth factors for continuous proliferation. When 5 X lo4 MOPC 104E cells/ml were cultured in the presence of growth factor, the approximate doubling time was - 12 hr and reached a cell density of -4-5 X lo5 cells/ml by 60 hr. Cells cultured in the presence 10% FCS media alone did not survive. Conditions for the Production of the Growth Factor in Serum-Free Media Normal or C. parvum-immune spleen cells were stimulated with ConA or PHA or incubated in the absence of mitogen in serum-free BSA-substituted medium. Normal spleen cells stimulated with ConA or unstimulated were unable to generate growth factors, whereas, when stimulated with PHA, normal spleen cells were able to generate significant levels of growth factor activity (Fig. 2). Spleen cells from C, parvum-immune animals, on the other hand, were able to generate considerable amounts of growth factors without stimulation with T-cell mitogen. Addition of T-cell mitogen to the spleen cells from C. parvum-stimulated animals resulted in an increase in the growth-promoting activity of the supernatant. Proliferation Is Dose Dependent When serial doubling dilutions of the growth factor preparations (concentrated and desalted ConA-BSA or PHA-BSA supernatant) were used to quantitate proliferative response, a dose-dependent uptake of [3H]thymidine was observed (Fig. 3). Dose-responsewas converted into percentagemaximum responseand quantitated using probit analysis as described for quantitation of T-cell growth factor (11). Concentrated standard preparations were diluted 1O-fold and doubling dilutions
6-
0
24 lncubatlan
I 46 72 Time (hr)
FIG. 1. Dependency of MOPC 104E cultured line on growth factors. Growth of MOPC 104E cells suspended in media containing 25% ConA-stimulated culture supematant (0) and growth of MOPC 104E suspended in media control (0). Quadruplicate cultures were examined at 36 and 60 hr after initiation of the cultures.
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SHRESTHA, HIRAMOTO,
AND GHANTA
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DILUTION
FIG. 2. Growth factor activity in different preparations of spleen cell culture supematant prepared in BSA-substituted media; normal spleen cells (Cl); C. parrum-immune spleen cells (A); C. parrum-immune spleen cells stimulated with 10 &ml ConA (A); normal spleen cells stimulated with 5 pg/ml PHA (0); C. parrum-immune spleen cells stimulated with 5 &ml PHA (0); and normal spleen cells stimulated with 10 &ml of ConA (m).
were assayed (Fig. 4). Similarly, doubling dilutions of 24- and 36-hr PHA-BSA crude preparations were also assayed. The doubling dilution (log 2) of control growth factor at which the curve crossesthe 50% maximum activity (probit value of 5) corresponds to 1 unit of growth factor. Thus, the growth factor activity of test samples were obtained by dividing dilution (x-axis) of test samples at probit value of 5 by dilution of the control growth factor corresponding to 1 unit. The 24-hr preparation contained 1.65 units/ml and 36-hr preparations had 2.1 units/ml activity. The 36-hr preparation contained more growth factor than the 24-hr preparation. 60-
60-
40P P *
30-
B I
20-
10-
0 1:2
1:4
I:6
I:16
I:32
1 I:64
DILUTION
FIG. 3. Response of MOPC 104E to the concentrated and desalted preparations of growth factors generated from C. purvum-immune spleen cells by stimulation with 10 &ml ConA (A) and 5 &ml PHA (0) in BSA-substituted media.
29
MOPC 104E CELL LINE
I
2
3
4
5
6
7
LOG2 DILUTION OF FACTOR SAMPLE
FIG. 4. Probit analysis of proliferative activity present in C. purvum-immune spleen cells stimulated with 5 &ml PHA. Concentrated standard (7-8X) diluted IO-fold (0); 24-hr (A) and 36-hr (0) culture supematant.
Growth Factor Activity in Adherent Peritoneal Exudate Cell Culture Supernatant Factors generated in the culture supernatant of adherent peritoneal exudate cells were also able to support the growth of MOPC 104E cells. The proliferative response to the peritoneal adherent cell culture supematant is shown in Fig. 5. The growth promoting activity was completely retained by Diaflo XM 100 membrane, indicating that the factor is unable to pass through the membrane with a retention capacity of 100,000 Da. The proliferative response was dose dependent. In Vivo Growth of Cultured Line We have previously shown that the growth of MOPC 104E in vivo can be monitored by quantitating tumor IgM in the sera of tumor-bearing mice. Thus, the in vivo growth of the in vitro line was monitored by determining tumor IgM in the sera of the tumor-bearing mice (Fig. 6). Logarithmic growth of the MOPC 104E
DILUTION
FIG. 5. Growth factor activity generated by C. purvum-immune adherent peritoneal cells. Crude (A), Diaflo XM 100 membrane-retained (0), and fihrate from DiaIIo XM 100 membrane retained in YMlO Diaflo membrane (0). Both XM 100 and YM 10 concentrate were brought back to original volume prior to the assay.
30
SHRESTHA, HIRAMOTO,
AND GHANTA
was observed as determined by the MOPC 104E IgM. The results indicate that the factor-dependent line is capable of growing in vivu and the clonal expansion of MOPC 104E is supported by the host. DISCUSSION Myelomas are difficult to maintain in continuous culture. However, once established, they assume autonomous growth and show poor immunoresponsiveness (12-18). We have established a MOPC 104E cell line in continuous culture that shows complete dependency on the growth factors generated by lymphoreticular cells. This line was developed using a combination of feeder layer and cytokine(s) generated from C. pawurn-immune spleen cells stimulated with T-cell mitogens. Once established, this line showed growth factor-dependent proliferative response to the growth stimuli generated from a variety of sources, such as PHA-stimulated spleen cells and C. parvum-activated peritoneal macrophages, but not normal spleen cells or normal spleen cells stimulated with ConA in serum-free media. The growth is rapid, with a doubling time of - 12 hr, and the absence of factors results in cell death. Namba and Hanaoka (19) have previously described the development of a MOPC 104E cell line that gave proliferative response to a macrophage-derived factor of 50K Da. However, the factors generated from the adherent cells in our hands did not pass through the XM 100 untrafiltration membrane, indicating them to be of high molecular mass; or, conversely, they could be aggregatein nature or associated with BSA and, thus, unable to pass through the ultrafiltration membrane. Factor production from C. panturn-stimulated spleen cells was increased by the addition of T-cell mitogen, which indicates active involvement of T-cells. There may be, however, two possible reasons for this. First, properly activated (PHA or C. parvum) T cells might be directly involved in the production of the factors, or,
’ IO
20
30
40
, 50
DAYS
FIG. 6. Growth of MOPC 104E in BALB/c mice.. Five mice were given 1 X 106 MOFC 104E-D iv, and tumor IgM was quantitated in the sera every 4 to 5 days. The values are mean k SD.
MOPC 104E CELL LINE
31
T cells produce macrophage-stimulatory factors for the production of macrophagederived growth factors. Factor-dependent proliferation can be quantitated using [3H]thymidine uptake assay, and thus can be used for rapid quantitation of B-cell growth-promoting activity. In recent years B-cell growth-promoting activity has been identified and quantitated in [3H]thymidine uptake assay using short-term proliferative response of preactivated normal B cells (3-6), or by a BCLl cell line whose growth in vitro is enhanced by the presence of the factors (20). Howard et al. (1) have shown that the growth of a continuous culture of normal murine B-cells can be supported by growth factors from a variety of sources, including T cells, macrophages, or T-cellmitogen-stimulated spleen cell culture supematants. Thus, the growth-promoting activity seen in the various culture supematants for MOPC 104E growth is not surprising; it only indicates that the B-cell clonal expansion can be induced by a variety of factors. However, the nature of the factor(s) and its mode of action might be different, but still result in clonal expansion of a responsive population. This MOPC 104E line grows in vivo and secretes immunoglobulin. When lo6 cells were given iv, three out of five mice died within 45 to 50 days with a large tumor burden, indicating that the cultured myeloma cell has retained its tumorigenicity. In conclusion, we have established a MOPC 104E cell line that is dependent on growth factors for its proliferation. We believe this line could be a very useful tool for the study of biological and biochemical properties of factors involved in the clonal expansion of immune B cells. ACKNOWLEDGMENT We thank Dr. Phillipa Marrack, National Jewish Hospital and Research Center, Denver, Colorado, for critical reading of the manuscript.
REFERENCES 1. Howard, M., Kessler, S., Chused, T., and Paul, W. E., Proc. N&l. Acad. Sci. USA 78, 5788, 1981. 2. Sredni, B., Sieckmann, D. G., Kumagai, S., House, S., Green, I., and Paul, W. E., J. Exp. Med. 154, 1500, 1981. 3. Muraguchi, A., Kasahara, T., Oppenheim, J. J., and Fauci, A. S., J. Immunol. 129, 2488, 1982. 4. Garchon, H., Levi-Strauss, M., Camaud, C., and Bach, J., J. Immunol. 130, 777-780, 1983. 5. Muraguchi, A., and Fauci, A. S., .I. Immunol. 129, 1104, 1982. 6. Howard, M., Farrar, J., Hilfiker, M., Johnson, B., Torkatsu, K., and Paul, W. E., J. Exp. Med. 155, 914, 1982. 7. Lynch, R. G., Rohrer, J. W., Gdermatt, B., Giebel, H. M., Autry, J. R., and Hoover, R. G., Zmmunol. Rev. 48,4S, 1979. 8. Ghanta, V. K., and Hiramoto, R. N., J. Natl. Cancer Inst. 52, 1199, 1974. 9. Hiramoto, R. N., Ghan& V. K., McGhee, J. R., and Hamlin, N. M., J. Zmmunol. 111, 1546, 1973. 10. Ghan@ V. K., Hamlin, N. M., and Hiramoto, R. N., J. Immunol. 109, 810, 1972. 11. Gillis, S., Fenn, M. M., Ou, Winny, and Smith, K. A., J. Immunol. 120, 2027, 1978. 12. Hamburger, A., and Salmon, S. E., J. Clin. Invest. 60, 846, 1977. 13. Horibata, A., and Harris, A. W., Exp. Cell. Res. 60, 61, 1970. 14. Laskov, R., and Schati, M. D., J. Exp. Med. 131, 515, 1970. 15. Loachim, H. L., Osserman, E. F., and Agar, H. A., Fed. Proc. 25 (Abstr. 419), 1301, 1966. 16. Cohn, M., Sympos. Quant. Biol.. 32, 211, 1967. 17. Periman, P., .I. Natl. Cancer Inst. 46, 403, 1971. 18. Pettergill, S. G., and Sorensen, G. D., Exp. Cell. Res. 47, 608, 1967. 19. Namba, Y., and Hanaoka, M., J. Immunol. 109, 1193, 1972. 20. Thoman, M. L., and Weigle, W. O., J. Immunol. 130, 233, 1983.