cell culture have resulted in the derivation of a line of ... monolayer cultures from the peripheral blood of adult ..... definition of the nutritional requirements of.
CONTINUOUS C U L T U R E OF HUMAN LYMPHOBLASTS FROM PERIPHERAL BLOOD OF A CHILD WITH A C U T E LEUKEMIA GEORGE E. FOLEY*, HERBERT LAZARUS, SIDNEYFARBER, BETTYGEREN UZMAN, BARBARA A. BOONE,AND ROBERT E. MCCARTHY
H
UMAN
BONE
MARROW
AND
PERIPHERAL
blood buffy coat specimens derived from leukemic and non-leukemic patients have been studied extensively by various methods of cell culture with and without “feeder layers.”l, 4, 6 , 1 6 , 2 1 Recent studies employing modifications of these methods of stationary or monolayer cell culture have resulted in the derivation of a line of monocytic cells from the buffy coat of an adult leukemic patient.11 In these laboratories (and ekewhere, so far as is known at this time) experience with a variety of stationary or monolayer cultures initiated with cells obtained from bone marrow and peripheral blood buffy coat of children with acute leukemia during the past 10 or more years has been unsuccessful. The studies reported by Osgood and Brooke17 indicating that the derivation of monolayer cultures from the peripheral blood of adult patients with leukemia did not require the participation of “fixed tissue” elements provided by the usual “feeder layer”together with more recent observations in these laboratories and elsewhere29 3 that mononuclear cells occasionally detach from such monolayers and “grow” for varying periods of time in the fluid portion of the culturessuggested that, given appropriate experimental conditions, such cells might be isolated directly in suspension culture. This, together with consideration of the phenomenon of population
From The Children’s Cancer Research Foundation and the Department of Pathology, Harvard Medical School, at the Children’s Hospital, Boston, Mass. +Holds Research Career Award I-K6-CA-22,150from the National Cancer Institute, National Institutes of Health. These studies were supported in part by research grant C-6516 and FR-05526 from the National Cancer Institute, National Institutes of Health, U. S . Public Health Service: Legacy of Loula D. Lasker, New York City: and the Albert and Mary Lasker Foundation. Received for publication February 22, 1965.
522
dependence as evidenced by differences in the nutritiona1 requirements of small and large populations of cells of the same established cell line,gs 13 led to attempts to cultivate large populations of buf€y coat cells in relatively small volumes of substrate directly in suspension culture. This communication reports the isolation of a continuous line of human lymphoblasts from such primary suspension cultures of the peripheral blood buffy coat of a child with acute leukemia. SOURCEOF SPECIMENS
C.E.M., a female, at the age of 2 11/12 years developed a mediastinal mass which was diagnosed as lymphosarcoma by cervical lymph node biopsy in another hospital in November 1963. Following a course of x-irradiation therapy, she (first was seen in the Tumor Therapy Clinic of this Foundation in December 1963, at which time her disease already had progressed to acute leukemia. At that time her peripheral WBC was 492,000, of which 87% were lymphoblasts (Fig. 1A). Although temporary hematological and clinical remissions were achieved on a regimen of total care and chemotherapy with methotrexate and prednisone, followed subsequently by 6-mercaptopurine, vincristine and cytoxan, her disease continued to progress, requiring repeated hospital admissions. Intervening episodes of central nervous system leukemia were treated with intrathecal methotrexate and x-irradiation. At the age of 3 10/12 years she was re-admitted to the Tumor Therapy Clinic of this Foundation in relapse on October 28, 1964 with a peripheral WBC of 177,000, of which 98% were lymphoblasts. Despite total care and vigorous chemotherapy with cytoxan and one course of intrathecal methotrexate, she continued in relapse. Prior to expiration on November 13, 1964, four 20-ml. specimens of
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TABLE1 PERIPHERAL BLOOD SPECIMENS (CEM) SEEDED I N PRIMARY SUSPENSION CULTURES
% Specimen
Date
Total WBC/Cu. mm.
Ivrnphoblasts .~
1
11/5/64 11/9/64 11/11/64 11/ 12/64
545,000 1,180,000 1,000,000 990,000
99 100 98 97
2 3 4
peripheral blood were drawn for cell culture in sterile centrifuge tubes containing sodium citrate (5.0 mg./lO ml. blood), as indicated in Table 1. CULTURE METHODS T h e buffy coat was separated immediately from red cells by aspiration following centrifugation (10 min. at 500 to 600 rpm) and from plasma following recentrifugation. T h e sedimented cells were suspended in a small volume of Eagle's minimum essential mediums supplemented with 10% whole fetal calf serum (EMEM) and enumerated by hemocytometer counts. Viability was estimated by the usual dye exclusion technique utilizing 0.0570.aqueous nigrosin.12 T h e cell suspension was drluted to a final volume of 100 ml. with EMEM in non-siliconized 500 ml. Erlenmeyer flasks containing a Teflon-coated magnetized bar and incubated at 37" on a magnetic stirrer. T h e initial viable cell population densities in these cultures are indicated in Table 1. T h e cultures were fed by the daily addition of 100 ml. of EMEM; resulting in 100, 50 and 33% increases in volume, respectively, during the first 72 hours. Total and viable cell counts were done daily on all cultures. RESULTS T h e culture initiated from specimen 2, seeded at a relatively low population density (Table I), failed to grow. Following the usual decline in viable cells during the first 48 hours, the number of viable cells in the cultures initiated from specimens one, 3 and 4 increased between the second and fourth days, as illustrated in Figure 2. On the fourth day, 200 ml. were withdrawn from the cultures and replaced with 200 ml. of EMEM. T h e viable cell population remaining in the cultures again grew steadily and on the sixth day 200 ml. of culture again were withdrawn and replaced
Viable cells/rnl. seeded in 100 ml. cultures
x x 1.5 x 1.5 x 5 5
106 106 107 107
with 200 ml. of EMEM, as indicated in Figure 2. At varying intervals these cultures were expanded to larger volumes in Erlenmeyer flasks on magnetic stirrers, conventional spinner culture vessels or the chemostat apparatus described by Cohen and Eagle6 by the transfer of 100 to 200 ml. of culture fed with a half or equal volume of EMEM on the day of transfer and maintained by subsequent withdrawal of culture and replacement with medium as described previously. T h e atmosphere in the vessels was replaced with either 5% CO, in air or 7% CO, and 10% 0, in nitrogen on the day of transfer. Otherwise, the cultures were maintained at circa p H 7.0 by the daily addition of appropriate volumes of 5% sodium bicarbonate. Since their initiation, the cells from 20 liters of these cultures, with a n average density of 1.5 x 106 cells/ml., have been harvested for biological and biochemical studies. All cultures from these initial isolations appeared to be identical in all respects; hence the culture (CCRF-CEM) derived from specimen one was selected as the permanent stock line and those derived from specimens 3 and 4 were expanded to largevolume cultures in conventional spinner flasks from which the cells were harvested for biological and biochemical studies. Replicate stock cultures of CCRF-CEM are maintained in Erlenmeyer flasks on magnetic stirrers or in spinner flasks as described previously except that the rate of growth in cultures maintained at circa pH 7.0 by the daily addition of sodium bicarbonate is such that 200 ml. with an average density of 1.8 x 106 cells/ml. can be withdrawn from a culture maintained a t a constant volume of 750 ml. by the daily replacement of these 200 ml. of media. As indicated in Figure 3, log-phase growth is re-initiated with each addition of fresh medium. T h e generation time as computed from such growth curves is circa 46 hours. Essentially similar harvests of cells can
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be obtained from “steady-state” cultures in a chemostat. Morphology: The cells in these cultures exhibit the characteristic morphology of lymphoblastic cells (Fig. 1B) and in general are remarkably similar to the lymphoblasts seen in direct smears of the patient’s peripheral blood.
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T h e nuclei are characteristically densely stained, frequently contain deep indentations and prominent nucleoli and in some cells are of rather bizarre morphology. The cytoplasm is scanty and faintly staining, appearing as a faint “rim” of cytoplasm around the densely stained nucleus in many cells. There is an
FIG. 1. A, Peripheral blood, patient CEM (Wright’s stain, x 1,000). B, CCRF-CEM cells after 1,000). C, Metaphase plate, CCRF-CEM, from preparations made after 3% months in continuous culture (Acetic carmine stain, x 2,000).
3y2 months in continuous culture (Wright’s stain, x
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average of one or 2 mitotic cells per high 15power field in smears prepared from log-phase ; cultures. These cells are pyronin positive and 0 peroxidase negative. Electron microscopy: Postmortem specimens v, and pellets of buffy coat and cells from cul- J ture were prepared by fixation in chromeosmium fluid as recommended by Dalton,T 0 followed by staining and post-fixation in 12 10% formalin containing uranyl acetate after m dehydration in a graded series of acetones. The specimens were then embedded in a mixture of epon, DDSA, araldite, dibutyl thalate and DMP-30 in the ratio (2.5:5.5:1.5:0.3:0.2) ‘0as described by M01lenhauer.l~Sections were cut with glass knives on an LKB ultratone, stained with lead citrate by a further dilution of the stain as described by Reynolds22 and examined in an RCA EMU-3G equipped with a 50-micra objective aperture and operated at 100 KV. The black line in each electron micrograph indicates one miI I I I I cron. 0 1 2 3 4 5 A cell typical of those in the peripheral blood buffy coat of the patient (specimen 1) DAYS OF INCUBATION depicted in Figure 4’ There are FIG. 3. Growth of human IymDhoblastic cells (CCRF-
4
&
4
12(4001
II10 9-
a7-
DAYS OF INCUBATION FIG. 2. Growth of human lymphoblastic cells in primary suspension cultures seeded with peripheral blood buffy coat, CEM, specimen I. Numbers in ( ) indicate increasing volume as culture was fed successively on days one to 3. Broken lines indicate withdrawal of 200 ml. of culture which were with ml. of fresh medium.
CEM) after 3% months in coniinuous suspensibn culture. Broken lines indicate withdrawal of 200 ml. of culture which were replaced with 200 ml. of fresh medium, maintaining constant volume of 750 ml. of culture.
indentations of the nucleus, sparse cytoplasm and a small number of cytoplasmic organelles. The homogeneous appearance of the nucleus, with small, patchy aggregates of larger chromatin granules, is typical of these cells. Nucleoli, although not visualized in this cell, were prominent in many other similar cells (see Fig. 6). A cell typical of the infiltrating cells observed in postmortem sections of rib bone marrow is illustrated in Figure 5. Similar cells were observed as infiltrates in electron micrographs of sections of the liver, spleen, salivary gland, meninges and lymph nodes. The plane of section in Figure 5 includes a poorIy developed Golgi zone and a few mitochondria. The cytoplasmic membrane has been damaged by postmortem autolysis. The similarities between these cells and those in Figures 4 and 6 are quite apparent. Sections of two cells of CCRF-CEM in a section made on the seventh day of culture are represented in Figure 6. The upper cell in the electron microgaph illustrates the nuclear indentation, scanty cytoplasm, diffuse distribution of chromatin and the patchy distribution
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FIG. 4. Lymphoblastic cell typical of those in buffy coat, specimen 1, CEM ( X 16,000). FIG. 5. Lymphoblastic cell from postmortem rib bone ma rro w . T h i s cell also is typical of those seen in visceral infiltrates, CEM ( x 16,000).
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of large granules characteristic of these cells in culture. T h e lower cell in the electron micrograph has a somewhat larger amount of cytoplasm containing a geographically-shaped lipid-type inclusion (lower right), which is not uncommon in these cells. Many of the cells in the preparation contain nuclear bridges (arrows, Fig. 6), the significance of which is obscure. Although the cells in culture contain somewhat more cytoplasm, more numerous and larger mitochondria than do the cells in the buffy coat or postmortem specimens, there are unquestionable similarities in over-all appearance among these cells. Virus-like particles have been visualized in these cells and in thin-section preparations of pellets sedimented by high-speed ultracentrifugation of the supernates of these cell cultures. These studies are described in detail elsewhere.26 Chromosomes: Preliminary chromosome analyses have been done on circa 100 metaphase cells prepared by a modification of the air-drying technique.25 Cells incubated 16 to 18 hours in EMEM containing Velban (0.01 ylml.) were resuspended in 0.7% sodium citrate and incubated 20 minutes at 37”. The cells then were fixed in Carnoy’s solution for 30 minutes to 2 hours and were spread, air-dried and stained with 1% carmine dissolved in 45% acetic acid. T h e chromosome complement of these cells after 3 months in continuous culture is illustrated in Figure 1C. The chromosome number ranged from 44 to 48 in 90% of the cells counted, with a mode of 46 (35% of the cells counted), indicating that this is a near-diploid cell. Detailed karyotypic analyses will be reported elsewhere. DISCUSSION The isolation of these continuous cell lines from cultures initiated with high density populations and the failure of the relatively lowdensity culture to grow lends support to the hypotheses upon which the present experiments were predicated. The ultimate validity and general applicability of these hypotheses for the isolation of cells from buffy coats can be established only by additional experiments. Similar experiments already have been undertaken with comparable buffy coat specimens obtained from 2 other children with acute leukemia. The culture initiated from one of these failed to grow while the behavior of the other thus far has been remarkably similar to that
Ul.
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of the cultures described herein. In addition, a system of “miniature” suspension culturels based upon the phenomenon of population dependence for use with smaller numbers of buffy coat or bone marrow cells is being developed to further examine these hypotheses. Thus far these cells will grow only in suspension cultures. The use of siliconized glassware or suspending agents in the medium has not been necessary; indeed, it has been difficult to attach these cells to glass for cytological studies without marked morphologic distortion or damage. These cells do not seem to tolerate freezing in glycerol-containing media24 but preliminary evidence from short-term experiments indicates that they may be preserved in media containing 15% dimethyl~ulfoxide~~ when frozen as described by Stulberg et al.,24 followed by storage in liquid nitrogen. The CCRF-CEM line of lymphoblastic cells already is yielding sufficient cells for studies now in progress which are concerned with the possible virus etiology of acute leukemia, and with the he terotransplantation to experimental animals, nucleic acid content, metabolic activity, nutritional requirements, drug responses and chromosomal and cytochemical characteristics of these cells. The yield of cells can be increased as necessary by expanding the volume of culture and the yield per volume of culture perhaps can be improved further by definition of the nutritional requirements of these cells, since there is as yet no evidence that the medium now in use is the optimal substrate. It appears that the majority of human leukemic lymphocytes differ from the majority of human normal lymphocytes in that they do not respond to the blastogenic stimulus of phytohemagglutinin and consequently do not undergo mitosis, at least in short-term cell ~ulture.2~ It is not uncommon, however, to observe mitotic cells in smears of the peripheral blood of patients with acute leukemia. Whether or not mitosis is initiated in the peripheral blood, or these are precursor cells which have been liberated even more prematurely from the bone marrow than the typical leukemic lymphoblast, is not known. As evidenced by the present experiments, lymphoblastic cells capable of autonomous growth and mitosis can be isolated from the peripheral blood of children with acute leukemia. In this respect the behavior of the CCRF-CEM cell lines, like the cells derived from adult leu-
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CANCER April 1965
kemiall and Burkitt’s lymphomalor 19, 2o resemble that of normal lymphocytic cells “transformed” by treatment with phytohem-
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agglutinin.*O It is interesting to speculate that the autonomous growth of the CCRF-CEM lymphoblastic cells may be the result of
FIG. 6. Lymphoblastic cells CCRF-CEM in suspension culture. Section of pellet of cells harvested on seventh day of culture (x 16,000).
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“transformation” induced in vivo by some blastogenic factor, perhaps virus (or other) antigen.
SUMMARY
A continuous line of lymphoblastic cells has been isolated from the peripheral blood of a child with acute leukemia by the cultivation of buffy coats directly in suspension cultures in Eagle’s minimum essential medium supplemented with 10% whole fetal calf serum. As adjudged by light and electron microscopic studies, these cultured cells are essentially similar to those present in the original buffy coat specimens, and to the infiltrating cells seen in electron micrographs of thin-section preparations obtained from the patient at autopsy. These cells are near-diploid and have a generation time of circa 46 hours after 3% months in continuous culture. Volumes of 200 ml. with an average of 1.8 x lo6 cells/ml. are being harvested daily from an ordinary spinner culture for biological and biochemical studies. Virus-like particles have been visualized in these cultures. REFERENCES 1. Ang, B., Jaross, L., and McAllister, R. M.: Studies of fibroblast-like cells from the bone marrow of leukemic and non-leukemic children. Proc. SOC. Exp. Biol. Med. 109: 467-471, 1962. 2. Bain, B., Vas, M. R., and Lowenstein, L.: T h e development of large immature mononuclear cells in mixed leukocyte cultures. Blood 23: 108-116, 1964. 3. Benyesh-Melnick, M., Fernbach, D. J., and Lewis, R. T.: Studies on human leukemia-I. Spontaneous lymphoblastoid transformation of fibroblastic bone marrow cultures derived from leukemic and non leukemic children. J . N a t . Cancer Znst. 31: 1311-1325, 1963. 4. Berg, R. B., and Rosenthal, M. S.: Studies of fibroblastic cells cultivated from bone marrow of leukemic and nonleukemic patients. Proc. SOC. Exp. Biol. M e d . 106: 614-617, 1961. 5. Berman, L., Stulberg, C. S., and Ruddle, F. H.: Long term tissue culture of human bone marrow-I. Report of isolation of a strain of cells resembling epithelial cells from bone marrow of a patient with carcinoma of the lung. Blood 10: 896-911, 1955.
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6. Cohen, E. P., and Eagle, H.: A simplified chemostat for the growth of mammalian cells-Characteristics of cell growth in continuous culture. J . E x p . Med. 113: 467-74, 1961. 7. Dalton, A. J.: Personal communication to Dr. B. G. Uzman. 8. Eagle, H.: Amino acid metabolism in mammalian cell cultures. Science 130: 432-437, 1959. 9. Eagle, H., and Piez, K.: T h e popuIation dependent requirements by cultured mammalian cells for metabolites which they can synthesize. J . E x p . Med. 116: 29-43, 1962. 10. Epstein, M. A., and Barr, Y. M.: Cultivation in vitro of human lymphoblasts from Burkitt’s malignant lymphoma. Lancet 1: 252-253, 1964. 11. Iwakata, S., and Grace, J. T., Jr.: Cultivation in vitro of myeloblasts from human leukemia. N.Y. State J. Med. 64: 2279-2282, 1964. 12. Kaltenbach, J. P., Kaltenbach, M. H., and Lyons, W. B.: Nigrosin as a dye for differentiating live and dead ascites cells. Exp. Cell Res. 15: 112-117, 1958. 13. Lockart, R. Z., Jr., and Eagle, H.: Requirements for growth of single human cells. Science 129: 252-254, 1959. 14. Lovelock, J. W., and Bishop, M. W. H.: Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183: 1394-1395, 1959. 15. Mollenhauer, H. H.: Plastic embedding mixtures for use in electron microscopy. Stain Tech. 39: 111-114, 1964. 16. Osgood, E. E.: Tissue culture in the study of leukocytic functions. Ann. N . Y . Acad. Sci. 59: 806-814, 1958. 17. Osgood, E. E., and Brooke, J. H.: Continuous tissue culture of leukocytes from human leukemic bloods by application of “gradient” principles. Blood 10: 1010-1022, 1955. 18. Osgood, E. E., and Brownlee, I. E.: Culture of human marrow-Details of a simple method. JAMA 108: 1793-1796, 1937. 19. Pulvertaft, R. J. V.: Cytology of Burkitt’s tumour (African lymphoma). Lancet 1: 238-240, 1964. 20. Ibid: Phytohemagglutinin in relation to Burkitt’s tumour (African lymphoma). Lancet 2: 552-555, 1964. 21. Reisner, E. H., Jr.: Tissue culture of bone marrow. Ann. N.Y. Acad. Sci. 71: 487-500, 1959. 22. Reynolds, E. W.: T h e use of lead citrate at high p H as an electron opaque stain i n electron microscopy. J . Cell Biol. 17: 208 212, 1963. 23. Robbins, J. H.: Tissue culture studies of the human lymphocyte. Science 146: 1648-1654, 1964. 24. Stulberg, C. S., Peterson, W. D., Jr., and Berman, L.: Quantitative and qualitative preservation of cellstrain characteristics. N a t . Cancer Inst. Monograph #7: 17-31, 1962. 25. Todaro, G. J., Wolman, S. R., and Green, H.: Rapid transformation of human fibroblasts with low growth potential into established cell lines by SV,. J . Cell. Comp. Physiol. 62: 257-266, 1963. 26. Uzman, B. G., et al.: T o be published.
