Int. J . Cancer: 15, 321-341 (1975)
CLASSIFICATION AND BIOLOGICAL NATURE OF ESTABLISHED HUMAN HEMATOPOIETIC CELL LINES
Kenneth NILSONand Jan P O N T ~ N Department of Pathology and Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden
Over 200 established human hernatopoietic cell lines of normal and malignant origin have been investigated by morphological and functional parameters. Employing morphology as the overriding parameter four types of lines were identified. ( I ) Lymphoblastoid cell lines, derived from normal and neoplastic hematopoietic tissue, were characterized by the wide morphologic flexibility of individual lymphoblastoid cells, constant association with Epstein-Barr virus ( E B V ), polyclorial derivation, differentiation for immunoglobulin production (secretion) and their diploids. ( 2 ) Lymphoma cell lines. This type of line was established at a high frequency from Burkitt 's lymphoma and rarely from other types of lymphoma, but never from patients without malignancy or with non-lymphoma malignancies. Important characteristics were morphologic stereotypia within each line, monoclonal derivation, common but not obligatory association with E B V,variability in the expression of Ig synthesis (noproduction, or membrane bound Ig, or secretion) and aneuploidy. ( 3 ) Myeloma cell lines could only rarely be obtained from patients with myeloma. The basis for classification of these lines is their production of Ig identical to the myeloma protein in vitro. Other important distinguishing features were: plasma cell morphology, absence of E B V and aneuploidy. ( 4 ) The leukemia cell line ( M O L T 4 ) was the only line with T-cell characteristics and was easily distinguished from the other types. Important characteristics were a typicalsurface ultrastructure, absence of E B V and absence of imrnunoglobulin production. Individual lymphoblastoid lines were in principle identical whereas each line of the other three types had its own characteristic profile. The phenotypic characteristics of the lymphoblastoid lines were very stable during prolonged serial cultivation. Only in a few cases were secondary chromosomal, functional or morphologic alterations noted. W e conclude that EB V-carrying lymphoblastoid lines can be obtained from non-neoplastic precursor cells from healthy as well as from diseased individuals. Lymphoma, myeloma and leukemia lines are only obtained from the respective neoplastic tissue but generally at a low frequency. With the exception of Burkitt's lymphoma, malignant hematopoietic tissue and leukemia frequently give rise to established cell lines in vitro of the lymphoblastoid type rather than lines derived from the neoplastic cells.
Decades of experience have shown that longterm serial cultivation is only possible for a few types of cells derived from normal human tissues. The three most intensively studied examples are fibroblasts (Hayflick, 1965), glia cells (Ponten and
Macintyre, 1968) and lymphoblastoid cells (for reviews see Moore and Minowada, 1969; Miller, 1971 ; Nilsson, 1971a). The first two-after a period of luxuriant growth-invariably enter a degenerative stage culminating in loss of the
Received: July 24, and in revised form October 14, 1974.
321
NILSSON AND P O N T ~ N
cultures. Lymphoblastoid cells, on the other hand, can be transferred apparently indefinitely and a population of such cells is thus immortal. In the mouse and rat the situation is quite different. Fibroblasts of normal origin will enter a degenerative stage similar to that of human fibroblasts, but they will recover from this to form immortal heteroploid lines, which generally are also tumorigenic. Explants from normal lymphoid tissue will, on the other hand, deteriorate and murine lymphoblastoid lines analogous to those of human origin are unknown. Since spontaneous formation of immortal rodent fibroblast lines is strongly correlated with tumorigenicity, it has often been conjectured that established human lymphoblastoid lines are examples of spontaneous malignant cell transformation in vifro. This view is indirectly supported by the fact that immortal human fibroblast or glia lines can only be obtained either by cultivation of spontaneous sarcomas and gliomas or by transformation by an oncogenic virus. In fact, the best criterion of growth of, for instance, neoplastic glia cells rather than normal stroma glia cells is the capacity of a tumor explant t o form an infinitely multiplying cell line in culture (for review see PontCn, 1971). Permanent human hematopoietic lines have not only been obtained from normal individuals but also to a large extent from patients with malignant lymphomas, leukemias and myelomas. Particularly intensive studies have been made on lines derived from African Burkitt's lymphoma.
Terminology. Permanent (or established) line = a population of cells which in culture has a capacity for infinite multiplication. Hematopoietic line = a permanent cell line composed of any of the specific elements of blood-cell-forming tissue (myelopoietic cells, erythropoietic cells, megakaryocytes, histiocytes or monocytes, lymphoid cells). Lymphoid line = a permanent cell line composed of elements of lymphopoietic tissue (histiocytes, lymphoblasts, lymphocytes, plasma blasts, plasma cells). Lymphoblastoid line = a permanent cell line composed of elements conforming with the criteria of Table I. Lymphoma line = a cell line composed of the neoplastic elements of a malignant lymphoma. Leukemia line = a cell line composed of the neoplastic cells of a leukemia. Myeloma line = a cell line composed of the neoplastic cells of a myeloma. Secondary alteration of a lymphoblastoid line = a stable change of any of the " defining characteristics" in Table I, which occurs spontaneously during serial passage in vituo.
322
Non-neoplastic cells are inevitably present as contaminants in any such explants. Since lymphoblastoid lines may grow out from normal tissue it does not-in contrast to sarcomas and gliomas-automatically follow that a cell line from a human hematopoietic malignancy is of neoplastic cell origin (Nilsson et al., 1970). The main issue of this article is to find out if there are any reliable ways of distinguishing between hematopoietic lines of normal or neoplastic cell origin. Previous attempts to make such a distinction have only been partly successful for reasonselaborated on in our '' Discussion ", A successful discrimination would be important for an understanding of the biology of established human lymphoid lines, including studies on basic differences between normal and malignant cells. This survey is based on over 9 years of experience with a variety of human lymphoid explants and the derived lines and on representative studies of morphological and function parameters.
MATERIAL A N D METHODS
Cell lines Approximately 200 cell lines have been studied. The majority were established from normal and malignant hematopoietic tissue in our laboratory by the grid organ culture described by Nilsson (19716). Lines have also been obtained from Dr. G. Klein, Institute for Tumor Biology, Karolinska Institute, Stockholm. Cross-examinations have been excluded by the use of appropriate markers (HL-A) (Lindblom and Nilsson, 1973); immunoglobulin production (Nilsson et a/., 1968 ; Nilsson, 19716; BCchet et al., 1972, 1973); G6-PD and PGM (BCchet et al., 1975; Fialkow, personal communication) and rate of B2-microglobulin production (Nilsson et al., 1974). All lines have been maintained under identical tissue-culture conditions, i.e. incubation in a humidified 5 % C0,-in air atmosphere at 37" C in Ham's F-10 medium (Ham, 1963; GIBCO, New York) supplemented by 10% new-born calf serum and antibiotics (100 IU penicillin/ml, 50 pg streptomycin/ml, 1.25 pg amphotericin B). Morhological studies
Morphological characteristics have been analysed by different techniques: (1) observations of
CLASSIFICATION OF HUMAN HEMATOPOIETIC CELL LINES
viable cells by inverted microscopy and by timelapse cinematography as reported (Nilsson et al., 1970; Nilsson, 1971~);(2) studies with light microscopy of fixed cells with different stains, including acridine orange (Nilsson, 1971~);(3) transmission (TEM) and scanning electron microscopy (SEM) as detailed (Nilsson and Sundstrom, 1974).
TABLE I DEFINING FEATURES OF LYMPHOBLASTOID LINES
Basic cell type
Lymphoblast
Growth pattern
Suspension Large cell clumps On feeder cells Isolated cells or in clumps strongly attached to feeder cells Mobility
Functional studies
The techniques for detecting surface-localized immunoglobulin and immunoglobulins secreted to the medium have been described (Philipson and PontCn, 1967; Nilsson, 19716; Nilsson and Sundstrom, 1974). &-Microglobulin was detected by radioimmunoassay (Evrin and Nilsson, 1974),
Mainly by uropods Suspension On feeder cells Rapid locomotion (peripolesis) Secretion of complete molecules Ig production into the medium Diversity
Between lines Within lines
Chromosome studies
The chromosomes have been studied in 16 cell lines.
EBV-genome Karyotype
None Wide spectrum of reversible morphologic modulations of one basic cell type Present Diploid
EB V studies Many of our lines have been examined for EBV-determined antigens (Nilsson et al., 1971) and in a few cases for EBV genome (Klein G . et al., 1974). HL-A- typing
Cell lines were examined by the microcytotoxicity test described by Kissmeyer-Nielsen and Kjerbye (1 967). RESULTS
By comparative morphology one kind of lymphoid line-the lymphoblastoid type-could be clearly separated from the non-lymphoblastoid types. The latter have been subdivided into the lymphoma, the leukemia and the myeloma types depending on their origin.
Lymphoblastoid cell lines The basic cell type, illustrated in Figures l ~ 3 , 4 , 5 and defined in Table I, can be described as a lymphoblast moderately rich in cytoplasm. It grows interchangeably either in clumps in nonstirred culture suspension or attached to feeder cells such as fibroblasts or glia (Fig. 1 ~ ) .For multiplication in suspension a rich medium such as F-10 or RPMI 1640 is required. With feeder cells present, growth is maintained also in poor media (e.g. Eagle’s MEM). The population
doubling time is 30-48 h and maximum cell density 1-1.3 x 106/ml. Individual lines have a markedly pleomorphic morphology. The spectrum of possible modulations is, however, the same from line to line. The shape of individual cells changes constantly as visualized by time-lapse cinematography (Fig. 2). Free-floating cells are round and villous all over the surface or elongated (“ hand-mirror ” shape) with the slender pseudopodia located at one end (the uropod). They show a strong tendency to form clumps with the uropods pointing away from the center. Lymphoblastoid cells become rapidly attached to feeder cells, where they exhibit a variety of interchangeable forms. Most elements spread out with lively locomotive mobility on the outside of the feeder cells (peripolesis) while others are only loosely attached and show rapid , bubbling movements of the cell surface. The mobility of the cell membrane is pronounced enough to be appreciated also by ordinary inverted microscopy, where parts can be seen to change position within only a few minutes. It can also be inferred from the presence of many markedly different cell shapes at any given instant. A few cells, particularly if they manage t o become attached to bare plastic, stop their movements and acquire a fibroblast-like shape
323
NILSSON A N D P O N T ~ N
324
CLASSIFICATION OF HUMAN HEMATOPOlETtC CELL LINES
which, however, is also interchangeable with the other forms. This highly variable morphology is also noted in the scanning-or transmission-electron microscope. The surface of some cells is shown in Figures 3 , 4 and 5. A few cells are round, villous, lamellated or bubbling. The most common cell configuration is an elongated or pear-shaped form with rather few broad and short villi. In transmission electron microscopy a small percentage of plasmablast-like cells may be seen, while the majority have a lymphoblastic morphology with an immature nucleus, pleomorphic nucleoli and a cytoplasm containing sparse endoplasmic reticulum, many polyribosomes and a primitive Golgi apparatus. The lymphoblastoid cells have only a restricted potential for colony formation in agar or agarose. Lymphoblastoid cells synthesize immunoglobulins which can be detected at the cell surface by immunofluorescence or as free molecules in the supernatant by immunochemical methods (Philipson and Ponten, 1967). When examined shortly after establishment, the production is polyclonal, i.e. Ig molecules of different heavy chain classes and type of light chains can be identified by monospecific antisera. With prolonged cultivation the pattern of Ig synthesis usually changes towards monoclonality indicating a selection in vitro of one immunoglobulinproducing clone or alternatively a restrictive change in a clone of multipotent cells (Nilsson, 1971b; BBchet et al., 1975). A few lines, however,
4
keep an oligoclonal Ig synthesis even after years of continuous cultivation. The synthesis of immunoglobulin is a stable phenotypic function. Our oldest ceIl Iines have maintained their production for 9 years. Cloning of one lymphoblastoid line showed production of identical type of Ig in all of 19 clones (Nilsson, 1971~).The capacity for Ig production is therefore presumably shared by all members of a lymphoblastoid population. Complete molecules are secreted at a rate of 1-3 pg/106 cells/24 h in optimally growing asynchronous cultures (Sjoquist et al., in preparation). All lymphoblastoid lines tested produced microglo globulin (Evrin and Nilsson, 1974; Nilsson et al., 1974) at a rate of ca 200-400 yg/ 5 x lo5 cells/65 h in logarithmically growing cultures. All of 21 tested lymphoblastoid lines have contained EBV (Nilsson et a/., 1971). Sixteen lines have been subjected to cytogenetic analysis. At least those in comparatively early passage were diploid (Saksela and PontBn, 1968) and normal as analyzed even with the sensitive quinacrine mustard banding technique (L. Zech, personal communication). Seventeen lines were tested with a panel of HL-A allo-antisera. In all instances the cells reacted strongly and exhibited more than four antigenic specificities. Two antisera (HL-A 28, W 10) reacted with all lines. In six cases the HL-A type of the donor could be compared and in n o instance were HL-A 28 and 10 found. Thus,
FIGURE1 A human lymphoblastoid cell line on feeder cells. The lymphoblastoid cells are mainly assembled in dense clumps. Between these, isolated cells engaged in peripolesis may be seen. Inverted microscope x 80. Secondarily altered lymphoblastoid line (NC 37) on feeder cells. Peripolesis, but no clumps. Non-adherent B. cells are mainly round. Inverted microscope x 75. C . Burkitt lymphoma cells (Daudi). Monomorphic morphology. No peripolesis. A few loose clumps. Inverted microscope x 100. D. Burkitt lymphoma cells (Raji). Picture almost identical to the secondarily altered lymphoblasts of Figure 1 B. Inverted microscope x 100. Burkitt lymphoma cells (U-47703 BL). Picture as lc and I D except for a higher rate of cell death and E. differences in cell size. A few adhering cells but no peripolesis. Inverted microscope x 100. F. Burkitt lymphoma line (U-626 BL). Light variations in cell size. A few adhering, but not peripoletic cells. Inverted microscope x 100. 0. Burkitt lymphoma line [U-604 BL (Seraphina)].This line is more strongly attached to feeder cells but few cells are peripoletic. Inverted microscope x 100. Myeloma line 266 BL. Round cells. No clumps. No adherence to feeder cells. Inverted microscope x 100. H. RPMI 8226 myeloma cells. Variations in cell size. No clumps. No adherence to feeder cells. Inverted I. microscope x 100. J. Leukemia line MOLT 4. Typical appearance after 48 h co-cultivation with feeder cells. A proportion of the cells adhere and elongate. The elongated cell is almost non-mobile. Inverted microscope x 100. A.
325
NILSSON AND F r e e floating c e l l ' -
PONTBN
L o a s l y a i t a c h r ~ d( e l l i
Stronsly a d h e r e d c e l l +
/c-c
\
-
/
Lou mobility
V e r y low rnobilify No direcrional
No directional Io~ornolion
l o ~ ~ n i ~ t i o n
Very few C e l l s .
L Y MPl l OMA CCll
----&Y Lou m o b i l i t y
Low mobilrty N o directtonal l o c o r n ~ l i ~ r i
F e w
CLASSIFICATION AND BIOLOGICAL NATURE OF ESTABLISHED HUMAN HEMATOPOIETIC CELL LINES
Kenneth NILSONand Jan P O N T ~ N Department of Pathology and Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden
Over 200 established human hernatopoietic cell lines of normal and malignant origin have been investigated by morphological and functional parameters. Employing morphology as the overriding parameter four types of lines were identified. ( I ) Lymphoblastoid cell lines, derived from normal and neoplastic hematopoietic tissue, were characterized by the wide morphologic flexibility of individual lymphoblastoid cells, constant association with Epstein-Barr virus ( E B V ), polyclorial derivation, differentiation for immunoglobulin production (secretion) and their diploids. ( 2 ) Lymphoma cell lines. This type of line was established at a high frequency from Burkitt 's lymphoma and rarely from other types of lymphoma, but never from patients without malignancy or with non-lymphoma malignancies. Important characteristics were morphologic stereotypia within each line, monoclonal derivation, common but not obligatory association with E B V,variability in the expression of Ig synthesis (noproduction, or membrane bound Ig, or secretion) and aneuploidy. ( 3 ) Myeloma cell lines could only rarely be obtained from patients with myeloma. The basis for classification of these lines is their production of Ig identical to the myeloma protein in vitro. Other important distinguishing features were: plasma cell morphology, absence of E B V and aneuploidy. ( 4 ) The leukemia cell line ( M O L T 4 ) was the only line with T-cell characteristics and was easily distinguished from the other types. Important characteristics were a typicalsurface ultrastructure, absence of E B V and absence of imrnunoglobulin production. Individual lymphoblastoid lines were in principle identical whereas each line of the other three types had its own characteristic profile. The phenotypic characteristics of the lymphoblastoid lines were very stable during prolonged serial cultivation. Only in a few cases were secondary chromosomal, functional or morphologic alterations noted. W e conclude that EB V-carrying lymphoblastoid lines can be obtained from non-neoplastic precursor cells from healthy as well as from diseased individuals. Lymphoma, myeloma and leukemia lines are only obtained from the respective neoplastic tissue but generally at a low frequency. With the exception of Burkitt's lymphoma, malignant hematopoietic tissue and leukemia frequently give rise to established cell lines in vitro of the lymphoblastoid type rather than lines derived from the neoplastic cells.
Decades of experience have shown that longterm serial cultivation is only possible for a few types of cells derived from normal human tissues. The three most intensively studied examples are fibroblasts (Hayflick, 1965), glia cells (Ponten and
Macintyre, 1968) and lymphoblastoid cells (for reviews see Moore and Minowada, 1969; Miller, 1971 ; Nilsson, 1971a). The first two-after a period of luxuriant growth-invariably enter a degenerative stage culminating in loss of the
Received: July 24, and in revised form October 14, 1974.
321
NILSSON AND P O N T ~ N
cultures. Lymphoblastoid cells, on the other hand, can be transferred apparently indefinitely and a population of such cells is thus immortal. In the mouse and rat the situation is quite different. Fibroblasts of normal origin will enter a degenerative stage similar to that of human fibroblasts, but they will recover from this to form immortal heteroploid lines, which generally are also tumorigenic. Explants from normal lymphoid tissue will, on the other hand, deteriorate and murine lymphoblastoid lines analogous to those of human origin are unknown. Since spontaneous formation of immortal rodent fibroblast lines is strongly correlated with tumorigenicity, it has often been conjectured that established human lymphoblastoid lines are examples of spontaneous malignant cell transformation in vifro. This view is indirectly supported by the fact that immortal human fibroblast or glia lines can only be obtained either by cultivation of spontaneous sarcomas and gliomas or by transformation by an oncogenic virus. In fact, the best criterion of growth of, for instance, neoplastic glia cells rather than normal stroma glia cells is the capacity of a tumor explant t o form an infinitely multiplying cell line in culture (for review see PontCn, 1971). Permanent human hematopoietic lines have not only been obtained from normal individuals but also to a large extent from patients with malignant lymphomas, leukemias and myelomas. Particularly intensive studies have been made on lines derived from African Burkitt's lymphoma.
Terminology. Permanent (or established) line = a population of cells which in culture has a capacity for infinite multiplication. Hematopoietic line = a permanent cell line composed of any of the specific elements of blood-cell-forming tissue (myelopoietic cells, erythropoietic cells, megakaryocytes, histiocytes or monocytes, lymphoid cells). Lymphoid line = a permanent cell line composed of elements of lymphopoietic tissue (histiocytes, lymphoblasts, lymphocytes, plasma blasts, plasma cells). Lymphoblastoid line = a permanent cell line composed of elements conforming with the criteria of Table I. Lymphoma line = a cell line composed of the neoplastic elements of a malignant lymphoma. Leukemia line = a cell line composed of the neoplastic cells of a leukemia. Myeloma line = a cell line composed of the neoplastic cells of a myeloma. Secondary alteration of a lymphoblastoid line = a stable change of any of the " defining characteristics" in Table I, which occurs spontaneously during serial passage in vituo.
322
Non-neoplastic cells are inevitably present as contaminants in any such explants. Since lymphoblastoid lines may grow out from normal tissue it does not-in contrast to sarcomas and gliomas-automatically follow that a cell line from a human hematopoietic malignancy is of neoplastic cell origin (Nilsson et al., 1970). The main issue of this article is to find out if there are any reliable ways of distinguishing between hematopoietic lines of normal or neoplastic cell origin. Previous attempts to make such a distinction have only been partly successful for reasonselaborated on in our '' Discussion ", A successful discrimination would be important for an understanding of the biology of established human lymphoid lines, including studies on basic differences between normal and malignant cells. This survey is based on over 9 years of experience with a variety of human lymphoid explants and the derived lines and on representative studies of morphological and function parameters.
MATERIAL A N D METHODS
Cell lines Approximately 200 cell lines have been studied. The majority were established from normal and malignant hematopoietic tissue in our laboratory by the grid organ culture described by Nilsson (19716). Lines have also been obtained from Dr. G. Klein, Institute for Tumor Biology, Karolinska Institute, Stockholm. Cross-examinations have been excluded by the use of appropriate markers (HL-A) (Lindblom and Nilsson, 1973); immunoglobulin production (Nilsson et a/., 1968 ; Nilsson, 19716; BCchet et al., 1972, 1973); G6-PD and PGM (BCchet et al., 1975; Fialkow, personal communication) and rate of B2-microglobulin production (Nilsson et al., 1974). All lines have been maintained under identical tissue-culture conditions, i.e. incubation in a humidified 5 % C0,-in air atmosphere at 37" C in Ham's F-10 medium (Ham, 1963; GIBCO, New York) supplemented by 10% new-born calf serum and antibiotics (100 IU penicillin/ml, 50 pg streptomycin/ml, 1.25 pg amphotericin B). Morhological studies
Morphological characteristics have been analysed by different techniques: (1) observations of
CLASSIFICATION OF HUMAN HEMATOPOIETIC CELL LINES
viable cells by inverted microscopy and by timelapse cinematography as reported (Nilsson et al., 1970; Nilsson, 1971~);(2) studies with light microscopy of fixed cells with different stains, including acridine orange (Nilsson, 1971~);(3) transmission (TEM) and scanning electron microscopy (SEM) as detailed (Nilsson and Sundstrom, 1974).
TABLE I DEFINING FEATURES OF LYMPHOBLASTOID LINES
Basic cell type
Lymphoblast
Growth pattern
Suspension Large cell clumps On feeder cells Isolated cells or in clumps strongly attached to feeder cells Mobility
Functional studies
The techniques for detecting surface-localized immunoglobulin and immunoglobulins secreted to the medium have been described (Philipson and PontCn, 1967; Nilsson, 19716; Nilsson and Sundstrom, 1974). &-Microglobulin was detected by radioimmunoassay (Evrin and Nilsson, 1974),
Mainly by uropods Suspension On feeder cells Rapid locomotion (peripolesis) Secretion of complete molecules Ig production into the medium Diversity
Between lines Within lines
Chromosome studies
The chromosomes have been studied in 16 cell lines.
EBV-genome Karyotype
None Wide spectrum of reversible morphologic modulations of one basic cell type Present Diploid
EB V studies Many of our lines have been examined for EBV-determined antigens (Nilsson et al., 1971) and in a few cases for EBV genome (Klein G . et al., 1974). HL-A- typing
Cell lines were examined by the microcytotoxicity test described by Kissmeyer-Nielsen and Kjerbye (1 967). RESULTS
By comparative morphology one kind of lymphoid line-the lymphoblastoid type-could be clearly separated from the non-lymphoblastoid types. The latter have been subdivided into the lymphoma, the leukemia and the myeloma types depending on their origin.
Lymphoblastoid cell lines The basic cell type, illustrated in Figures l ~ 3 , 4 , 5 and defined in Table I, can be described as a lymphoblast moderately rich in cytoplasm. It grows interchangeably either in clumps in nonstirred culture suspension or attached to feeder cells such as fibroblasts or glia (Fig. 1 ~ ) .For multiplication in suspension a rich medium such as F-10 or RPMI 1640 is required. With feeder cells present, growth is maintained also in poor media (e.g. Eagle’s MEM). The population
doubling time is 30-48 h and maximum cell density 1-1.3 x 106/ml. Individual lines have a markedly pleomorphic morphology. The spectrum of possible modulations is, however, the same from line to line. The shape of individual cells changes constantly as visualized by time-lapse cinematography (Fig. 2). Free-floating cells are round and villous all over the surface or elongated (“ hand-mirror ” shape) with the slender pseudopodia located at one end (the uropod). They show a strong tendency to form clumps with the uropods pointing away from the center. Lymphoblastoid cells become rapidly attached to feeder cells, where they exhibit a variety of interchangeable forms. Most elements spread out with lively locomotive mobility on the outside of the feeder cells (peripolesis) while others are only loosely attached and show rapid , bubbling movements of the cell surface. The mobility of the cell membrane is pronounced enough to be appreciated also by ordinary inverted microscopy, where parts can be seen to change position within only a few minutes. It can also be inferred from the presence of many markedly different cell shapes at any given instant. A few cells, particularly if they manage t o become attached to bare plastic, stop their movements and acquire a fibroblast-like shape
323
NILSSON A N D P O N T ~ N
324
CLASSIFICATION OF HUMAN HEMATOPOlETtC CELL LINES
which, however, is also interchangeable with the other forms. This highly variable morphology is also noted in the scanning-or transmission-electron microscope. The surface of some cells is shown in Figures 3 , 4 and 5. A few cells are round, villous, lamellated or bubbling. The most common cell configuration is an elongated or pear-shaped form with rather few broad and short villi. In transmission electron microscopy a small percentage of plasmablast-like cells may be seen, while the majority have a lymphoblastic morphology with an immature nucleus, pleomorphic nucleoli and a cytoplasm containing sparse endoplasmic reticulum, many polyribosomes and a primitive Golgi apparatus. The lymphoblastoid cells have only a restricted potential for colony formation in agar or agarose. Lymphoblastoid cells synthesize immunoglobulins which can be detected at the cell surface by immunofluorescence or as free molecules in the supernatant by immunochemical methods (Philipson and Ponten, 1967). When examined shortly after establishment, the production is polyclonal, i.e. Ig molecules of different heavy chain classes and type of light chains can be identified by monospecific antisera. With prolonged cultivation the pattern of Ig synthesis usually changes towards monoclonality indicating a selection in vitro of one immunoglobulinproducing clone or alternatively a restrictive change in a clone of multipotent cells (Nilsson, 1971b; BBchet et al., 1975). A few lines, however,
4
keep an oligoclonal Ig synthesis even after years of continuous cultivation. The synthesis of immunoglobulin is a stable phenotypic function. Our oldest ceIl Iines have maintained their production for 9 years. Cloning of one lymphoblastoid line showed production of identical type of Ig in all of 19 clones (Nilsson, 1971~).The capacity for Ig production is therefore presumably shared by all members of a lymphoblastoid population. Complete molecules are secreted at a rate of 1-3 pg/106 cells/24 h in optimally growing asynchronous cultures (Sjoquist et al., in preparation). All lymphoblastoid lines tested produced microglo globulin (Evrin and Nilsson, 1974; Nilsson et al., 1974) at a rate of ca 200-400 yg/ 5 x lo5 cells/65 h in logarithmically growing cultures. All of 21 tested lymphoblastoid lines have contained EBV (Nilsson et a/., 1971). Sixteen lines have been subjected to cytogenetic analysis. At least those in comparatively early passage were diploid (Saksela and PontBn, 1968) and normal as analyzed even with the sensitive quinacrine mustard banding technique (L. Zech, personal communication). Seventeen lines were tested with a panel of HL-A allo-antisera. In all instances the cells reacted strongly and exhibited more than four antigenic specificities. Two antisera (HL-A 28, W 10) reacted with all lines. In six cases the HL-A type of the donor could be compared and in n o instance were HL-A 28 and 10 found. Thus,
FIGURE1 A human lymphoblastoid cell line on feeder cells. The lymphoblastoid cells are mainly assembled in dense clumps. Between these, isolated cells engaged in peripolesis may be seen. Inverted microscope x 80. Secondarily altered lymphoblastoid line (NC 37) on feeder cells. Peripolesis, but no clumps. Non-adherent B. cells are mainly round. Inverted microscope x 75. C . Burkitt lymphoma cells (Daudi). Monomorphic morphology. No peripolesis. A few loose clumps. Inverted microscope x 100. D. Burkitt lymphoma cells (Raji). Picture almost identical to the secondarily altered lymphoblasts of Figure 1 B. Inverted microscope x 100. Burkitt lymphoma cells (U-47703 BL). Picture as lc and I D except for a higher rate of cell death and E. differences in cell size. A few adhering cells but no peripolesis. Inverted microscope x 100. F. Burkitt lymphoma line (U-626 BL). Light variations in cell size. A few adhering, but not peripoletic cells. Inverted microscope x 100. 0. Burkitt lymphoma line [U-604 BL (Seraphina)].This line is more strongly attached to feeder cells but few cells are peripoletic. Inverted microscope x 100. Myeloma line 266 BL. Round cells. No clumps. No adherence to feeder cells. Inverted microscope x 100. H. RPMI 8226 myeloma cells. Variations in cell size. No clumps. No adherence to feeder cells. Inverted I. microscope x 100. J. Leukemia line MOLT 4. Typical appearance after 48 h co-cultivation with feeder cells. A proportion of the cells adhere and elongate. The elongated cell is almost non-mobile. Inverted microscope x 100. A.
325
NILSSON AND F r e e floating c e l l ' -
PONTBN
L o a s l y a i t a c h r ~ d( e l l i
Stronsly a d h e r e d c e l l +
/c-c
\
-
/
Lou mobility
V e r y low rnobilify No direcrional
No directional Io~ornolion
l o ~ ~ n i ~ t i o n
Very few C e l l s .
L Y MPl l OMA CCll
----&Y Lou m o b i l i t y
Low mobilrty N o directtonal l o c o r n ~ l i ~ r i
F e w
