Acta path. microbiol. scand. Sect. A, 84: 79-84, 1976
SOME CHARACTERISTICS OF LYMPHOBLASTOID LINES DERIVED FROM HUMAN ASTROCYTOMATA ELIZABETH H. MACINTYRE, ANDERSLINDGREN and JANP O N T ~ N Group of Cell Biology, The Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden
Macintyre, E. H., Lindgren, A. & PontCn, J. Some characteristics of lymphoblastoid lines derived from human astrocytomata. Acta path. microbiol. scand. Sect. A, 84: 79-84, 1976. Two immunoglobulin-producing lines of normal lymphoblastoid cells have been established from cultures of two human astrocytornata. These had the same characteristics as lymphoblastoid lines derived from normal human lymphoid tissue or peripheral blood. I t is concluded that the lymphoblastoid lines are derivates of non-neoplastic lymphoid cells (perhaps of the B series), which were present as an infiltrate within the astrocytomata. Furthermore, the slow emergence of the lyrnphoblastoid cells in culture, their continuing requirement for feeder cells and their production of monoclonal rather that heteroclonal immunoglobulin are attributed to a n artefact of growth conditions. Key words: Astrocytoma, human; lymphoblastoid cell lines.
E. H . Macintyre, Division of Virology, National Institute for Medical Research, Mill Hill, London N.W.7 IAA, and Clinical Research Centre, Harrow, England.
Received 15.vi.75
Accepted 12.viii.75
A lengthy series of cultures in this laboratory from human lymphoid tissue and from peripheral blood was yielded lymphoblastoid cell lines in a high percentage of cases (Pontkn 1967, Philipson & Ponte'n 1967, Nilsson 1971). The characteristics of such lines have recently been defined and a clear distinction drawn between lymphoblastoid lines composed of normal cells and lymphoma lines consisting of tumour cells (Nilsson & Ponte'n 1975). We report here the culture of lymphoblastoid cell lines from two human brain tumours (astrocytomata). These lines will be shown to be equivalent to cells of lymphoblastoid lines derived from normal lymphoid tissue (Nilsson 1971). In this paper will be
discussed their source within the astrocytomata and the reasons for the rarity of lymphoblastoid line development in the extensive series of consecutive biopsies of brain tumours cultured in this laboratory.
MATERIALS AND METHODS Grid and direct cultures were made of 2 human brain tumours (astrocytomata laboratory numbers 119 and 401) following published techniques ( ] e n sen e t d. 1964, Ponte'n & Macintyre 1968). For tumour 119, samples for grid culture were taken fro the centre of the astrocytoma ( U - l I 9 M G ) and from a grossly visible vessel within the turnour (U-119MG). 1 mm cubes of tissue were set up on stainless steel grids covered by gelatin foam (Spongostan*) and contained in a petri dish whose
*
Obtained from A. B. Ferrosan, Malmo.
79
fluid-gas interface was a t $rid level ( j e n s e n et al. 1 9 6 4 ) . Grid transfers w ~ r rniadr when the ar ea iindrr the grid ( 2 m i 2 ) was covrred hy crlls; th e grid \\as rnovrd tiy forcrps to a new p r tr i dish an d served as a continuing soiircr of non-trypsinised material for srvrral months. Dir r ct preparations for cnltiirr \vrre rnadr without trypsiniaation 1)y arcding into a 50 mni dish a coarse siisprnsion ma de from small fragments of tu m o u r tiy vigorous pipetting. Cells \vere gro\vn on Falcon plastic\vare in Eagle's Minimiim Essential Medium ( M E M ) , snpplementrd by 10 per cr n t foetal calf serum, 100 units of penicillin per nil an d 5 0 pg streptomycin per ml. ' l h e rriltnres \\ere maintained at 3 7 " C in hrimidificd atmosphere containing 5 per r r n t C0,/95 per cent air. Cell transfers \vrrc m ad e at ronfliiencc using 0.25 per c r n t trypsin in phosphatr huffered saline. FID mrdiiim* \vas snlxtitiited for MEM in the gro\vth mediiini latr in the investigation. Similar procrdures Lvrrr follo\ved for th e other astrocytorna, 401, antl its ciiltiires drsignated U-401 MGI,. Cells used as feeders inclndrd U-I 1 9 M G astrocytes, U-2s normal hiiman skin fi1)roblasts (Pontdn K1 Saksela 1967) antl U-79 C G normal h u m an astrocytes (Ponte'n RC M u c i n t y r e 1968). For histology, solid tissiie was fixed in 10 per cent formalin overnight; cell\ in cu ltu r r werr rinsed, then fixed in methanol: acetic acid ( 3 : l ) for one hour. T h e routine stain for solid tissue was haematoxylin-eosin-van Girson an d for cells in culture May-Grunwald-Gienisa. Contamination by PPI.0 was looked for hy exposure of culttires in 60 m m Falcon petri dishes t o 5 pc :'H-thymidine for 24 hours a n d sut)seqiient autoradiography ( N a r done et ul. 1 9 6 5 ) . Inimnnoglol~iilin synthesis was tested rising douhle gel diffusion of concentrated cnltiire snpernatants; medium \vas harvested after three days' inciilmtion from cultures containing a total of 3 x 10' cells; crll-free sn p r r n atan t \vas concentrated t\venty fold an d \tored at 20" C. (Philipson eC; P o n t i n 1 9 6 7 ) . ' I h e preparation of cells for electron microscopy has been described in considcrat)le detail (.\.iucint y r e et al. 1972). Briefly, the cell3 werr fixed for 30 rninntes in a 1 : 2 ( v / v ) mixture of 2.5 per cr n t gliitaraldehyde a n d of 1 per cent osmium tetroxide in 0.1 M cacodylate Iiuffer at p H 7.4 on ice ( I f i r J c h ei Fedorko 1 9 6 8 ) . ~ l ' h einitial fixation was follotved by several rinses with 0.1 M cacodylate buffer at 0" C, then two rinses with cold 0.1 M acetate buffer p H 6.3 containing 0.25 per cent nranyl acetate ( H i r s c h & Fedorko 10681. T h e sample \vas left in contact \vith the final uranyl acetate solntion for 20 rriinntrs a t 0" C , then Lvashed \vith several changes of cacodylate lniffer,
*
O bta ine d from G r a n d Island Biological C o m pany, G r a n d Rapids, Michigan.
80
dehydrated through 70, 80 and 9 7 p r r cent hydroxypropylmethacrylate in \vater (allowing 5 minutes per co n cen t r at i o n ) , and finally infiltrated with a series of hydroxypropylmethacrylate an d Epon mixtnres an d emliedded in Ep o n . Srction\ of vario w preparations \\ere stained with lead acetatt: ( R e y n o l d s 1963 ) to improve contra\t.
R E S U I , '1's
1. Histology Sections of tumour 119 (Fig. 1 ) shokved a high grade astrocytoma: this had an unusually heavy infiltrate of round, mononuclear cells, which were concentrated in perivascular regions, had a single eccentric nucleus and moderately rich, deeply basophilic cytoplasm. Tuinour 401 was an astrocytorna of lesser grade of malignancy and had only a scattering of round, mononuclear cells. At autopsy, the lymphoreticular systems of the 2 patients (119 and 401) showed no abnormal proliferation. 2. Cell Cultures Grid cultures of U-11SMG and U11SMGL grew slowly; it took one month for seeded cells to cover the space under the grids. T h e initial outgrowth was of large, uninucleate cells ; these had abundant, palely staining cytoplasm which contained numerous neuroglial fibrils. They were classed as tumour-derived astrocytes ( S h c i n 1965). Three and a half months after primary explantation, groups of small, round cells allpeared in cultures derived from U-1 IShlGI,: these new cells grew initially in broad heaps of 5-50 cells attached to the astrocytes (Fig. 2 i . Mitoses were frequent within the heaps, and their attachment to the astrocytes was easily broken by vigorous pipetting. As thp size of the heaps increased, round viable cells detached into the medium and reached allpreciable dimensions in older cultures (1-2 x lO'/ml). These detached cells could attarh to feeder cell layers, but not to free surfaces, and did not survive without feeder cells. Cultures of the other grid, U-1 ISMG, and direct cultures of tumour 119 gave only tumour-derived astrocytes.
Fig. I . This section from the original biopsy of tumour shows a vessel, surrounded by many compact round cells and lying within the pleomorphic astrocytoma. T h e endothelial nuclei (arrow) are swollen and hyperplastic. (Van Gieson x 400.)
Fig. 2. Clusters of round refractile cells (arrow) are associated with much larger, but poorly-defined turnour astrocytes (double arrow). Live tissue culture oblique lighting (rnagn. x 40.) 6 Acta path. mictobiol. scand. Sect. A, 84, 1
81
3 Fi
SOME CHARACTERISTICS OF LYMPHOBLASTOID LINES DERIVED FROM HUMAN ASTROCYTOMATA ELIZABETH H. MACINTYRE, ANDERSLINDGREN and JANP O N T ~ N Group of Cell Biology, The Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden
Macintyre, E. H., Lindgren, A. & PontCn, J. Some characteristics of lymphoblastoid lines derived from human astrocytomata. Acta path. microbiol. scand. Sect. A, 84: 79-84, 1976. Two immunoglobulin-producing lines of normal lymphoblastoid cells have been established from cultures of two human astrocytornata. These had the same characteristics as lymphoblastoid lines derived from normal human lymphoid tissue or peripheral blood. I t is concluded that the lymphoblastoid lines are derivates of non-neoplastic lymphoid cells (perhaps of the B series), which were present as an infiltrate within the astrocytomata. Furthermore, the slow emergence of the lyrnphoblastoid cells in culture, their continuing requirement for feeder cells and their production of monoclonal rather that heteroclonal immunoglobulin are attributed to a n artefact of growth conditions. Key words: Astrocytoma, human; lymphoblastoid cell lines.
E. H . Macintyre, Division of Virology, National Institute for Medical Research, Mill Hill, London N.W.7 IAA, and Clinical Research Centre, Harrow, England.
Received 15.vi.75
Accepted 12.viii.75
A lengthy series of cultures in this laboratory from human lymphoid tissue and from peripheral blood was yielded lymphoblastoid cell lines in a high percentage of cases (Pontkn 1967, Philipson & Ponte'n 1967, Nilsson 1971). The characteristics of such lines have recently been defined and a clear distinction drawn between lymphoblastoid lines composed of normal cells and lymphoma lines consisting of tumour cells (Nilsson & Ponte'n 1975). We report here the culture of lymphoblastoid cell lines from two human brain tumours (astrocytomata). These lines will be shown to be equivalent to cells of lymphoblastoid lines derived from normal lymphoid tissue (Nilsson 1971). In this paper will be
discussed their source within the astrocytomata and the reasons for the rarity of lymphoblastoid line development in the extensive series of consecutive biopsies of brain tumours cultured in this laboratory.
MATERIALS AND METHODS Grid and direct cultures were made of 2 human brain tumours (astrocytomata laboratory numbers 119 and 401) following published techniques ( ] e n sen e t d. 1964, Ponte'n & Macintyre 1968). For tumour 119, samples for grid culture were taken fro the centre of the astrocytoma ( U - l I 9 M G ) and from a grossly visible vessel within the turnour (U-119MG). 1 mm cubes of tissue were set up on stainless steel grids covered by gelatin foam (Spongostan*) and contained in a petri dish whose
*
Obtained from A. B. Ferrosan, Malmo.
79
fluid-gas interface was a t $rid level ( j e n s e n et al. 1 9 6 4 ) . Grid transfers w ~ r rniadr when the ar ea iindrr the grid ( 2 m i 2 ) was covrred hy crlls; th e grid \\as rnovrd tiy forcrps to a new p r tr i dish an d served as a continuing soiircr of non-trypsinised material for srvrral months. Dir r ct preparations for cnltiirr \vrre rnadr without trypsiniaation 1)y arcding into a 50 mni dish a coarse siisprnsion ma de from small fragments of tu m o u r tiy vigorous pipetting. Cells \vere gro\vn on Falcon plastic\vare in Eagle's Minimiim Essential Medium ( M E M ) , snpplementrd by 10 per cr n t foetal calf serum, 100 units of penicillin per nil an d 5 0 pg streptomycin per ml. ' l h e rriltnres \\ere maintained at 3 7 " C in hrimidificd atmosphere containing 5 per r r n t C0,/95 per cent air. Cell transfers \vrrc m ad e at ronfliiencc using 0.25 per c r n t trypsin in phosphatr huffered saline. FID mrdiiim* \vas snlxtitiited for MEM in the gro\vth mediiini latr in the investigation. Similar procrdures Lvrrr follo\ved for th e other astrocytorna, 401, antl its ciiltiires drsignated U-401 MGI,. Cells used as feeders inclndrd U-I 1 9 M G astrocytes, U-2s normal hiiman skin fi1)roblasts (Pontdn K1 Saksela 1967) antl U-79 C G normal h u m an astrocytes (Ponte'n RC M u c i n t y r e 1968). For histology, solid tissiie was fixed in 10 per cent formalin overnight; cell\ in cu ltu r r werr rinsed, then fixed in methanol: acetic acid ( 3 : l ) for one hour. T h e routine stain for solid tissue was haematoxylin-eosin-van Girson an d for cells in culture May-Grunwald-Gienisa. Contamination by PPI.0 was looked for hy exposure of culttires in 60 m m Falcon petri dishes t o 5 pc :'H-thymidine for 24 hours a n d sut)seqiient autoradiography ( N a r done et ul. 1 9 6 5 ) . Inimnnoglol~iilin synthesis was tested rising douhle gel diffusion of concentrated cnltiire snpernatants; medium \vas harvested after three days' inciilmtion from cultures containing a total of 3 x 10' cells; crll-free sn p r r n atan t \vas concentrated t\venty fold an d \tored at 20" C. (Philipson eC; P o n t i n 1 9 6 7 ) . ' I h e preparation of cells for electron microscopy has been described in considcrat)le detail (.\.iucint y r e et al. 1972). Briefly, the cell3 werr fixed for 30 rninntes in a 1 : 2 ( v / v ) mixture of 2.5 per cr n t gliitaraldehyde a n d of 1 per cent osmium tetroxide in 0.1 M cacodylate Iiuffer at p H 7.4 on ice ( I f i r J c h ei Fedorko 1 9 6 8 ) . ~ l ' h einitial fixation was follotved by several rinses with 0.1 M cacodylate buffer at 0" C, then two rinses with cold 0.1 M acetate buffer p H 6.3 containing 0.25 per cent nranyl acetate ( H i r s c h & Fedorko 10681. T h e sample \vas left in contact \vith the final uranyl acetate solntion for 20 rriinntrs a t 0" C , then Lvashed \vith several changes of cacodylate lniffer,
*
O bta ine d from G r a n d Island Biological C o m pany, G r a n d Rapids, Michigan.
80
dehydrated through 70, 80 and 9 7 p r r cent hydroxypropylmethacrylate in \vater (allowing 5 minutes per co n cen t r at i o n ) , and finally infiltrated with a series of hydroxypropylmethacrylate an d Epon mixtnres an d emliedded in Ep o n . Srction\ of vario w preparations \\ere stained with lead acetatt: ( R e y n o l d s 1963 ) to improve contra\t.
R E S U I , '1's
1. Histology Sections of tumour 119 (Fig. 1 ) shokved a high grade astrocytoma: this had an unusually heavy infiltrate of round, mononuclear cells, which were concentrated in perivascular regions, had a single eccentric nucleus and moderately rich, deeply basophilic cytoplasm. Tuinour 401 was an astrocytorna of lesser grade of malignancy and had only a scattering of round, mononuclear cells. At autopsy, the lymphoreticular systems of the 2 patients (119 and 401) showed no abnormal proliferation. 2. Cell Cultures Grid cultures of U-11SMG and U11SMGL grew slowly; it took one month for seeded cells to cover the space under the grids. T h e initial outgrowth was of large, uninucleate cells ; these had abundant, palely staining cytoplasm which contained numerous neuroglial fibrils. They were classed as tumour-derived astrocytes ( S h c i n 1965). Three and a half months after primary explantation, groups of small, round cells allpeared in cultures derived from U-1 IShlGI,: these new cells grew initially in broad heaps of 5-50 cells attached to the astrocytes (Fig. 2 i . Mitoses were frequent within the heaps, and their attachment to the astrocytes was easily broken by vigorous pipetting. As thp size of the heaps increased, round viable cells detached into the medium and reached allpreciable dimensions in older cultures (1-2 x lO'/ml). These detached cells could attarh to feeder cell layers, but not to free surfaces, and did not survive without feeder cells. Cultures of the other grid, U-1 ISMG, and direct cultures of tumour 119 gave only tumour-derived astrocytes.
Fig. I . This section from the original biopsy of tumour shows a vessel, surrounded by many compact round cells and lying within the pleomorphic astrocytoma. T h e endothelial nuclei (arrow) are swollen and hyperplastic. (Van Gieson x 400.)
Fig. 2. Clusters of round refractile cells (arrow) are associated with much larger, but poorly-defined turnour astrocytes (double arrow). Live tissue culture oblique lighting (rnagn. x 40.) 6 Acta path. mictobiol. scand. Sect. A, 84, 1
81
3 Fi
