Journal of lmmunological Methods, 18 (1977) 123--132

123

© Elsevier/North-Holland Biomedical Press

A SIMPLIFIED ISOTOPE RELEASE ASSAY FOR CELL-MEDIATED CYTOTOXICITY AGAINST ANCHORAGE DEPENDENT TARGET CELLS

TUOMO TIMONEN ~ and EERO SAKSELA 2

Laboratory of Pathology, I-II Departments of Gynecology and Obstetrics, University Central Hospital, and III Department of Pathology, University of Helsinki, Helsinki, Finland (Received 17 March 1977, accepted 30 May 1977)

An assay for cell-mediated cytotoxicity has been developed in which anchoragedependent target cells are cultured on small plastic beads in suspension. Confluent target cells on the beads are handled by methods appropriate to suspension-grown cells and labelled with chromium-51, iodine-125 and [ 3H ]proline. Fetal human lung fibroblasts and HeLa cells were used as targets in model experiments measuring human natural killer cell activity. In 20 h experiments, chromium-51 was the most suitable isotope. In 40 h experiments, [3H]proline release assay was superior to chromium-51 and iodine-125 assays. The bead cytotoxicity assay offers a rapid and simple isotope release technique for anchorage dependent cells because no trypsinization and re-seeding of target cells is needed.

INTRODUCTION

In the assays for cell-mediated cytotoxicity, the isotope release techniques (Vainio et al., 1964; Holm and Perlman, 1967; Cohen et al., 1971; Bean et al., 1974; Seeger et al., 1974) are most widely used. Compared with the visual techniques (Hellstr6m, 1967; Takasugi and Klein, 1970; HellstrSm and Hellstr6m, 1971} they involve fewer variables, the danger of subjective errors is smaller and counting is less tedious. However, when anchorage-dependent monolayer cells are used as targets, the processing of cells, involving trypsinization and re-plating before effector cells are added, is laborious for large scale studies. We describe an assay for human cell mediated cytotoxicity against anchorage-dependent target cells, in which the plating of trypsinized target cells is unnecessary. MA TER I ALS AND METHODS

Growth m e d i u m

The cells were cultured in Ham's F10 (Flow Laboratories, Irvine, Scotland} supplemented with 10% heat inactivated fetal bovine serum (Flow), 0.29 1 Research fellow of the Finnish Academy, National Research Council for Medical Sciences. 2 Supported by grants from Sigrid Juselius Foundation and the Finnish Cancer Society.

124 mg/ml glutamine, 100 IU/ml penicillin and 10 pg/ml streptomycin. This is designated growth medium below. Culture conditions

All cells were cultured and experiments conducted in humidified air atmosphere with 5% CO2 at 37°C. Monolayer cultures

HeLa cells were cultured in plastic tissue culture flasks (Falcon 3024, Falcon plastics, CA, U.S.A.) and subcultured twice weekly. The methods for initiating fetal lung cell cultures was described previously (Timonen and Saksela, 1976). Briefly, 1 g of lung tissue from fetal autopsies was dissected into approximately 1 mm 3 pieces, suspended in 20 ml of phosphate buffered saline (PBS) with 0.25% trypsin and kept at 20°C on a magnetic stirrer for 45 min. The supernatant was collected and centrifuged, and the resulting pellet was suspended in growth medium and cultured as described above. Cultures on Degalan beads

Degalan beads (Degussa Wolfgang, Hanau, Germany) were placed in 0.1 M HC1 at 20°C for 1 h and subsequently washed extensively with boiling distilled water. The beads were stored in sterilized water. When confluent monolayers of HeLa and fetal lung cells had grown they were trypsinized, and 15 × 106 cells in 20 ml of growth medium were mixed with 2 ml of packed beads. The suspension was pipetted into Falcon 3024 tissue culture flasks and left u n t o u c h e d under culture conditions for three days. The flasks were then shaken to loosen adherent beads and the growth medium was changed. During the next three days the beads were intermittently pipetted into the tissue culture flasks to prevent clustering. After approximately one week the beads were confluently covered b y growing cells and could be used in cytotoxicity experiments. Subsequently the cultures could be maintained by trypsinizing the cells from the beads and adding an equal volume of new beads in fresh medium. This was repeated at approximately one-week intervals and thus a stock of bead-anchored cells was maintained. Effector cells

Forty ml samples of vanous blood from healthy male and female donors were collected into heparinized plastic syringes (Terumo, Jintan, Japan). Mononuclear cells were separated by Ficoll--Isopaque gradient centrifugation (BSyum, 1968), washed three times in PBS, suspended in growth medium and incubated in glass flasks (150 ml, Beatson, Clark & Co, U.K.)

125 for 1 h to remove glass adherent cells. Non-adherent cells were washed twice in growth medium and finally run through nylon wool columns (Julius et al., 1973). The concentration of effector cells was adjusted to 5 X 106/ml. Cytotoxicity experiments

Microcytotoxicity assay. The m e t h o d of Takasugi and Klein (1970) was used for comparison. Trypsinized target cells were seeded on microcytotoxicity plates (Falcon 3034) in the following concentrations: HeLa 100 cells/20 pl growth medium/well and fetal lung cells 200 cells/20 pl growth medium/well, which resulted in 50--150 attached target cells/well after overnight incubation. The effector cell suspension was diluted to give the concentrations of 10 X 104, 5 X 104, 2.5 X 104 and 1.25 X 104/well. Six wells at each effector cell concentration were prepared. The incubation times were 20 h and 40 h, after which the plates were washed three times with PBS to remove non-adherent cells, fixed in methanol, air dried, stained with Giemsa and counted under a light microscope. Per cent c y t o t o x i c i t y was calculated from the following formula: C X% -

MC-- T MC

X 100

where M C = the mean number of remaining cells/well without effector cells (medium control), and T = the mean number of remaining cells/well with effector cells. Plastic bead assay

SlCr-labelled cells as targets: Confluent target cells growing on beads were gently p i p e t t e d into 20 ml glass flasks and washed three times in PBS simply by allowing the beads to sediment by gravity. 100 pl of beads in two ml of PBS were labelled for 1.5 h with 20 pCi of sodium chromate solution (1 mCi Cr 51/ml, specific activity 100--350 pCi/mg, Radiochemical Centre, Amersham, U.K.). After labelling the beads were washed three times in growth medium as above and pipetted into a conical test tube, from which 4 pl volumes of beads were transferred into 5 ml glass test tubes containing either 0.2 ml growth medium (spontaneous and maximal release tubes) or 0.2 ml of the effector cell suspension described above. This gave an effector/ target cell ratio of 30--40/1. Each test was performed in triplicate. The incubation time was 18 h. The experiments were finished by adding 0.8 ml of growth medium to test tubes so that the beads detached from the bottom. The final volume of growth medium was 1 ml, from which after sedimentation of the beads 0.5 ml was pipetted into another test tube. Both tubes were then counted for 100 sec in an automatic gamma counter (Wallac, Turku, Finland). Percentage of released radioactivity (PRR) was calculated

126 f r o m the f o r m u l a : P R R =-

SN×2

SN + BT

× 100

w h e r e S N = c o u n t s in the t u b e with 0.5 ml g r o w t h m e d i u m , and B T = c o u n t s in t h e t u b e with beads and 0.5 ml g r o w t h m e d i u m . Average c y t o t o x i c i t y for t h e triplicate t u b e s was calculated f r o m the f o r m u l a : C ×%-

T--S M--S

× 100

where T = average P R R in tubes with e f f e c t o r cells; S = average P R R in tubes with n o e f f e c t o r cells ( s p o n t a n e o u s release), and M = average P R R in tubes to which had been a d d e d 50 ~1 o f 10% T r i t o n X-100 b e f o r e a d d i t i o n o f 0.8 ml g r o w t h m e d i u m ( m a x i m a l release). ~2SI-labelled ceils as targets: T h e cells were labelled a c c o r d i n g to the m e t h o d described b y Seeger et al. (1974). T h e target cells were washed three times in g r o w t h m e d i u m and p i p e t t e d i n t o a 20 ml d e c a n t e r glass. 100 pl of beads in t w o ml o f g r o w t h m e d i u m were i n c u b a t e d with 10 pCi 5-(~2sI) I o d o - 2 ' - d e o x y u r i d i n e (720 pCi/ml, specific activity 9 0 - - 1 1 0 gCi/mg, Radiochemical Centre, A m e r s h a m , England) for 16 hours. After labelling, the beads were washed t h r e e times in g r o w t h m e d i u m . T h e e x p e r i m e n t a l p r o c e d u r e s were the same as in the c h r o m i u m e x p e r i m e n t s e x c e p t t h a t the i n c u b a t i o n times were 20 h and 40 h. [ 3 H ] p r o l i n e labelled cells as targets: T h e m e t h o d described by Bean et al. ( 1 9 7 4 ) was followed. T h e target cells were washed t h r e e times in BME diploid m e d i u m (Orion, Mankkaa, Finland) s u p p l e m e n t e d with 10% heat inactivated d o n o r calf serum (Flow), 2 m g / m l NaHCO3 and antibiotics as in the g r o w t h m e d i u m . 100 pl beads in t w o ml o f BME diploid m e d i u m were labelled with 50 gCi o f L-(G-3H)proline (677 /~Ci/mmol, R a d i o c h e m i c a l Centre) f o r 16 h, a f t e r which t h e beads were washed t h r e e times in g r o w t h m e d i u m and h a n d l e d as in the iodine e x p e r i m e n t s . The c o n t e n t s o f the tubes were t r a n s f e r r e d into 20-ml scintillation c o u n t i n g t u b e s b y r e p e a t e d l y washing o u t with scintillation liquid (Insta-Gel, Packard I n s t r u m e n t s Co., IL, U.S.A.) the final v o l u m e being 10 ml. T h e t u b e s were c o u n t e d in an a u t o m a t i c liquid scintillation c o u n t e r (Wallac, T u r k u , Finland) for 120 sec. C y t o t o x i e i t y figures were calculated as above. Statistical handling o f the results: S t a n d a r d deviations were calculated for each triplicate in the bead assays and each set o f six wells in the m i c r o c y t o t o x i c i t y assays. T h e d i f f e r e n c e s of m e a n values were analysed with S t u d e n t ' s t-test. P a r t i c l e - b o u n d label as a p e r c e n t a g e o f the released radioactivity: In conv e n t i o n a l c h r o m i u m - 5 1 - r e l e a s e assays, released r a d i o a c t i v i t y is c o u n t e d in c e n t r i f u g e d m e d i u m free f r o m p a r t i c u l a t e material. T h e bead assay does n o t include c e n t r i f u g a t i o n : the p r o c e d u r e c o n c l u d e s with the a d d i t i o n o f excess g r o w t h m e d i u m so t h a t beads d e t a c h f r o m the b o t t o m and the released

127 isotope is dispersed homogenously into the medium. This procedure involves a danger of detachment of living cells from the beads. This possibility was investigated with HeLa cells and all isotopes used. The experiments were finished by serially washing beads with five 0.2-ml volumes of growth medium. The resulting 1 ml of washing medium was centrifuged and 0.5 ml of supernatant was transferred to another tube. The radioactivity in the pellet was calculated by subtracting the counts in the supernatant tube from the counts in the tube with the pellet. The average percentage of particlebound label (pellet) in the total released radioactivity was calculated. Cytotoxicities calculated for the centrifuged and non-centrifuged supernatants were also compared with each other. RESULTS Pilot experiments on effector target cell ratio

A series of experiments was performed to determine the effector cell dose dependence of the natural cytotoxicity in the bead assay. Fifteen replicate tubes containing 4 pl of beads coated with HeLa cells were trypsinized, diluted to 1 ml and the concentration of detached cells counted with a h e m o c y t o m e t e r . The number of cells per tube varied between 23,300 and 33,300 (mean 28,087, standard deviation 3193}. Effector cell donors were selected on the basis of previous experiments and were known to possess low but significant reactivity against fetal fibroblasts in microcytotoxicity experiments, at an effector cell concentration of 10 X 104/well. A dose response curve with one donor's effector cells in four concentrations against chromium-51-1abeled HeLa cells and fetal lung cells is shown in fig. 1. 1 X 106 cells/tube was chosen as a standard effector cell concentration in experiments below. Chromium-51-labelled HeLa cells as targets

The correlation of c y t o t o x i c i t y by the bead assay with that by the microc y t o t o x i c i t y assay is shown in table 1. In general, the effector/target cell ratio of 30--40/1 corresponded to 125/1--500/1 in the microcytotoxicity assay. In experiment no. IV, the high activity in the microcytotoxicity assay was not reflected in the bead assay. This was confirmed in a repeat experiment. The percentage of particle-bound label was investigated in three experiments. 13.5% of the spontaneously released radioactivity and 12.5% of the radioactivity released in the presence of effector cells were in the pellet after centrifugation. The c y t o t o x i c i t y figures calculated from uncentrifuged and centrifuged washing medium were practically identical. 12SI-labelled HeLa cells as targets: As shown in table 2, effector/target cell ratios 1000/1 and 500/1 in the microcytotoxicity assay gave closest agree-

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Fig. 1. The eytotoxic effect of one donor's effector cells against fetal fibroblasts (~ -') and HeLa Cells (o o) growing on 4 pl of beads coated with approximately 30,000 cells. The effector/target ratios are about 10/1--70/1.

m e n t with the results of the bead assay. The high c y t o t o x i c i t y figure at 20 h in e x p e r i m e n t no. I is due to the insufficient lysis of target cells in maximal release tubes. The percentage of particle-bound label was 60.5% of the spontaneously released activity and 67.2% of the radioactivity released in the presence of e f f e c t o r cells. This resulted in no apparent c y t o t o x i c i t y if calculations were based u p o n centrifuged supernatant instead of upon uncentrifuged supernatant. [3H]proline-labelled HeLa cells as targets: The correlation of the bead assay with the m i c r o c y t o t o x i c i t y assay is shown in table 3. E f f e c t o r / t a r g e t cell ratios 2 5 0 / 1 - - 5 0 0 / 1 in the m i c r o c y t o t o x i c i t y assay gave closest agreem e n t with the bead assay with the standard e f f e c t o r / t a r g e t cell ratio. A trend o f relative insensitiveness o f the bead assay with highly active effect or cells (see tables 1 and 2) can be seen in e x p e r i m e n t no. II. The percentages of particle-bound label in the released radioactivity in the spontaneous release tubes and tubes with e f f e c t o r cells were 17.1 and 28.1, respectively. This had no appreciable effect on the c y t o t o x i c i t y figures calculated either for the uncentrifuged or centrifuged washing medium.

Chromiurn-51-labelled fetal fibroblasts as targets The correlation of the bead assay with the m i c r o c y t o t o x i c i t y assay is shown in table 4. Low activity in the bead assay (experiments I--II) corre-

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sponded to an effector/target cell ratio of 1000/1 in the microcytotoxicity assay, whereas high activity (experiments II--IV) was nearest the ratio 250/1. The same trend, although less clear, can be seen in the HeLa cell experiments in table 1. DISCUSSION We have utilized human natural cell-mediated cytotoxicity (Oldham et al., 1973; Takasugi et al., 1973; Peter et al., 1975; Kiuchi and Takasugi, 1976) to develop a convenient technique for isotope release assay using anchoragedependent target cells. This is based on the use of 20--200 pm Degalan beads as growth substrates for the cells. The cells attach readily to the beads and continuous stock cultures can be maintained over extended periods of time by weekly splitting of the cultures by trypsinization and addition of new beads and fresh medium. The main advantage of the method is the ease of preparing replicate target cell tubes without trypsinization and subsequent incubation to allow reattachment. The labelling of the cells with isotopes is simple and effective as demonstrated for 51Cr sodium chromate, 12sI UDR and [3H]proline. Spontaneous release during 18--20 h incubation varied between 26 and 59%, in accordance with previous methods. 12sI UDR and [3H]proline could also be used in 40 h cytotoxicity experiments, the spontaneous release being 47-67%. In our hands modifications involving direct incubation of labelled and trypsinized anchorage-dependent target cells with effector cells in 'forced suspension' has resulted in unacceptably high (70--90%) spontaneous release of radioactivity. The modification presented here has all the practical advantages of similar assays but ensures low spontaneous release, thus allowing a much better resolution of the cytotoxicity effects measured. An additional advantage is the omission of centrifugation at the end of the assay. The possibility that the final procedure might cause detachment of living cells from the beads was investigated by serially washing the beads after standard cytotoxicity experiment with growth medium and centrifuging the resulting washing medium. The sedimentable radioactivity was negligible in the chromium-51 and 3H experiments, but it constituted a major part of the released radioactivity (60--67%) in the 12sI UDR experiments. The sedimentable radioactivity here probably represented ~2SI-labelled intact nuclei of lysed cells, since few viable cells were found in the pellet. The only disadvantage of the bead assay is the relatively inaccurate estimate of effector/target cell ratios. This source of variation is minimized by calculating the percent release of radioactivity separately for each replicate tube. Furthermore, if confluent growth beads are used, the number of target cells is reasonably consistent as demonstrated in the trypsinization experiments: the standard deviation was around 10% of the mean of the number of cells attached to the standard dose of 4 pl of the beads. This source of variation is not greater than that caused by variation in cloning

132

e f f i c i e n c y w h e n t a r g e t cells are seeded in relatively l o w densities on t h e b o t t o m o f micro-wells in s t a n d a r d m i c r o c y t o t o x i c i t y e x p e r i m e n t s . A l t h o u g h t h e b e a d assay c o r r e l a t e d well w i t h t h e m i c r o c y t o t o x i c i t y assay as a w h o l e , in o n e e x p e r i m e n t a high lyric activity in t h e m i c r o c y t o t o x i c i t y assay was n o t r e f l e c t e d in t h e b e a d assay. Also in o t h e r e x p e r i m e n t s the b e a d assay was slightly less sensitive w i t h highly active e f f e c t o r cells t h a n the m i c r o c y t o t o x i c i t y assays. O n e r e a s o n f o r this d i s c r e p a n c y m a y be the fact t h a t i s o t o p e release assays d o n o t m e a s u r e g r o w t h i n h i b i t i o n , a p h e n o m e n o n also involved in t h e e x p r e s s i o n o f c y t o t o x i c i t y against p r o l i f e r a t i n g cells w h e n tests b a s e d on r e m a i n i n g cell n u m b e r s are used (Seeger et al., 1974). It is also possible t h a t e f f e c t o r cells do n o t gain u n i f o r m c o n t a c t with target cells g r o w i n g on all sides o f t h e b e a d s w h e n static i n c u b a t i o n is used so t h a t o p t i m a l l y high c y t o t o x i c i t y is n o t o b t a i n e d . E x p e r i m e n t s on a r o c k i n g s y s t e m m a y t h r o w light on this aspect. We find t h e c h r o m i u m - 5 1 - r e l e a s e assay m o s t useful in 20 h e x p e r i m e n t s b e c a u s e o f t h e s h o r t labeling t i m e r e q u i r e d , t h e h o m o g e n o u s dispersion o f released i s o t o p e into t h e m e d i u m (Mertz, 1 9 7 6 ) a n d t h e ease o f g a m m a c o u n t i n g . In 40 h e x p e r i m e n t s , [ 3 H ] p r o l i n e assay is p r e f e r a b l e b e c a u s e o f l o w s p o n t a n e o u s release a n d b e c a u s e v e r y little o f t h e released r a d i o a c t i v i t y is in a p a r t i c u l a t e f o r m . REFERENCES Bean, M.A., H. Pees, J.E. Fogh, H. Grabstald and H.F. Oettgen, 1974, Int. J. Cancer 13, 697. BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21, supplement 97, 77. Cohen, A.M., J.F. Burdick and A.S. Ketcham, 1971, J. Immunol. 107,895. Hellstr6m, I., 1967, Int. J. Cancer 2, 65. Hellstr6m, I. and K.E. HellstrSm, 1971, In vitro methods in cell mediated immunity (B.R. Bloom and P.R. Glade, eds.) Academic Press, N.Y., p. 409. Holm, G. and P. Perlman, 1967, Immunology 12, 525. Julius, M.H., E. Simpson and L.A. Herzenberg, 1973, Eur. J. Immunol. 3,645. Kiuchi, M. and M. Takasugi, 1976, J. Nat. Cancer Inst. 56, 575. Mertz, E., 1976, Cell. Immunol. 26, 313. Oldham, R.K., D. Siwarski, I.L. McCoy, E.I. Plata and R.B. Verberman, 1973, I. Nat. Cancer Inst. Monogr. 37, 49. Peter, H.H., J. Pavie-Fischer, W.H. Fridman, C. Aubert, J.P. Cesarini, R. Roubin and F.M. Kourilsky, 1975, J. Immunol. 115,539. Seeger, R.C., S.A. Rayner and J.J.T. Owen, 1974, Int. J. Cancer 13,697. Takasugi, M. and E. Klein, 1970, Transplantation 9, 219. Takasugi, M., M.R. Mickey and P.I. Terasaki, 1973, Cancer Res. 33, 2898. Timonen, T. and E. Saksela, 1976, Clin. Exp. Immunol. 23,462. Vainio, T., O. Koskimies, H. Perlman and G. Klein, 1964, Nature Lond. 204,453.