A new ultra-microculture system. I. Stimulation of human T lymphocytes by phytohemagglutinin (PHA)

Journal oflmmunologicalMethods, 68 (1984) 285-295

285

Elsevier JIM03013

A New Ultra-Microculture System. I. Stimulation of Human T Lymphocytes by Phytohemagglutinin (PHA) A.J. U l m e r , W. Scholz a n d H . - D . F l a d Forschungsinstitut Borstel, D- 2061 Borstel, F.R.G.

(Received 18 July 1983, accepted 1 December 1983)

We have developed an ultra-microtechnique for culturing lymphocytes in glass capillary tubes at a final culture volume of I #1 or 2 ~1. The advantage of the method is that a substantially lower number of cells and minute amounts of culture medium are required. The cultures are premixed in microtubes, sucked into glass capillary tubes and incubated for an appropriate culture period. For determination of [3H]thymidine ([3H]TdR) incorporation, the cells are transferred into the wells of microtiter plates. Some special accessories have been developed which allow routine use of this system for large numbers of cultures. Optimal culture conditions for stimulation of human T lymphocytes by PHA are described. Key words: ultra-microculture - T lymphocytes - phytohemagglutinin

Introduction During the last 10 years lymphocyte culture techniques have been progressively minified. In the early '70s in vitro culture of lymphocytes was performed in glass or plastic tubes in volumes of 1-3 ml. Later the use of multiwell microtiter plates for lymphocyte cultures provided a culture system using volumes of 100-200 ~1 (Brody and Huntley, 1965; Hartzmann et al., 1971). In Terasaki microtest plates lymphocytes may be cultured in volumes of 10-20 #1 (Pena-Martinez and Festenstein, 1975; O'Brien et al., 1979). We have now further reduced the culture volume to 1 #1 by the use of glass capillary tubes as culture vessels. This ultra-microculture has the advantage that much less chemical and biological material is required. Furthermore, we believe this ultra-microculture system is the first described that offers opportunities to investigate accessory cell functions under limiting dilution conditions without the presence of feeder cells (Ulmer et al., 1983). A disadvantage is that it is more time consuming than other micromethods. This paper deals with the development of ultra-microculture of phytohemagg0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

286 lutinin (PHA) stimulated human peripheral blood mononuclear cells (MNC) or human E-rosette forming cells (E-RFC) as responder cells, and [3H]thymidine ([3H]TdR) incorporation for measurement of proliferative responses.

Materials and Methods

Materials RPMI 1640 medium and Hanks' BSS were prepared from powdered media (Seromed, Berlin). RPMI 1640 medium was supplemented with 100 U / m l penicillin and 100 #g/ml streptomycin. Fetal calf serum was obtained from Seromed (Berlin). The serum was inactivated by heating at 56°C for 30 min. Percoll and Ficoll 400 were purchased from Deutsche Pharmacia (Freiburg), and PHA (reagent grade) from Wellcome (Beckenham, Kent). Microtiter plates (M24A) and 'Vitatron' microtubes were supplied by Greiner (N0rtingen). Most of the experiments were performed with glass capillary tubes (disposable capillary pipettes, 20 #1) (Assistent, Sondheim/Rhrn, or R. Brand, Wertheim/Main). These capillary tubes were washed in an ultrasonic bath at 80°C for 1/2 h with Neodisher UW (Chemische Fabrik Dr. Weigert, Hamburg), rinsed with tap and purified water and heated again in an ultrasonic bath at 80°C for 1/2 h without detergent. After rinsing with water purified by reverse osmosis and deionization, the tubes were dried at 70°C and sterilized at 180°C for 2 h.

Accessories for the ultra-microculture system Ultra micropipettes To suck the cell suspension into the glass capillary tubes, we used 1 #1 or 2 #1 Oxford ultra-micropipette (Lancer, Munich) bored open (tip and barrel insert) so that glass capillaries could be inserted. For larger volumes we used a microtiter pipette (Gilson Pipetman P 20, Ahmed, DOsseldorf) with a tip made of silicon tubing (3.0 mm x 5.0 mm) with a silicon tube insert (1.5 mm x 4.0 mm). Capillary holder The glass capillaries were inserted for culture into the grooves of multiple capillary racks made of plexiglass as shown in Fig. 1. Perforated cover for microtiter plate To transfer the culture from the glass capillaries to the microtiter plate, we prepared a special cover device by piercing 2 mm holes over the positions of the wells. Adapter for multiple microtiter pipettes To blow the cell cultures out of the glass capillaries into the microtiter plate with a multiple microtiter pipette (Flow Laboratories, Meckenheim), a special adapter

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Cells Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood by density gradient centrifugation on Percoll (d = 1.077 g/ml). E-rosette forming cells (E-RFC) and adherent cells were isolated from PBMC as described previously (Ulmer and Flad, 1982). Cell culture The cell cultures were premixed in 'Vitatron' microtubes (Greiner, Niirtingen) at volumes of 10-100 td. Under standard culture conditions 5-50 × 105 ceUs/ml were suspended in RPMI 1640 medium containing 8 td/ml PHA and 20% heat-inactivated FCS. Aliquots of 1 ttl or 2/~1 were sucked into glass capillaries by means of the adapted Oxford ultra-micropipette. The cell suspensions were sucked further inside the tubes on removing the capillaries from the pipette. The medium-contaminated tip of the glass capillaries was wiped clean with a sterile swab and sealed with hematocrit sealing clay. Although the other end of the tubes was not sealed, the cultures remained sterile presumably due to the small diameter of the capillaries and the localization of the culture volume away from the end of the capillary. The capillary cultures were inserted in groups of 12 into the special racks (Fig. 1) and incubated for different culture periods in a horizontal position at 37°C in a humidified atmosphere of 5% CO 2.

Determination of 3HTdR incorporation For determining [3H]TdR incorporation, the cultures had to be transferred into the wells of a microtiter plate. The sealed ends of the capillary tubes were cut off with a glass cutting knife and the capillary tubes (still in the rack) were inserted through the perforated cover into the wells of 1 row of a microtiter plate, each well containing 100 gl of RPMI 1640 medium + 10% FCS. With a 12-channel microtiter pipette (Flow, Meckenheim) with the special adapter device (Fig. 2) the cells were blown out, and the capillary tubes washed out by 10 strokes of the pipette. The cells were pulsed with [3H]TdR (0.2/~Ci/culture, 2 Ci/mM) for 5 h and processed for measurement of [3H]TdR incorporation as described elsewhere (Ulmer and Flad, 1982).

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Fig. 3. Selection of capillary tubes for culture of human T lymphocytes. T cells (1 X 105/ml up to 10 x 105/ml) were stimulated with PHA at a volume of 1/~1 in various capillary tubes (capillary pipettes 10 /~1 and capillary pipettes 20 FI, both from B. Brand, capillary pipettes 50/100 /~1 from Labora, Mannheim). [3H]TdR incorporation was determined after 4 days culture. Each value is the mean ± SD of 3 cultures.

Results

Selection of type of glass capillaries In p r e l i m i n a r y e x p e r i m e n t s we tested various glass c a p i l l a r y p i p e t t e s (including the d i s p o s a b l e plastic c a p i l l a r y tip of the O x f o r d u l t r a - m i c r o p i p e t t e ) for culturing h u m a n T l y m p h o c y t e s in a m i n i m a l v o l u m e a n d using a m i n i m u m n u m b e r of cells. T h e results of I representative e x p e r i m e n t are given in Fig. 3. T h e o p t i m a l r e s p o n s e 15

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290

was obtained with 20/~1 capillary pipettes. With the different types of capillary tubes used it was not possible to reduce the cell concentration necessary for stimulation of T lymphocytes to less than 5 × 105 cells/ml. Because 20/~1 capillary pipettes gave the best results and were also most practicable to handle, we selected them for the ultra-microculture procedure. As shown in Fig. 4, T lymphocytes may be stimulated in these tubes at culture volumes of 1 #1 or 2 #1. Higher culture volumes (5 #1 or 10 ~tl) resulted in reduction of the proliferative response. Cell culture conditions P H A concentration. T lymphocytes were stimulated by PHA at various concentrations in ultra-microculture. As shown in Fig. 5, the optimal response of T lymphocytes to PHA was seen at a concentration of 8 #1 P H A / m l . The cells were cultured at concentrations of 10 × 105/ml or 20 × 10S/ml. At 5 × 105 T lymphocytes/ml, the responses to 4 - 1 6 / ~ g / m l of PHA were the same. Again, no response to any PHA concentration tested was found at a cell concentration of 2 × 105/ml. From these results we selected a PHA concentration of 8/~l/ml for standard culture conditions. Serum concentration. T lymphocytes were stimulated with PHA at various concentrations of FCS. The results show (Fig. 6) that higher numbers of cells required a lower concentration of FCS for optimal proliferative response. For stimulation of T cells at a concentration of 50 × 105/ml, addition of serum was n o t necessary. Because the addition of 20% FCS resulted in an almost optimal response to PHA at all cell concentrations tested, we chose this amount of FCS for use under standard culture conditions. Cell concentration and kinetics. As Fig. 3 and Fig. 5 show no response to PHA was observed after stimulation of T lymphocytes at a concentration of less than 5 × 105 cells/ml. Stimulation of the lymphocytes by PHA at 5 X 105 cells/ml or

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292

more resulted in characteristic kinetics of responses depending on the type of cells and the number of cells cultured (Fig. 7). First, it was found that the higher the number of cells stimulated by PHA in the ultra-microculture, the shorter the culture period giving a peak response of DNA synthesis. Second, the peak response of the cells depended on the number of monocytes or adherent cells present during activation; MNC or T lymphocytes supplemented with 20% of adherent cells responded maximally after 2 days culture at a cell concentration of 50 × 105 cells/ml and maximally after 5 days culture at 5 × 105 cells/ml. On the other hand, the kinetics of DNA synthesis of purified T lymphocytes cultured at different cell concentrations were only slightly different (Fig. 7). Optimal responses were noted at day 4 of culture (10 × 105 cells/ml up to 50 × 105 cells/ml) or at day 5 (5 × 105 cells/ml).

Proliferation of the cells T lymphocytes were cultured in ultra-microculture and the number of viable (trypan blue excluding) cells counted daily (Fig. 8). After setting up 10 × 105 cells/ml (2000 cells/culture) without PHA, the number of living cells in the culture remained constant until culture day 4 and then declined at a linear rate almost to zero by culture day 10. In the presence of PHA the number of living cells decreased

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293 somewhat during the first 3 days of culture but increased during the next 3 days, giving the highest number of living cells at culture day 6. Different results were observed when culturing T cells at a concentration of 50 × 105 cells/ml (10000 cells/culture). In the presence of PHA the number of viable cells in the ultra-microculture decreased rapidly during the first days to less than 4000 living cells per culture and further decreased during the following culture period. In the absence of PHA no alteration of the number of living cells was observed at any day of culture tested.

Observation of the cells in the capillary tube In ultra-microculture, T lymphocytes could be directly observed with an inverted microscope. In the absence of PHA the cells lay in a line at the bottom of the capillary tube (Fig. 9a), but in presence of PHA the cells formed aggregates (Fig. 9b). For higher magnification (e.g., 40 X objective) immersion oil has to be put between the objective and the capillary tube for distortion-free observation (data not given).

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294

Discussion

We have shown that human T lymphocytes may be cultured in capillary tubes in a volume of 1 /~1 at 1000 cells per culture or even less. A culture volume in the capillary tube of up to 2 ttl was optimal, higher volumes resulting in a reduction of cell growth. This may be due to insufficient gas exchange at the sealed end of the capillary tube. The cells should not be cultured for periods longer than 7-10 days, since the cultures may disintegrate for unknown reasons. With the special equipment as described, routine work became possible. We have run experiments with up to 600 capillary tube cultures. Kinetic studies on the stimulation of DNA synthesis of T lymphocytes by PHA at different cell concentrations showed that the thymidine uptake curve was affected by several parameters (Fig. 7). Thus we found that both higher numbers of cells and higher numbers of adherent or phagocytic cells in the culture shortened the culture period necessary for maximal response and accelerated the decline of [3H]TdR uptake following the peak response. These inhibitory effects are presumably due to monocytes but we have not observed them in cultures in microtiter plates. This suppressive effect cannot be abolished by the addition of indomethacin (data not given), indicating that it is not mediated by prostaglandins. Other inhibitory effects mediated by monocytes/macrophages have been summarized by Allison (1978). Which of these is active in ultra-microculture remains to be investigated. As well as DNA synthesis, cell survival and proliferation were followed after stimulation of T lymphocytes by PHA in the ultra-microculture. Pappenheim staining of PHA-stimulated T lymphocytes after 4 days culture showed some cells in mitosis (data not given). In cultures of 2000 PHA stimulated T lymphocytes the number of living cells doubled (Fig. 8). The kinetics of cell proliferation are compatible with the kinetics of DNA synthesis (Fig. 7). However, in cultures of 104 PHA stimulated T lymphocytes the number of viable cells did not increase. At first sight this seems to be incompatible with the results in Fig. 7 but may be due to the number of dying cells being higher than the number of proliferating cells. The morphology and number of cells may be observed without distortion in the capillary tube by the use of an inverted microscope and immersion oil, providing an opportunity to follow their growth in culture. Because the number of cells is low (about 1000 per culture), each individual cell in a culture may be examined. In the presence of PHA the cells form aggregates resembling T lymphocyte colonies in agar culture (Ulmer and Flad, 1979). Presumably these aggregates were due to the agglutinating property of the lectin, and enlarged through cell proliferation. In summary, this paper deals with the handling of a new ultra-microculture system for assaying stimulation of human T lymphocytes by PHA. This system, which can be performed in a volume of only 1/~1, is convenient when the relevant biological or chemical material is limited. Whether it is also suitable for culturing cells other than T lymphocytes remains to be investigated.

295

Acknowledgements The skilful technical assistance of Mrs. A. Vorreiter and Mrs. S. Finnern is gratefully acknowledged. We thank Mrs. R. Hinz for typing the manuscript and Mrs. M. Lohs for drawing the graphs. This work was supported by Deutsche Forschungsgemeinschaft (F1 104/4-1).

References Allison, A.C., 1978, Immunol. Rev. 40, 4 Brody, J.A. and B. Huntley, 1965, Nature (London) 208, 1232 Hartzmann, R.J., M. Segail, M.L. Bach and F.H. Bach, 1971, Transplantation 11,268 O'Brien, J., S. Knight, N.A. Quick, E.H. Moore and A.S. Platt, 1979, J. Immunol. Methods 27, 219 Pena-Martinez, J. and H. Festenstein, 1975, Transplantation 20, 26 Ulmer, A.J. and H.-D. Flad, 1979, Immunology 38, 393 Ulmer, A.J. and H.-D. Flad, 1982, Immunobiology 161,476 Ulmer, A. and H.R. Maurer, 1978, Immunology 34, 919 Ulmer, A.J., H.-D. Flad and H.-G. Opitz, 1981, J. Immunol. Methods 40, 27 Ulmer, A.J., W. Scholz, M. Ernst and H.-D. Flad, 1983, Eur. J. Cell. Biol. Suppl. 2, 42