13, 134-141 (1976)
Freezing of Rat Lymphocytes. I. The Effects of Dimethyl Sulfoxide and Freezing on the Phytohemagglutininand Pokeweed Mitogen-Responding Lymphocyte Subpopulations EINAR
The Norwegian Radium Hospital, Oslo 3, Norway
Deep freezing and prolonged storage of human peripheral lymphocytes are now routine procedures, e.g., for use in histocompatibility testing. When evaluated by dye exclusion, post-thaw viability of SO95% has been reported (7, 8, 16, 32). Greater variations have been demonstrated by functional viability tests (2, 3, 6, 21, 23, 30, 31). Somewhat lower figures have been obtained on freezing mouse (1, 1315)) guinea pig (24, 25) and dog lymphocytes (5). There are at least two basic populations of lymphocytes, both in various stages of development. It is therefore important to determine whether these subpopulations are equally sensitive to the freezing procedure, if a group of cells is selectively destroyed, and if the techniques are of significance in this respect. In this laboratory various functions and circulation kinetics of rat lymphocytes have been studied both before and after freezing. This report describes the finding of a higher response of spleen lymphocytes to phytohemagglutinin (PHA) after freezing and a decreased response of the lymph node lymphocytes after freezing. This difference might be explained by the inactivation by freezing of a suppressor cell or cell complex present in the spleen. MATERIALS
Spleen and lymph nodes were collected from our Institute’s inbred strain of hooded Received May 28, 1975.
rats. Three to five animals, of the same sex, 9-11 weeks old, were used in each experiment. The animals in ether anesthesia were killed by exsanguination from the aorta. Spleen and lymph nodes were removed and single cell suspensions were prepared under sterile conditions after fat and adhering tissue had been stripped off. Spleen cells. A needle was inserted into the spleen and slow perfusion with phosphate-buffered saline (PBS, Flow Laboratories, Irvine, Scotland) was continued until the spleens were blanched. They were then gently teased with fine forceps and the released cells suspended in PBS, filtered through fine nylon-gauze mesh and finally separated in a Ficoll/Hypaque gradient (Lymphoprep sp. w. 1077, Nyco, Oslo, Norway) (4). The cells were then washed twice with PBS and once with RPM1 medium 1640 with the standard bicarbonate buffer (RPM1 1640, Flow Laboratories). Each time centrifugation was performed at 140g for 10 min. Lymph node cells. Cervical, axillary, brachial, and mesenterial nodes were prepared the same way as the spleens but with omitting the gradient separation. In both the spleen and lymph node preparations, differential counts revealed more than 99% mononuclear cells. Cell counting was performed by celloscope. Freezing procedure. The samples were frozen in aliquots of 5 ml at a concentralS4
Copyright 1976 by Academic Press, Inc. All rights o8 reproduction in any form reserved.
tion of 10s cells/ml injected into pinholed Teflon/Kapton bags, inner space 50 X 130 mm (Habia KB, Knivstad, Sweden). The freezing medium consisted of RPM1 1640 supplemented with a 25% fresh rat serum (FRS ) . The appropriate amount of dimethyl sulfoxide (DMSO), 50% in RPM1 1640, was added just before heat sealing the bags (330°C for 20 set). They were immediately placed between aluminum plates and immersed in a methanol bath at 0°C in a Hetofrig freezing unit (A/S Heto, Birker$d, Denmark) and cooled at a rate of 1”CJmin until a temperature of -60°C had been reached. The plates were then transferred to liquid nitrogen for 10 min. Temperature recordings were performed with a thermocouple inserted into one control bag with the same freezing medium and cells. The crystallization of water-ice took place between -9 and -12°C elevating the temperature about 7°C for some seconds. However, due to the great surface of this bag, the freezing curve returned to the original slope within 40 set without any elaborate procedure to neutralize the heat effusion. Thawing. The plates were immersed in a water bath at 37°C. Visible ice disappeared within 15 sec. Ten milliliter of cold RPM1 1640 was immediately added to each bag and the samples were centrifugated at 1OOg for 5 min, resuspended in the culture medium, and counted. Viability. This was assessed by [methyl3H] thymidine incorporation after 2 days in culture with mitogens. Mitogens. Lyophilized phytohaemagglutinin-P (PHA-P, Difco, Detroit, Mich. ) was reconstituted with 5 ml of RPM1 1640 and further diluted 1:24 with the same medium. Lyophilized pokeweed mitogen (PWM, Gibco, Grand Island, N.Y.) was reconstituted with 5 ml of medium and further diluted 2:3. The solutions were split into l-ml samples and stored at -25°C until used.
Culture conditions. The cells were resuspended in the culture medium, RPM1 1640 containing penicillin, 100 IU/ml; streptomycin, 100 pg/ml; glutamine, 2 mmol/ml; and 2.5% pooled fresh rat serum (FRS). Cultures of 2 ml containing 0.9-1.1 X 10G fresh or frozen-thawed nuclear cells per milliliter were maintained in a Linbro tissue culture tray (FB-16-24 TC, Linbro, New Haven, Conn. ) . Fifty microliters of the appropriate mitogen was added to give a final concentration of 0.1 and 1% of the reconstituted PHA-P and PWM, respectively. Cultures were always prepared in triplicate and incubated at 37°C in 100% humidity, 92% air and 8% COZ atmosphere. Removal of spleen adherent cells. Twenty milliliters containing 2-2.5 x 10’ cells/ml in the culture medium were incubated at 37°C for 0.5 hr in a 25-cm2 tissue culture flask (Falcon Plastics, California). Thereafter the cells were passed freely through a loosely packed glass wool column of 3 to 4 cm in a 5-ml syringe. (In these experiments RPM1 1640 from Gibcobio-cult, Glasgow, Scotland, was used.) Harvesting. After 45 hr of incubation, 2.5 &i [methyZ-3H]thymidine, specific activity 5 Ci/mmol (Radiochemical Centre, Amersham, England) was added to each sample, and the cultures were maintained for an additional 3 hr. The cultures were then harvested on 0.45-pm Millipore filters and washed four times with cold saline. DNA was precipitated with 5% trichloroacetic acid (TCA) for 30 min and given four additional washes with 5% TCA. The filters were put in plastic scintillation vials and dried at 80°C for 1 hr. Radioactivity was measured by liquid scintillation counting. The data are calculated as mean counts per minute (cpm) per 2 x lo6 nucleated cells in triplicate cultures. The final values are reported as the mean of three or four repeated experiments performed on separate days, plus or minus one standard deviation ( 21 SD).
136 50 t
i 20 ,r F IO t
. F&h No DMSO
caxontralim w-f.) freezing nwdkml
d DMSO in thz
FIG. 1. pH]thymidine incorporation (counts per minute) by PHA-stimulated (-¤-) or PWMstimulated (-A-) and unstimulated (-•-) spleen lymphocytes, fresh or frozen with 5, 10, or 15% DMSO. Verticle bars represent f 1 standard deviation ( * 1 SD ) .
higher [3H]thymidine uptake in the PHAstimulated spleen lymphocytes after freezing. (Fig. 1). In contrast, the frozen PWMstimulated spleen cells gave a lower [3H]thymidine uptake. Lymph node cells gave a different result (Fig. 2) : PHA-stimulated cultures showed lower [ 3H] thymidine uptake after freezing than the fresh cultures. The response to PWM, which was studied only with 10% DMSO, was even more reduced (Fig. 2). The effect of various concentrations of DMSO in the medium during stimulation with PHA and PWM is shown in Figs. 3 and 4. The PHA-stimulated spleen cell cultures showed a higher [3H]thymidine incorporation than their controls when DMSO was added to the medium. With concentration of 1.5% DMSO the average uptake was about five times that of the controls (Fig. 3). The same trend was observed for the lymph node cells but the
The cells were counted before injection into the freezing bag and after the postthaw washing. The numerical recovery of cells was 66-70% with no difference between spleen and lymph node cells and the concentrations of DMSO used. Figures 1 and 2 express the [3H]thymidine incorporation of the spleen and lymph node cell cultures, fresh and frozen with various concentrations of DMSO and with PHA or PWM. The fresh spleen cells cultured with PHA gave somewhat higher [SH]thymidine uptake than those cultured with PWM. This difference between PHA and PWM responses was even more pronounced in the fresh lymph node cultures. The PHA response of frozen cells with various concentrations of DMSO shows that the optimal protective concentration of DMSO was 5-10s for the spleen (Fig. 1) as well as for the lymph node cells (Fig. 2). (In preliminary experiments spleen cells were frozen with 2.5% DMSO which gave very poor protection. ) With an optimal concentration of DMSO there is a
FIG. 2. [‘Hlthymidine incorporation (counts per minute) by PHA-stimulated (-¤-) or PWMstimulated (-A-) and unstimulated (-•-) lymph node lymphocytes, fresh or frozen with 5, 7.5, 10, or 12.5% DMSO. Vertical bars represent -C-1SD.
E 8 soB .g608 g
.r 7 2 1 s-0
Per cent DMSO Ivlv%l in the culture medium
FE. 3. The effect of various concentrations of DMSO on [3H]thynlidine incorporation of unstimulated spleen cell cultures (-•-) and cultures stimulated with PHA (-w-) or PWM (-A-). Vertical bars represent f 1 SD. difference from the controls was not significant (Fig. 4). Exposure of the spleen lymphocytes to 10% DMSO for 5 min at ice-water temperatures followed by one wash with RPM1 1640 gave no increase in [3K]thymidine incorporation after stimulation with PHA or PWM (Fig. 5). PHA-stimulated
FIG. 5. [3H]thymidine incorporation (as perof untreated controls) of spleen lymphocytes exposedto 10% DMSO at ice-water temperacentage
tures for 5 min and stimulated with PHA or PWM. The DMSO was washed out before addition of the mitogens. Vertical bars represent * 1 SD.
cultures of spleen cells where the adherent cell had been removed (Fig. 6) gave more than two times the increase in [gH]thymidine incorporation compared to the control. The [3H]thymidine uptake after PWM stimulation seemed to be unaltered by this procedure. After freezing the nonadherent spleen cells, [ 3H] thymidine incorporation of the PHA-stimulated cultures was about 65% that of the fresh ones. The responses of PWM-stimulated cultures were even more reduced. DISCUSSION
The response of two different sources of rat lymphocytes to the mitogens PHA and PWM have been studied before and after freezing. In the present experiments the cells were kept in liquid nitrogen for only 0
L-+-~~. f-y- 0’25 0: 5 0
10 min to assure a close similarity in the .
Per cent DMSOWv%) in the culture medium FIG. 4. The effect of various concentrations of DMSO on the [SHlthymidine incorporation of un-
stimulated lymph node cell cultures (-•-) and cultures stimulated with PHA (-¤-) or PWM
Vertical bars represent f 1 SD.
thawed lymphocytes. There seems to be general agreement that the procedures of freezing and thawing are of far greater significance for the post-thaw viability than
the time spent at -196°C. In mice, the mitogens PHA and PWM are
Original spleen cells
Frozen non-adkronl spleen cells
FIG. 6. t3H]thymidine incorporation (counts per minute) by unstimulated (open bars), F’HA-stimulated (solid bars) and PWM-stimulated (striped bars) original spleen cells, nonadherent spleen cells and frozen-thawed nonadherent spleen cells. Vertical lines represent 1 SD. -
known to stimulate T and B Iymphocytes, respectively, probably with some degree of overlapping ( 17, 18, 27, 28). Their effect on rat lymphocytes remains more obscure, mainly due to the lack of appropriate Tand B-cell markers in this species. However, several investigations have given convincing evidence for the same effect of these mitogens in the rat as in mice (12, 19, 22, 26). The findings reported here show that the response to PWM is rather similar in fresh cultures of spleen and lymph node cells. Assuming a correlation between the number of responsive cells and the [3H]thymidine incorporation, it might be concluded that fresh spleen and lymph node cultures contain about the same number of PWMresponsive cells. The great difference seen in the response to PHA in fresh cultures from these two cell sources are eliminated after freezing. As expected there is a diminished [SH Jthymidine uptake in the lymph node cultures after freezing. The un-
foreseen increased response in the spleen cell cultures may be explained in several ways: 1. In the present experiment it was found that certain concentrations of DMSO in the medium augmented the PHA response as also found by others using murine spleen cells (29). In the frozen-thawed cultures the DMSO concentration never exceeded 0.08% (v/v). This is far below the concentrations which in our experiments gave a significant increase in the PHA response. The small concentration of DMSO remaining in the culture medium can therefore not explain the higher PHA response of the frozen-thawed cells. This is also supported by the finding that the spleen cells exposed to 10% DMSO, followed by the usual washing procedure, did not show any marked differences in the reactions to PHA or PWM. It seems therefore unlikely that the DMSO is the sole reason for this effect. 2. Another possible explanation would be that more B lymphocytes than T lympho-
RESPONSE AFTER FREEZING
cytes had been killed by freezing, leading to a relative increase of T lymphocytes. In this connection it should be emphasized that the same number of cells has been stimulated in all experiments. Work with prestimulated human peripheral lymphocytes (the cells were exposed to mitogens or specific antigens for 24 hr in culture) has shown that manipulations with the cooling rate can be used to destroy T or B lymphocytes selectively (9, 20). However, as the response to PHA in lymph node cell cultures, having about the same number of PWM-responsive cells (B lymphocytes?) as the spleen cell cultures, are greatly diminished after freezing, enrichment of T cells can not explain the doubled [3H]-thymidine incorporation of the PHA-stimulated spleen cells after freezing. 3. Recent reports have called attention to a suppressor activity of adherent cells in the rat spleen diminishing the PHA response ( 10, 11). It is known from experiments with murine spleen cells that adherent cells are extensively destroyed in the freezing process ( 14). One could therefore postulate that the suppressor activity had been reduced by freezing giving a higher post-thaw response of spleen cells to PHA even though some of the T cells also are damaged in the freezing process. Since this suppressor activity is far less pronounced in the lymph node suspensions (11, 17) it may be assumed that inactivation of the suppressor activity by freezing lymph node cells will not be sufficient to counterbalance the destruction of lymphocytes during this procedure. Accordingly the net result would be a higher [3H]thymidine uptake in the spleen cell cultures in response to PHA after freezing and a lower uptake in the lymph node cell cultures. Our experiments with spleen cells depleted of adherent cells supports this postulation provided the inhibition of PHA response of fresh spleen cells is caused by a small proportion of adherent cells. Since the PHA response after freezing and thaw-
ing does not differ significantly between spleen and lymph node cells, one has to assume (if the above-mentioned correlation between the number of responding cells and the [SH]thymidine incorporation exists here) that after freezing and thawing spleen and lymph node cultures contain the same number of PHA-responsive cells. As there is no reason to assume that PHAresponsive cells from spleen and from lymph nodes would suffer differently from freezing and thawing, this would indicate that fresh spleen and lymph node cell cultures should contain similar numbers of PHA-responsive cells (and of PWM-responsive cells, see above), the spleen cells contaminated with some inhibiting cells, however. The fact that this is only a small proportion is also supported by the fact that removal of adherent cells does not alter the (relative) number of PWM-responsive cells significantly (Fig. 6). If a large proportion of inhibiting cells had been removed selectively by adherence, the relative number of PWM-responsive cells would be considerably increased, resulting in higher counts per minute. This would also be in agreement with the fact that the PHA response of fresh lymph node cells seems not to differ significantly from fresh nonadherent spleen cells (although the experiments given in Fig. 6 are performed at a later time and obviously not fully comparable to those of Figs. 1 and 2). The effect observed with DMSO alone, when added to the culture medium, has also been reported by Strong et al. (29) in a murine cell system. Whether DMSO also has a depressive effect on suppressor cell populations in rat spleen can only be revealed by further studies. SUMMARY
Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide at l”C/min and stored at (DMSO) -196°C for 10 min. The functional recovery of the cell populations was moni-
tored by the mitogenic response (uptake of [ SH] thymidine ) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozenthawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures. A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than lo-fold higher than that present in the culture medium after freezing and thawing. Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations. REFERENCES 1. Ashwood-Smith, M. J. Low temperature preservation of mouse lymphocytes with dimethyl sulfoxide. Blood 23, 494-501 ( 1964). 2. Bouroncle, B. A. Preservation of human normal and leukemic cells with dimethyl sulfoxide at -80°C. Cryobiology 3, 445455 (1967). 3. Brody, J. A., Harlem, M. M., Plank, C. R., and White, L. R. Freezing human peripheral lymphocytes and a technique for culture in monolayers. Proc. Sot. Exp. Biol. Med. 129, 968-972 ( 19F8 ). 4. Bfiyum, A. Separation of leukocytes from blood and bone marrow. Stand. J. Clin. Lab. Znvest. 21, Suppl. 97 (1968). 5. Cavins, J. A., Scheer, S. C., Thomas, E. D., and Ferrebee, J. W. The recovery of lethally irradiated dogs given infusions of autologous leukocytes preserved at -80°C. Bloorl
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