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Cryopreservation of equine mononuclear cells for immunological studies
Veterinary Immunology and lmmunopathology, 25 (1990) 139-153 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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Cryopreservation of Equine Mononuclear Cells for Immunological Studies
R.E. TRUAX ~, M.D. POWELL ~, R.C. MONTELARO 2, C.J. ISSEL ~':~and M.J. NEWMAN TM
1Veterinary Microbiology and Parasitology, School o[ Veterinary Medicine and 2Department o[ Biochemistry, College of Basic Sciences, Louisiana State University and :~Department of Veterinary Science, Louisiana Agricultural Experiment Station, Baton Rouge, LA 70803 (U.S.A.) (Accepted 23 November 1989)
ABSTRACT Truax, R.E., Powell, M.D., Montelaro, R.C., Issel, C.J. and Newman, M.J., 1990. Cryopreservation of equine mononuclear cells for immunological studies. Vet. Immunol. Immunopathol., 25: 139153. A rapid and simple technique for the cryopreservation and recover)' of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in au~)logous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved huffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time. 4present address and to whom all correspondence should be sent: Cambridge BioScience Corporation, 365 Plantation Street, Worcester, MA 01605 (U.S.A.).
0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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INTRODUCTION Cryopreservation of human mononuclear cells is done routinely as a standard part of biomedical research programs. The most common parameters of various cryopreservation methods are the use of cryo-protectant compounds, such as dimethylsulphoxide (DMSO), and a controlled slow rate of freezing. Frozen cellular preparations are routinely stored in the vapor phase in liquidnitrogen freezers or in ultra-low temperature ( - 135 °C) electric freezers. Cryopreservation procedures were primarily developed for use with human blood mononuclear cells. This methodology has allowed investigators to obtain cellular materials from patients and control volunteers when they are available and to use them for in vitro assays at later times (Sears and Rosenberg, 1975; Jewett et al., 1976). These techniques have been used most extensively for studies in the area of clinical immunology where cellular immune functions have been measured successfully using cryopreserved peripheral blood mononuclear cells. Cryopreserved human mononuclear cells retain their ability to express histocompatibility antigens (Strong et al., 1977, 1978) and subset specific differentiation antigens (Slease et al., 1980; Strong et al., 1982; Fujiwara et al., 1986; Prince and Lee, 1986 ). Cell-mediated immune parameters retained by cryopreserved mononuclear cells included lymphocyte proliferation in response to mitogens, lymphokines and antigens (Factor et al., 1975; Strong et al., 1975; Strong, 1976; Pappas et al., 1979; Gramatzki et al., 1982), NK-cell and antigen specific cell-mediated cytotoxicity (Factor et al., 1975; Strong et al., 1982 ), antibody dependent cell-mediated cytotoxicity (Strong et al., 1982 ), chemotaxis (Dean and Strong, 1977 ) and the production of cytokines (Munoz et al., 1987). The use of cryopreserved mononuclear cells for research with domestic animals has been very limited. Gradient-purified lymphocytes have been cryopreserved for use in histocompatibility testing systems with the dog (Netzel et al., 1975; Weaver et al., 1975) and the sheep (Stear et al., 1982). Bovine lymphocytes have been cryopreserved and shown to retain responsiveness to mitogens (Kleinschuster et al., 1979; Kuil, 1984). However, all of these studies have been very limited in scope and the use of cryopreserved cellular materials has not gained widespread acceptance. The use of this methodology would be of great value because cells could be stored for multiple controlled testing and exchanged with other investigators. We have successfully used the methods of cryopreservation, that were developed for use with human cells, to freeze gradient-purified equine lymphocytes. However, the purification of mononuclear cells from peripheral blood is laborious and time-consuming and this has limited the number of samples that we can process routinely. We have therefore developed a technique for cryopreserving equine leukocytes as buffy-coat leukocyte preparations. Our method has proven to be rapid and technically simple, thus allowing one person to process a large number of blood samples.
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Mononuclear cells were routinely recovered in a viable state, capable of responding to mitogens and antigens. The development and characterization of this method is described in this report. MATERIALSAND METHODS
Test animals and collection of blood samples Eight healthy Shetland ponies, 1-3 years of age, were used for the study. They were maintained on pasture with free choice hay, water and mineral supplementation. All ponies were vaccinated against anthrax, encephalitis, equine herpes virus-l, strangles and influenza (Equi-1 and Equi-2 ) prior to inclusion into the study. Anthelmintic treatments were done at 12-week intervals. Whole blood samples, 100 ml, were collected from ponies at two different times using standard aseptic venipuncture techniques and preservative free heparin ( Sigma Chemical Co., St. Louis, MO) at the final concentration of 10 U/ml. Blood samples were processed within 4 h of collection.
Cryopreservation of bully-coat leukocytes Blood samples were centrifuged at 400g at room temperature for 15 min. All but 1 ml of the plasma was removed using sterile techniques and an aliquot was mixed with dimethyl sulfoxide {DMSO; Sigma) to a final concentration of 20% (v/v) DMSO and 80% {v/v) autologous plasma. One ml of this DMSOautologous plasma mixture was prepared for every 20 ml of whole blood and placed on ice. The buffy-coat leukocyte layer was recovered in the remaining autologous plasma, resuspended and put on ice for 30 min. Equal quantities of buffy-coat leukocytes and DMSO-autologous plasma were mixed and dispensed into polypropylene vials (Sarstedt, Princeton, NJ), all materials and vials were kept on ice. Each vial contained 1 ml of cell suspension from 10 ml of whole blood in autologous plasma plus 10% {v/v) DMSO. Leukocyte samples were frozen using a programmable controlled rate freezer (KRYO-10; T.S. Scientific, Quakertown, PA) and several different freezing rates, as described (Strong et al., 1982; Fujiwara et al., 1986; Prince and Lee, 1986; Munoz et al., 1987). Frozen samples were stored for up to 14 months in the vapour phase in a liquid-nitrogen freezer until thawed for experimental purposes. Leukocyte samples were thawed quickly by placing vials in a 37°C water bath. Thawed samples were immediately transferred to a 15-ml cell-culture tube and diluted slowly to a total volume of 10 ml using cold Ca ~+ and Mg ~+ free Hank's balanced salt solution (HBSS, pH 7.4). The diluted leukocyte mixture was layered onto 4 ml of Histopaque-1077 (Sigma) and the gradients centrifuged at 400g for 30 min at room temperature. The mononuclear cell layer was recovered from the Histopaque-HBSS interface and the cells washed three times at room temperature using HBSS. Mononuclear cells were resus-
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pended at a concentration of 1-2>< 106 cells/ml in RPMI-1640 buffered with NaHCO3 and supplemented with 10% (v/v) fetal bovine serum (GIBCO, Grand Island, NY ), 2 m M L-glutamine, 10 pJV/2-mercaptoethanol and 50 ]~g/ml gentamicin sulfate (Schering Corp., Kenilworth, NJ). Cell viability was assessed using standard trypan exclusion criteria.
Measurement of mitogen and antigen induced cellular proliferation The ability of mononuclear cells to respond to polyclonal stimulation was compared using standard in vitro assays and three mitogens, phytohemagglutinin (PHA; Sigma), concanavalin A (Con A; Sigma) and pokeweed mitogen (PWM; Sigma). Mononuclear cells were cultured at a concentration of 1 × 106/ ml in supplemented RPMI-1640 cell culture media using round-bottom 96-well microculture plates (CoStar, Cambridge, MA) and 200 ~1 total culture volumes, were used for all culture experiments. Cultures were maintained at 37 °C in a humid incubator with 5% CO,~ in air. The concentration of mitogens was varied; PHA and P W M were used at concentrations of 1, 2, 4 and 8 ttg/ml, Con A was used at concentrations of 2.5, 5, 10 and 20 ttg/ml. Total culture durations of 72, 96, 120 and 144 h were used. One ~Ci of [3H]thymidine (ICN Radiochemicals, Irvine, CA) was added to all wells 16 h prior to the termination of cultures. Cellular proliferation was quantitated by determining the amount of isotope incorporated into dividing mononuclear cells by liquid scintillation counting. All tests were run using quadruplicate cultures and experiments were run twice at different times. The ability of cryopreserved and fresh mononuclear cells to profilerate in response to specific antigens was assessed using a similar assay system and equine influenza-2 (Equi-2, A/equine/Miami/63; American Type Culture Collection, Rockville, MD) as the test antigen. The Equi-2 antigen preparation used for our in vitro testing consisted of intact virus particles, grown in 11-day-old embryonated hens' eggs and purified from allantoic fluid using a 14-40% {w/v) sucrose gradient (Hinshaw et al., 1983). Virus titers were determined by standard hemagglutination assay using chicken erythrocytes (Habel and Salzman, 1969; Hinshaw et al., 1983) and were expressed as hemagglutination units (HA units). The Equi-2 preparation was used at concentrations of 5, 2.5, 1.25, 0.61 and 0.3 HA units/ml in 200 ~l cultures containing mononuclear cells at a final concentration of 2 ><106 cells/ml.
Flow cytometric quantitation of cell-surface expressed antigens The expression of cell "type-specific" or differentiation antigens on fresh and cryopreserved equine mononuclear cells was assessed using monoclonal antibodies and fluorescence activated flow cytometry. Selected routine monoclonal antibodies which bind to mature equine T-lymphocytes (EqT-2 and EqT-3) and immature T-lymphocytes (EqT-7 and EqT-12) were purchased commercially (VMRD, Pullman WA). The production and characterization
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of these monoclonal antibodies is reviewed elsewhere (Magnuson et al., 1987; Wyatt et al., 1988). The F-39.2 monoclonal antibody was produced in a rat and recognizes equine major histocompatibility (ELA) class-II antigens (Newman et al., 1984; Crepaldi et al., 1986). Gradient-purified mononuclear cells were resuspended in phosphate-buffered saline (PBS), pH 7.4, containing 10% (v/v) normal goat serum (NGS), to a concentration of 107 cells/ml and placed on ice for 20 min. Optimally diluted monoclonal antibodies were added to 100/~l aliquots of the cells and incubated for 1 h on ice. The cells were washed twice using cold PBS and then resuspended in 100 pl of optimal dilutions of FITC-conjugated anti-immunoglobulin reagents (Sigma) e.g., affinity purified goat IgG specific to either mouse IgG (H and L chains) or to rat IgG (H and L chains). The cells were incubated for 1 h on ice, washed twice and resuspended in 200/~l of 2% (w/v) paraformaldehyde in PBS. Antibody labeled mononuclear cells were analyzed using a fluorescence activated cell sorter (FACS-440; Becton-Dickinson, San Jose, CA). Data collection through the FACS-440 was triggered using only forward light scatter but list mode data were collected for forward light scatter, 90 ° light scatter and green fluorescence emission parameters. The data were analyzed using a DEC Micro-VAX II workstation (Digital Equipment Corporation, Westminster, MA ) and the Consort-40 flow cytometry program package (Becton-Dickinson).
Histocompatability typing of mononuclear cells Equine histocompatibility class-I antigen types (ELA) were determined using alloantisera defined in international workshops (Bernoco et al., 1987; Lazary et al., 1988) and the microlymphocytotoxicity test (Newman and Antczak, 1983). Both fresh and cryopreserved mononuclear cells were typed to determine the extent to which cryopreservation alters ELA class-I antigen expression. RESULTS
Initial tests were run to identify the optimal freezing rate for equine mononuclear cells in our system by determining the total recovery and viability of mononuclear cells from cryopreserved samples after using the various freezing rates identified in the literature. Cellular viability was generally 95% or greater with no significant differences between different freezing rates and fresh blood. The total numbers of mononuclear cells which were recovered did vary greatly when different freezing rates were used. The freezing rate which consistently gave the highest recovery rates was as follows: (1) samples were held at 4 ° C for 5 min, (2) the temperature was dropped 0.5 ° C/min until the sample temperature reached - 4 0 ° C, (3) the temperature was dropped 1.5 °C/min until the sample temperature reached - 60 ° C, (4 } samples were then placed directly
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into the vapor phase in a liquid-nitrogen storage freezer, approximate temperature of - 120 °C. This freezing procedure was used for all subsequent testing. Using this method we could routinely recover 1-4 × 106 mononuclear cells from each ml of whole blood. Controlled comparison of these numbers to those obtained using fresh whole blood demonstrated that 6-18% fewer cells were recovered from the cryopreserved samples. Cryopreserved mononuclear cells proliferated in response to all of the mitogens used. Maximum proliferative responses were generated in cultures of both fresh and cryopreserved cells using the same concentrations of mitogens. Significant differences between the responses of fresh and cryopreserved samples were not common. Representative proliferation data for five ponies and the three mitogens are shown in Figs. 1-3. Analysis of the mitogen induced proliferation data with respect to culture duration demonstrated that cryoprePONY ID *~ 7 1 6 0 717&
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Fig. 1. Proliferative responses of fresh and cryopreserved equine mononuclear cells from five representative ponies in response to various concentrations of PHA. Data are expressed as counts/ rain {log), actual data points represent the mean +_ 1 standard deviation of quadruplicate tests. Total culture time was 72 h. The results shown were obtained using cells from a single blood collection. Samples were processed within 4 h for cryopreservation and for cell culture. The assays using cryopreserved cells were run 2-6 months later. Fig. 2. Proliferative responses of fresh and cryopreserved equine mononuclear cells from five representative ponies in response to various concentrations of Con A. Data are represented as described in Fig. 1. Total culture time was 96 h.
CRYOPRESERVATIONOF EQUINEMONONUCLEARCELLSFOR IMMUNOLOGICALSTUDIES
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served mononuclear cells responded more slowly than fresh cells. Generally, an additional day in culture was required for cryopreserved cells to reach the highest level of proliferation for all of the mitogens tested. This is shown in Fig. 4 for two representative ponies. Cryopreserved mononuclear cells were also fully capable of proliferating in response to specific antigens. Data from two representative animals are shown in Fig. 5. Interestingly, there was variation in the total response levels using fresh mononuclear cells from different ponies. In Fig. 5 we showed data from a high responder pony (834) and a low responder pony {841 ). This same difference in response levels was seen using the cryopreserved samples. Analysis of the flow cytometric data demonstrated that cryopreserved mononuclear cells retained all of the basic properties which were assessed. This included light scatter properties and the expression of cell-surface antigens, detected using fluorescent antibody techniques. Histograms showing light scatter and fluorescence data from matched fresh and cryopreserved samples 10'
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Fig. 5. Proliferative responses of fresh and cryopreserved equine mononuclear cells from two representative ponies in response to various concentrations of purified Equi-2 influenza virus. Data are expressed as described in Fig. 1. Hens' egg albumin was a minor c o n t a m i n a n t of both the influenza vaccines and our Equi-2 preparations. Proliferative responses of the test mononuclear cell preparations to egg albumin (5/xg/ml ) are shown as the ()pen symbols. Total culture time was 144 h.
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Fig. 6. Comparison of flow cytometric data obtained using fresh and cryopreserved mononuclear cells from one representative pony. Data are shown as simple histogram with the vertical scale representing numbers of cells/channel and the horizontal scale representing intensity of the signal for each channel. These histograms are shown with a linear horizontal scale for measurement of light scatter parameters (forward scatter and 90 ° scatter } and with a h)garithmic horizontal scale for those portions of the experiment involving fluorescence measurements (FITC control, E q T 2, E q T - 3 a n d E L A - I I ).
are shown in Fig. 6. The histograms for the forward light scatter and the fluorescent data do not demonstrate any significant alteration of physical properties for these cell samples. The 90 ° scatter profiles were usually altered slightly for the cryopreserved samples. This altered scatter profile represents an increase in the area under the curve where monocytes and large granular lymphocytes would be expected to fall, channel 60-180. The data obtained using three representative ponies and the entire monoclonal antibody panel are summarized in Table 1. The percentages ofcryopreserved mononuclear cells exp.ressing each of theT-lymphocyte markers(EqT2, 3, 7 and 12 ) class-II ELA antigens is decreased. This decrease was slight, 615%, and the total percentages of antigen positive cells did not fall outside of the normal range for fresh mononuclear cells (data not shown). We repro-
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TABLE 1 Flow cytometric data obtained using fresh and cryopreserved mononuclear cells from three representative ponies and monoclonal antibodies to cell-surface antigens Antigen designation
Pony 87 % pos/mean fl"
Pony 811 % p o s / m e a n fl
Fresh
Frozen
Fresh
0/56/141 39/116 1/2/68/145
1/ -
1/ -
1/ -
1/ -
43/141 38/126 2/3/48/144
36/137 61/137 1/2/57/141
29/140 50/130 1/3/39/139
48/134 62/132 2/1/73/145
0/56/129 44/115 1/ 1/67/138
0/53/131 47/125 1/ l/63/141
34/128 69/136 0/1/54/134
Frozen
Pony 826 % pos/mean fl Fresh
Frozen
Raw data Neg control EqT-2
EqT-3 EqT-7 EqT-12 ELA-class II Reprocessed data Neg control EqT-2
EqT-3 EqT-7 EqT-12 EIJA-class Ii
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30/129 66/129 1/ 1/49/133
0/-
48/125 72/139 1/ 0/80/139
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39/135 48/130 2/6/59/142 0/-
44/125 65/129 0/2/74/135
'~Results are expressed as percent positive/mean fluorescence. Percent positive represents the number of cells within a sample with fluorescence intensity significantly greater than the negative control sample for the same pony. Mean fluorescence values are a measure of the intensity of the population of cells determined to be positive for any given marker. Mean fluorescence values arc expressed in the tbrm of channel numbers (see Fig. 6) and are an arbitrary measure of fluorescence.
cessed the data using 90 ° scatter measurements to remove the more granular cells from the analysis, channels 60-256. In the cryopreserved samples, the percentages of cells expressing T-lymphocyte and class-II ELA antigens increased slightly following this reprocessing step (Table 1 ). Analysis of the cell numbers of channels 60-180 revealed that 11-28% of the cells from cryopreserved samples were in these channels while only 7-19% of the cells from fresh samples fell into this range. These data suggest that the cryopreservation and/ or the purification steps select for monocytes since these cell types do not express the T-lymphocyte markers analyzed in this study. The density of the T-lymphocyte and class-II ELA antigens was determined using "mean fluorescence intensity" as a measure of antigen density on a per cell basis. The mean fluorescence intensity obtained using cryopreserved mononuclear cells was similar to that obtained using the same monoclonal antibodies and fresh cells (Table 1 ). This can also be seen in Fig. 6; the shape of the fluorescence histograms were the same for both the fresh and frozen samples. These data demonstrate that actual cell-surface antigen expression was not altered by cryopreservation.
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The ELA class-I antigen types of cryopreserved cells were not altered by the procedure. The absolute negative reaction, or background control, in the ELA typing test using fresh cellular samples was routinely estimated to be less than 5% dead cells. This increased to 10-15% when cryopreserved mononuclear cells were used. However, this slight increase in background cytotoxicity did not interfere with the analysis of the typing data and the assignment of ELA types; positive typing reactions were routinely represented by tests where 100% of the target cells are killed. DISCUSSION The use of cryopreserved peripheral blood mononuclear cells for experimentation involving immune functions has a number of distinct advantages when compared to the use of only fresh blood samples. Large numbers of cells can be stored and tested when new assays are developed or retested at later times when questions arise about technical problems. Additionally, studies in which multiple samples are collected at different time points can become difficult to run and to control experimentally unless samples can be saved and tested together in large batches (Sears and Rosenberg, 1975; Jewett et al., 1976). The use of cryopreserved mononuclear cells has become an important tool for retaining samples from unique groups or populations of individuals (Fujiwara et al., 1986). We have developed a simple and inexpensive technique which allows for the cryopreservation of buffy-coat leukocytes and eliminates the need for prior purification of mononuclear cells from samples of whole blood. Mononuclear cells are then purified from the samples using a standard discontinuous-gradient method and commercially available Histopaque after thawing. We tested the functional ability of the cryopreserved cells using standard in vitro assays. The data demonstrated that mononuclear cells from cryopreserved samples proliferated in response to mitogens and proteins from Equio2 influenza (Fig. 1-3,5). Using kinetic studies, we demonstrated that mononuclear cells from cryopreserved samples reached their maximum levels of proliferation a day later than cells from fresh blood (Fig. 4). When these data are analyzed together, it appears that mononuclear cells are fully capable of responding to mitogen stimulation. Cryopreserved samples of human and bovine origin have been tested similarly and shown to retain the ability to proliferate in response to mitogens (Sears and Rosenberg, 1975; Strong et al., 1975; Kleinschuster et al., 1979; Kuil, 1984). The results presented here compare the responses of cells from the same blood samples but the in vitro assays were run on different days. The variation observed comparing the fresh to the cryopreserved samples was usually less than the variation observed using fresh cells taken from individual ponies at different times (data not shown). We have successfully recovered viable and
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functional mononuclear cells from these samples after 14 months of storage and we are currently using cryopreserved samples for separate studies that have been in storage for over 2 years. Analysis of cryopreserved mononuclear cells using flow cytometric techniques demonstrated that these cells expressed the same concentration of antigens on their cells surface as fresh cells (Fig. 6 and Table 1 ). However, a slight difference in the representation of different cell types was observed. The cryopreserved samples appeared to contain 4%-9% more cells with light scatter properties which suggested that they were monocytes. Slight differences in the representation of mononuclear cell subpopulations has been reported for cryopreserved human cells. Prince and Lee (1986) reported a slight but insignificant positive selection for cells with characteristic NK-cell surface antigens. However, Strong et al. (1982) reported results suggesting a slight loss of large granular leukocytes in their cryopreserved samples and Fujiwara et al. (1986) found no changes. The apparent changes in the numbers of different cell types did not alter the ability of equine cryopreserved cells to proliferate in response to mitogens or the Equi-2 and as such, the significance of this observation is questionable. Cryopreserved equine mononuclear cells were found to express ELA antigens, both class-II (Table 1) and class-I. This observation is consistent with published reports for cryopreserved human mononuclear cells (Strong et al., 1977, 1978). The cryopreserved cells responded normally in proliferation assays and this response requires interaction between cells; an interaction which is mediated through cell-surface expressed antigens such as ELA antigens. Therefore, the fact that cryopreserved cells express ELA antigens and celltype specific antigens, as EqT-3 and EqT-2, on their cell surface in concentrations similar to fresh cells would be expected. We have not evaluated the cell-mediated cytotoxicity parameters of equine cryopreserved mononuclear cells, primarily because the assays have not yet been established for fresh equine cells. However, cryopreserved human mononuclear cells do retain cytotoxic activity {Factor et al., 1975; Strong et al., 1975; Fujiwara et al., 1986). Cryopreserved equine cells appear to proliferate normally in response to influenza virus (Fig. 5) and this fact leads us to believe that cytotoxic responses are likely to be maintained by these same cell preparations. Studies are underway currently to evaluate this questior. The main advantage to our technique is that the purification of mononuclear cells is done after the cryopreservation step, not before. This alteration in the sequence of technical steps has three distinct advantages over the methods developed primarily for use with human perpheral blood samples: (1) large amounts of blood can be processed for freezing very rapidly and using very little technical time or effort since the technique is very simple; {2 ) the technique is inexpensive because leukocytes are cryopreserved in autologous plasma, cell culture media and fetal calf serum are not used; (3) mononuclear cells are
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purified after thawing and this purification step removes cells which have died during the cryopreservation and thawing steps thus providing a very viable product. Therefore, the viability and i m m u n e responsiveness of mononuclear cell samples purified from cryopreserved buffy-coat leukocyte preparations is very similar to t h a t which can be at t ai ned using fresh whole blood samples. T h e only disadvantage t h a t we have observed using our cryopreservation system is t h a t fewer m o n o n u c l e a r cells are recovered from cryopreserved samples when compared to fresh blood. However, the total numbers of cells which we recovered from cryopreserved samples was usually greater t han 80% of the numbers obtained from fresh whole blood. Since one of the advantages of working with large domestic animals is t h a t relatively large quantities of blood can be obtained routinely, limitations in processing samples is usually the problem not the a m o u n t of sample available. Using our system, large numbers of samples can be processed and a second or third vial can be thawed if and when it is needed. In general, the advantages of the system greatly out weigh the minor loss of cells. ACKNOWLEDGEMENTS T h e authors express their sincere t h a n k s to Mr. W.V. Adams for his work with the blood collection from the ponies, to Ms. M.A. Dietrich for the flow c y t o m e t r y work and to Dr. J.J. McClure for the E L A typing antisera. This research study was supported in par t by a grant from the Public Health Service, National Institutes of H e a l t h (A125850).
REFERENCES Bernoco, D., Antczak, D.F., Bailey, E., Bell, K., Bull, R.W., Byrns, G., Guerin, G., I,azary, S., McClure, J., Templeton, J. and Varewyck, H., 1987. Joint report of the fourth international workshop on lymphocytealloantigens of the horse, Lexington, Kentucky, 12-22 October 1985. Anim. Genet.. 18: 81-94. Crepaldi, T., Crump, A., Newman, M., Ferrone, S. and Antczak, D.F., 1986. Equine T-lymphoc.vtes express MHC class II antigens. J. Immunogenet., 13: 349-360. Dean, D.A. and Strong, D.M., 1977. Improved assay for monocyte chemotaxis using frozen stored responder cells. J. Immunol. Methods, 14: 65-72. Factor, M.A., Strong, D.M., Miller, J.L. and Sell, K.W., 1975. Cell-mediatedlymphocy~otoxicity followingin vitro sensitization of frozenstored human mononuclearcells in heterologousserum. Cryobiology,12:521-529. Fujiwara, S., Akiyama, M., Yamakido, M., Seyama, T., Kobuke, K., Hakoda, M., Kyoizumi, ,q. and Jones, S.L., 1986. Cryopreservation of human lymphocytesfor assessment of lymphocyte subsets and natural killer cytotoxicity. J. Immunol. Methods, 90: 265-273. Gramatzki, M., Strong, D.M., Grove, S.B. and Bonnard, G.D., 1982. Cryopreservedhuman cultured T-cells as responder cells for the quantitative measurement of interleukin-2: Improvement of the assay. J. Immunol. Methods, 53: 209-220.
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Habel, H. and Salzman, N.P., 1969. Fundamental Techniques in Virology. Academic Press, New York, NY, pp. 82-86. Hinshaw, V.S., Nacre, C.W., Webster, R.G., Douglas, A., Skehel, J.J. and Bryans, J., 1983. Anal)'sis of antigenic variation in equine 2 influenza A virus. Bull. W. H. O., 61: 153-158. Jewett, M.A.S., Gupta, S., Hansen, J.A., Cunningham-Rundles, S., Siegel, F.P., Good, R.A. and Dupont, B., 1976. The use of cryopreserved lymphocytes for longitudinal studies of immune function and enumeration of subpopulations. Clin. Exp. Immunol., 25: 449-454. Kleinschuster. S.J., van Kampen, K.R., Spendlove, R., Atluru, D., Zupancic, M. and Muscoplat, C.C., 1979. Cryopreservation of bovine mononuclear leukocytes. Am. J. Vet. Res., 40: 16491651. Kuil, H., 1984. In vitro reactivity to concanavalin A of bovine lymphocytes after cryopreservation. Vet. Immunol. Immunopathol., 7: 89-98. Lazary, S., Antczak, D.F., Bailey, E., Bell, T.K., Bernoco, D., Byrns, G. and McClure, J.J., 1988. Joint Report of the fifth international workshop on lymphocyte alloantigens of the horse, Baton Rouge, Louisiana, 31 October-1 November 1987. Anim. Genet., 19: 447-456. Magnuson, N.S., Perryman, L.E., Wyatt, C.R., Mason, P.H. and Talmadge, J.E., 1987. l,arge granular lymphocytes from SCID horses develop potent cytotoxic activity after treatment with human recombinant interleukin 2. J. Immunol., 139:61-67. Munoz. C.G., Keusch, G.T. and Dinarello, C.A., 1987. A new micromethod for determination of interleukin-1 production from frozen human mononuclear cells. J. Immunol. Methods, 99: 123-127. Netzel, B., Grosse-Wilde, H. and Mempel, W., 1975. MLC reactions with dog lymphocytes frozen in microtiter plates. Trans. Proc., 7: 703-405. Newman, M.J. and Antczak, D.F., 1983. Histocompatibility systems in domestic animals. Adv. Vet. Sci. Comp. Med., 27: 1-76. Newman, M.J., Beegie, K.H. and Antczak, D.F., 1984. Xenogeneic monoclonal antibodies to cell surface antigens of equine lymphocytes. Am. J. Vet. Res., 45: 626-632. Pappas, F., Bonnard, G.D., Hartzman, R.J. and Strong, D.M., 1979. Human PL cells grown as continued T-cell cultures. Trans. Proc., 11: 1986-1989. Prince, H.E. and Lee, C.D., 1986. Cryopreservation and short-term storage of human lymphocytes tbr surface marker analysis: Comparison of three methods. J. Immunol. Methods, 93: 15-.18. Sears, H.F. and Rosenberg, S.A., 1975. Advantages of cryopreservation of lymphocytes fl)r sequential evaluation of human immune competence. I. Mitogen stimulation. J. Natl. Cancer Inst., 58: 183-187. Slease, R.B., Strong, D.M., Wistar, R., Budd, R.E. and Scher, I., 1980. Determination of surface immunoglobulin density on frozen-thawed mononuclear cells analyzed by the fluorescenceactivated cell sorter. Cryobiology, 17: 523-529. Stear, M.J., Allen, D. and Spooner, R.L., 1982. A method for freezing sheep lymphocytes prior to cytotoxicity testing. Tissue Antigens, 19: 134-139. Strong, D.M., 1976. Cryobiologic approaches to the recovery of immunologic responsiveness of murine and human mononuclear cells. Trans. Proc., 8: 203-208. Strong, D.M., Woody, J.N., Factor, M.A., Ahmed, A. and Sell, K.W., 1975. Immunological responsiveness of frozen-thawed human lymphocytes. Clin. Exp. Immunol., 21: 442-455. Strong, D.M., Knudsen, B.B., Mann, D.L. and Sell, K.W., 1977. Frozen-thawed mononuclear cells as a source of B-lymphocytes for anitbody screening. Tissue Antigens, l 0:108-113. Strong, D.M., Hartzman, R.J. and Light, J.A., 1978. Lymphocyte cryopreservation fi)r histocompatibility testing. Dial. Transplant., 7: 508-533. Strong, D.M., Ortaldo, J.R., Pandolfi, F., Maluish, A. and Herberman, R.B., 1982. Cryopreservation of human mononuclear cells for quality control in clinical immunology. I. Correlation in recovery of K- and NK-functions, surface markers and morphology. J. Clin. Immunol., 2: 214221.
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Weaver, W.J., Weber, F.S., Holmes, K., Jacob, S.W. and Templeton, J.W., 1975. Response of frozen and stored canine lymphocytes in culture after thawing. Trans. Proc., 14: 65-72. Wyatt, C.R., Davis, W.C., McGuire, T.C. and Perryman, L.E., 1988. T-lymphocyte characterization in horses. I. Characterization of monoclonal antibodies identifying three stages of Tlymphocyte differentiation. Vet. Immunol. Immunopathol., ! 8: 3-18.
139
Cryopreservation of Equine Mononuclear Cells for Immunological Studies
R.E. TRUAX ~, M.D. POWELL ~, R.C. MONTELARO 2, C.J. ISSEL ~':~and M.J. NEWMAN TM
1Veterinary Microbiology and Parasitology, School o[ Veterinary Medicine and 2Department o[ Biochemistry, College of Basic Sciences, Louisiana State University and :~Department of Veterinary Science, Louisiana Agricultural Experiment Station, Baton Rouge, LA 70803 (U.S.A.) (Accepted 23 November 1989)
ABSTRACT Truax, R.E., Powell, M.D., Montelaro, R.C., Issel, C.J. and Newman, M.J., 1990. Cryopreservation of equine mononuclear cells for immunological studies. Vet. Immunol. Immunopathol., 25: 139153. A rapid and simple technique for the cryopreservation and recover)' of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in au~)logous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved huffy coat preparations were compared with those of mononuclear cells from fresh samples by measuring the ability of cells to proliferate in response to mitogens and specific antigens. Cell-surface antigen expression was measured using monoclonal antibodies in conjunction with flow cytometric techniques and alloantisera in a complement mediated cytotoxicity assay. Cryopreserved mononuclear cells were capable of proliferating normally when stimulated with several mitogens, pokeweed mitogen, phytohemagglutinin and concanavalin A, and a single specific antigen preparation, equine influenza-2 (Equi-2) proteins. The maximum levels of proliferation induced by varying the concentrations of mitogens or the Equi-2 proteins were the same for both the fresh and cryopreserved cells. However, the cryopreserved cells usually required one more day in culture to attain maximum proliferation levels. Flow cytometric analysis of the samples demonstrated that the relative proportions of different lymphocyte populations were not altered by the cryopreservation step. Similarly, MHS alloantigen expression was not altered. The simplicity of the technique coupled with the retained functional properties allows for the cryopreservation of large numbers of leukocytes and the ability to assay various immune functions at a later time. 4present address and to whom all correspondence should be sent: Cambridge BioScience Corporation, 365 Plantation Street, Worcester, MA 01605 (U.S.A.).
0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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INTRODUCTION Cryopreservation of human mononuclear cells is done routinely as a standard part of biomedical research programs. The most common parameters of various cryopreservation methods are the use of cryo-protectant compounds, such as dimethylsulphoxide (DMSO), and a controlled slow rate of freezing. Frozen cellular preparations are routinely stored in the vapor phase in liquidnitrogen freezers or in ultra-low temperature ( - 135 °C) electric freezers. Cryopreservation procedures were primarily developed for use with human blood mononuclear cells. This methodology has allowed investigators to obtain cellular materials from patients and control volunteers when they are available and to use them for in vitro assays at later times (Sears and Rosenberg, 1975; Jewett et al., 1976). These techniques have been used most extensively for studies in the area of clinical immunology where cellular immune functions have been measured successfully using cryopreserved peripheral blood mononuclear cells. Cryopreserved human mononuclear cells retain their ability to express histocompatibility antigens (Strong et al., 1977, 1978) and subset specific differentiation antigens (Slease et al., 1980; Strong et al., 1982; Fujiwara et al., 1986; Prince and Lee, 1986 ). Cell-mediated immune parameters retained by cryopreserved mononuclear cells included lymphocyte proliferation in response to mitogens, lymphokines and antigens (Factor et al., 1975; Strong et al., 1975; Strong, 1976; Pappas et al., 1979; Gramatzki et al., 1982), NK-cell and antigen specific cell-mediated cytotoxicity (Factor et al., 1975; Strong et al., 1982 ), antibody dependent cell-mediated cytotoxicity (Strong et al., 1982 ), chemotaxis (Dean and Strong, 1977 ) and the production of cytokines (Munoz et al., 1987). The use of cryopreserved mononuclear cells for research with domestic animals has been very limited. Gradient-purified lymphocytes have been cryopreserved for use in histocompatibility testing systems with the dog (Netzel et al., 1975; Weaver et al., 1975) and the sheep (Stear et al., 1982). Bovine lymphocytes have been cryopreserved and shown to retain responsiveness to mitogens (Kleinschuster et al., 1979; Kuil, 1984). However, all of these studies have been very limited in scope and the use of cryopreserved cellular materials has not gained widespread acceptance. The use of this methodology would be of great value because cells could be stored for multiple controlled testing and exchanged with other investigators. We have successfully used the methods of cryopreservation, that were developed for use with human cells, to freeze gradient-purified equine lymphocytes. However, the purification of mononuclear cells from peripheral blood is laborious and time-consuming and this has limited the number of samples that we can process routinely. We have therefore developed a technique for cryopreserving equine leukocytes as buffy-coat leukocyte preparations. Our method has proven to be rapid and technically simple, thus allowing one person to process a large number of blood samples.
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Mononuclear cells were routinely recovered in a viable state, capable of responding to mitogens and antigens. The development and characterization of this method is described in this report. MATERIALSAND METHODS
Test animals and collection of blood samples Eight healthy Shetland ponies, 1-3 years of age, were used for the study. They were maintained on pasture with free choice hay, water and mineral supplementation. All ponies were vaccinated against anthrax, encephalitis, equine herpes virus-l, strangles and influenza (Equi-1 and Equi-2 ) prior to inclusion into the study. Anthelmintic treatments were done at 12-week intervals. Whole blood samples, 100 ml, were collected from ponies at two different times using standard aseptic venipuncture techniques and preservative free heparin ( Sigma Chemical Co., St. Louis, MO) at the final concentration of 10 U/ml. Blood samples were processed within 4 h of collection.
Cryopreservation of bully-coat leukocytes Blood samples were centrifuged at 400g at room temperature for 15 min. All but 1 ml of the plasma was removed using sterile techniques and an aliquot was mixed with dimethyl sulfoxide {DMSO; Sigma) to a final concentration of 20% (v/v) DMSO and 80% {v/v) autologous plasma. One ml of this DMSOautologous plasma mixture was prepared for every 20 ml of whole blood and placed on ice. The buffy-coat leukocyte layer was recovered in the remaining autologous plasma, resuspended and put on ice for 30 min. Equal quantities of buffy-coat leukocytes and DMSO-autologous plasma were mixed and dispensed into polypropylene vials (Sarstedt, Princeton, NJ), all materials and vials were kept on ice. Each vial contained 1 ml of cell suspension from 10 ml of whole blood in autologous plasma plus 10% {v/v) DMSO. Leukocyte samples were frozen using a programmable controlled rate freezer (KRYO-10; T.S. Scientific, Quakertown, PA) and several different freezing rates, as described (Strong et al., 1982; Fujiwara et al., 1986; Prince and Lee, 1986; Munoz et al., 1987). Frozen samples were stored for up to 14 months in the vapour phase in a liquid-nitrogen freezer until thawed for experimental purposes. Leukocyte samples were thawed quickly by placing vials in a 37°C water bath. Thawed samples were immediately transferred to a 15-ml cell-culture tube and diluted slowly to a total volume of 10 ml using cold Ca ~+ and Mg ~+ free Hank's balanced salt solution (HBSS, pH 7.4). The diluted leukocyte mixture was layered onto 4 ml of Histopaque-1077 (Sigma) and the gradients centrifuged at 400g for 30 min at room temperature. The mononuclear cell layer was recovered from the Histopaque-HBSS interface and the cells washed three times at room temperature using HBSS. Mononuclear cells were resus-
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pended at a concentration of 1-2>< 106 cells/ml in RPMI-1640 buffered with NaHCO3 and supplemented with 10% (v/v) fetal bovine serum (GIBCO, Grand Island, NY ), 2 m M L-glutamine, 10 pJV/2-mercaptoethanol and 50 ]~g/ml gentamicin sulfate (Schering Corp., Kenilworth, NJ). Cell viability was assessed using standard trypan exclusion criteria.
Measurement of mitogen and antigen induced cellular proliferation The ability of mononuclear cells to respond to polyclonal stimulation was compared using standard in vitro assays and three mitogens, phytohemagglutinin (PHA; Sigma), concanavalin A (Con A; Sigma) and pokeweed mitogen (PWM; Sigma). Mononuclear cells were cultured at a concentration of 1 × 106/ ml in supplemented RPMI-1640 cell culture media using round-bottom 96-well microculture plates (CoStar, Cambridge, MA) and 200 ~1 total culture volumes, were used for all culture experiments. Cultures were maintained at 37 °C in a humid incubator with 5% CO,~ in air. The concentration of mitogens was varied; PHA and P W M were used at concentrations of 1, 2, 4 and 8 ttg/ml, Con A was used at concentrations of 2.5, 5, 10 and 20 ttg/ml. Total culture durations of 72, 96, 120 and 144 h were used. One ~Ci of [3H]thymidine (ICN Radiochemicals, Irvine, CA) was added to all wells 16 h prior to the termination of cultures. Cellular proliferation was quantitated by determining the amount of isotope incorporated into dividing mononuclear cells by liquid scintillation counting. All tests were run using quadruplicate cultures and experiments were run twice at different times. The ability of cryopreserved and fresh mononuclear cells to profilerate in response to specific antigens was assessed using a similar assay system and equine influenza-2 (Equi-2, A/equine/Miami/63; American Type Culture Collection, Rockville, MD) as the test antigen. The Equi-2 antigen preparation used for our in vitro testing consisted of intact virus particles, grown in 11-day-old embryonated hens' eggs and purified from allantoic fluid using a 14-40% {w/v) sucrose gradient (Hinshaw et al., 1983). Virus titers were determined by standard hemagglutination assay using chicken erythrocytes (Habel and Salzman, 1969; Hinshaw et al., 1983) and were expressed as hemagglutination units (HA units). The Equi-2 preparation was used at concentrations of 5, 2.5, 1.25, 0.61 and 0.3 HA units/ml in 200 ~l cultures containing mononuclear cells at a final concentration of 2 ><106 cells/ml.
Flow cytometric quantitation of cell-surface expressed antigens The expression of cell "type-specific" or differentiation antigens on fresh and cryopreserved equine mononuclear cells was assessed using monoclonal antibodies and fluorescence activated flow cytometry. Selected routine monoclonal antibodies which bind to mature equine T-lymphocytes (EqT-2 and EqT-3) and immature T-lymphocytes (EqT-7 and EqT-12) were purchased commercially (VMRD, Pullman WA). The production and characterization
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of these monoclonal antibodies is reviewed elsewhere (Magnuson et al., 1987; Wyatt et al., 1988). The F-39.2 monoclonal antibody was produced in a rat and recognizes equine major histocompatibility (ELA) class-II antigens (Newman et al., 1984; Crepaldi et al., 1986). Gradient-purified mononuclear cells were resuspended in phosphate-buffered saline (PBS), pH 7.4, containing 10% (v/v) normal goat serum (NGS), to a concentration of 107 cells/ml and placed on ice for 20 min. Optimally diluted monoclonal antibodies were added to 100/~l aliquots of the cells and incubated for 1 h on ice. The cells were washed twice using cold PBS and then resuspended in 100 pl of optimal dilutions of FITC-conjugated anti-immunoglobulin reagents (Sigma) e.g., affinity purified goat IgG specific to either mouse IgG (H and L chains) or to rat IgG (H and L chains). The cells were incubated for 1 h on ice, washed twice and resuspended in 200/~l of 2% (w/v) paraformaldehyde in PBS. Antibody labeled mononuclear cells were analyzed using a fluorescence activated cell sorter (FACS-440; Becton-Dickinson, San Jose, CA). Data collection through the FACS-440 was triggered using only forward light scatter but list mode data were collected for forward light scatter, 90 ° light scatter and green fluorescence emission parameters. The data were analyzed using a DEC Micro-VAX II workstation (Digital Equipment Corporation, Westminster, MA ) and the Consort-40 flow cytometry program package (Becton-Dickinson).
Histocompatability typing of mononuclear cells Equine histocompatibility class-I antigen types (ELA) were determined using alloantisera defined in international workshops (Bernoco et al., 1987; Lazary et al., 1988) and the microlymphocytotoxicity test (Newman and Antczak, 1983). Both fresh and cryopreserved mononuclear cells were typed to determine the extent to which cryopreservation alters ELA class-I antigen expression. RESULTS
Initial tests were run to identify the optimal freezing rate for equine mononuclear cells in our system by determining the total recovery and viability of mononuclear cells from cryopreserved samples after using the various freezing rates identified in the literature. Cellular viability was generally 95% or greater with no significant differences between different freezing rates and fresh blood. The total numbers of mononuclear cells which were recovered did vary greatly when different freezing rates were used. The freezing rate which consistently gave the highest recovery rates was as follows: (1) samples were held at 4 ° C for 5 min, (2) the temperature was dropped 0.5 ° C/min until the sample temperature reached - 4 0 ° C, (3) the temperature was dropped 1.5 °C/min until the sample temperature reached - 60 ° C, (4 } samples were then placed directly
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into the vapor phase in a liquid-nitrogen storage freezer, approximate temperature of - 120 °C. This freezing procedure was used for all subsequent testing. Using this method we could routinely recover 1-4 × 106 mononuclear cells from each ml of whole blood. Controlled comparison of these numbers to those obtained using fresh whole blood demonstrated that 6-18% fewer cells were recovered from the cryopreserved samples. Cryopreserved mononuclear cells proliferated in response to all of the mitogens used. Maximum proliferative responses were generated in cultures of both fresh and cryopreserved cells using the same concentrations of mitogens. Significant differences between the responses of fresh and cryopreserved samples were not common. Representative proliferation data for five ponies and the three mitogens are shown in Figs. 1-3. Analysis of the mitogen induced proliferation data with respect to culture duration demonstrated that cryoprePONY ID *~ 7 1 6 0 717&
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Fig. 1. Proliferative responses of fresh and cryopreserved equine mononuclear cells from five representative ponies in response to various concentrations of PHA. Data are expressed as counts/ rain {log), actual data points represent the mean +_ 1 standard deviation of quadruplicate tests. Total culture time was 72 h. The results shown were obtained using cells from a single blood collection. Samples were processed within 4 h for cryopreservation and for cell culture. The assays using cryopreserved cells were run 2-6 months later. Fig. 2. Proliferative responses of fresh and cryopreserved equine mononuclear cells from five representative ponies in response to various concentrations of Con A. Data are represented as described in Fig. 1. Total culture time was 96 h.
CRYOPRESERVATIONOF EQUINEMONONUCLEARCELLSFOR IMMUNOLOGICALSTUDIES
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served mononuclear cells responded more slowly than fresh cells. Generally, an additional day in culture was required for cryopreserved cells to reach the highest level of proliferation for all of the mitogens tested. This is shown in Fig. 4 for two representative ponies. Cryopreserved mononuclear cells were also fully capable of proliferating in response to specific antigens. Data from two representative animals are shown in Fig. 5. Interestingly, there was variation in the total response levels using fresh mononuclear cells from different ponies. In Fig. 5 we showed data from a high responder pony (834) and a low responder pony {841 ). This same difference in response levels was seen using the cryopreserved samples. Analysis of the flow cytometric data demonstrated that cryopreserved mononuclear cells retained all of the basic properties which were assessed. This included light scatter properties and the expression of cell-surface antigens, detected using fluorescent antibody techniques. Histograms showing light scatter and fluorescence data from matched fresh and cryopreserved samples 10'
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Fig. 5. Proliferative responses of fresh and cryopreserved equine mononuclear cells from two representative ponies in response to various concentrations of purified Equi-2 influenza virus. Data are expressed as described in Fig. 1. Hens' egg albumin was a minor c o n t a m i n a n t of both the influenza vaccines and our Equi-2 preparations. Proliferative responses of the test mononuclear cell preparations to egg albumin (5/xg/ml ) are shown as the ()pen symbols. Total culture time was 144 h.
CRYOPRESERVATION OF EQUINE MONONUCLEAR CELLS FOR IMMUNOLOGICAL STUDIES
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are shown in Fig. 6. The histograms for the forward light scatter and the fluorescent data do not demonstrate any significant alteration of physical properties for these cell samples. The 90 ° scatter profiles were usually altered slightly for the cryopreserved samples. This altered scatter profile represents an increase in the area under the curve where monocytes and large granular lymphocytes would be expected to fall, channel 60-180. The data obtained using three representative ponies and the entire monoclonal antibody panel are summarized in Table 1. The percentages ofcryopreserved mononuclear cells exp.ressing each of theT-lymphocyte markers(EqT2, 3, 7 and 12 ) class-II ELA antigens is decreased. This decrease was slight, 615%, and the total percentages of antigen positive cells did not fall outside of the normal range for fresh mononuclear cells (data not shown). We repro-
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TABLE 1 Flow cytometric data obtained using fresh and cryopreserved mononuclear cells from three representative ponies and monoclonal antibodies to cell-surface antigens Antigen designation
Pony 87 % pos/mean fl"
Pony 811 % p o s / m e a n fl
Fresh
Frozen
Fresh
0/56/141 39/116 1/2/68/145
1/ -
1/ -
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1/ -
43/141 38/126 2/3/48/144
36/137 61/137 1/2/57/141
29/140 50/130 1/3/39/139
48/134 62/132 2/1/73/145
0/56/129 44/115 1/ 1/67/138
0/53/131 47/125 1/ l/63/141
34/128 69/136 0/1/54/134
Frozen
Pony 826 % pos/mean fl Fresh
Frozen
Raw data Neg control EqT-2
EqT-3 EqT-7 EqT-12 ELA-class II Reprocessed data Neg control EqT-2
EqT-3 EqT-7 EqT-12 EIJA-class Ii
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l/ -
30/129 66/129 1/ 1/49/133
0/-
48/125 72/139 1/ 0/80/139
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39/135 48/130 2/6/59/142 0/-
44/125 65/129 0/2/74/135
'~Results are expressed as percent positive/mean fluorescence. Percent positive represents the number of cells within a sample with fluorescence intensity significantly greater than the negative control sample for the same pony. Mean fluorescence values are a measure of the intensity of the population of cells determined to be positive for any given marker. Mean fluorescence values arc expressed in the tbrm of channel numbers (see Fig. 6) and are an arbitrary measure of fluorescence.
cessed the data using 90 ° scatter measurements to remove the more granular cells from the analysis, channels 60-256. In the cryopreserved samples, the percentages of cells expressing T-lymphocyte and class-II ELA antigens increased slightly following this reprocessing step (Table 1 ). Analysis of the cell numbers of channels 60-180 revealed that 11-28% of the cells from cryopreserved samples were in these channels while only 7-19% of the cells from fresh samples fell into this range. These data suggest that the cryopreservation and/ or the purification steps select for monocytes since these cell types do not express the T-lymphocyte markers analyzed in this study. The density of the T-lymphocyte and class-II ELA antigens was determined using "mean fluorescence intensity" as a measure of antigen density on a per cell basis. The mean fluorescence intensity obtained using cryopreserved mononuclear cells was similar to that obtained using the same monoclonal antibodies and fresh cells (Table 1 ). This can also be seen in Fig. 6; the shape of the fluorescence histograms were the same for both the fresh and frozen samples. These data demonstrate that actual cell-surface antigen expression was not altered by cryopreservation.
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The ELA class-I antigen types of cryopreserved cells were not altered by the procedure. The absolute negative reaction, or background control, in the ELA typing test using fresh cellular samples was routinely estimated to be less than 5% dead cells. This increased to 10-15% when cryopreserved mononuclear cells were used. However, this slight increase in background cytotoxicity did not interfere with the analysis of the typing data and the assignment of ELA types; positive typing reactions were routinely represented by tests where 100% of the target cells are killed. DISCUSSION The use of cryopreserved peripheral blood mononuclear cells for experimentation involving immune functions has a number of distinct advantages when compared to the use of only fresh blood samples. Large numbers of cells can be stored and tested when new assays are developed or retested at later times when questions arise about technical problems. Additionally, studies in which multiple samples are collected at different time points can become difficult to run and to control experimentally unless samples can be saved and tested together in large batches (Sears and Rosenberg, 1975; Jewett et al., 1976). The use of cryopreserved mononuclear cells has become an important tool for retaining samples from unique groups or populations of individuals (Fujiwara et al., 1986). We have developed a simple and inexpensive technique which allows for the cryopreservation of buffy-coat leukocytes and eliminates the need for prior purification of mononuclear cells from samples of whole blood. Mononuclear cells are then purified from the samples using a standard discontinuous-gradient method and commercially available Histopaque after thawing. We tested the functional ability of the cryopreserved cells using standard in vitro assays. The data demonstrated that mononuclear cells from cryopreserved samples proliferated in response to mitogens and proteins from Equio2 influenza (Fig. 1-3,5). Using kinetic studies, we demonstrated that mononuclear cells from cryopreserved samples reached their maximum levels of proliferation a day later than cells from fresh blood (Fig. 4). When these data are analyzed together, it appears that mononuclear cells are fully capable of responding to mitogen stimulation. Cryopreserved samples of human and bovine origin have been tested similarly and shown to retain the ability to proliferate in response to mitogens (Sears and Rosenberg, 1975; Strong et al., 1975; Kleinschuster et al., 1979; Kuil, 1984). The results presented here compare the responses of cells from the same blood samples but the in vitro assays were run on different days. The variation observed comparing the fresh to the cryopreserved samples was usually less than the variation observed using fresh cells taken from individual ponies at different times (data not shown). We have successfully recovered viable and
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functional mononuclear cells from these samples after 14 months of storage and we are currently using cryopreserved samples for separate studies that have been in storage for over 2 years. Analysis of cryopreserved mononuclear cells using flow cytometric techniques demonstrated that these cells expressed the same concentration of antigens on their cells surface as fresh cells (Fig. 6 and Table 1 ). However, a slight difference in the representation of different cell types was observed. The cryopreserved samples appeared to contain 4%-9% more cells with light scatter properties which suggested that they were monocytes. Slight differences in the representation of mononuclear cell subpopulations has been reported for cryopreserved human cells. Prince and Lee (1986) reported a slight but insignificant positive selection for cells with characteristic NK-cell surface antigens. However, Strong et al. (1982) reported results suggesting a slight loss of large granular leukocytes in their cryopreserved samples and Fujiwara et al. (1986) found no changes. The apparent changes in the numbers of different cell types did not alter the ability of equine cryopreserved cells to proliferate in response to mitogens or the Equi-2 and as such, the significance of this observation is questionable. Cryopreserved equine mononuclear cells were found to express ELA antigens, both class-II (Table 1) and class-I. This observation is consistent with published reports for cryopreserved human mononuclear cells (Strong et al., 1977, 1978). The cryopreserved cells responded normally in proliferation assays and this response requires interaction between cells; an interaction which is mediated through cell-surface expressed antigens such as ELA antigens. Therefore, the fact that cryopreserved cells express ELA antigens and celltype specific antigens, as EqT-3 and EqT-2, on their cell surface in concentrations similar to fresh cells would be expected. We have not evaluated the cell-mediated cytotoxicity parameters of equine cryopreserved mononuclear cells, primarily because the assays have not yet been established for fresh equine cells. However, cryopreserved human mononuclear cells do retain cytotoxic activity {Factor et al., 1975; Strong et al., 1975; Fujiwara et al., 1986). Cryopreserved equine cells appear to proliferate normally in response to influenza virus (Fig. 5) and this fact leads us to believe that cytotoxic responses are likely to be maintained by these same cell preparations. Studies are underway currently to evaluate this questior. The main advantage to our technique is that the purification of mononuclear cells is done after the cryopreservation step, not before. This alteration in the sequence of technical steps has three distinct advantages over the methods developed primarily for use with human perpheral blood samples: (1) large amounts of blood can be processed for freezing very rapidly and using very little technical time or effort since the technique is very simple; {2 ) the technique is inexpensive because leukocytes are cryopreserved in autologous plasma, cell culture media and fetal calf serum are not used; (3) mononuclear cells are
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purified after thawing and this purification step removes cells which have died during the cryopreservation and thawing steps thus providing a very viable product. Therefore, the viability and i m m u n e responsiveness of mononuclear cell samples purified from cryopreserved buffy-coat leukocyte preparations is very similar to t h a t which can be at t ai ned using fresh whole blood samples. T h e only disadvantage t h a t we have observed using our cryopreservation system is t h a t fewer m o n o n u c l e a r cells are recovered from cryopreserved samples when compared to fresh blood. However, the total numbers of cells which we recovered from cryopreserved samples was usually greater t han 80% of the numbers obtained from fresh whole blood. Since one of the advantages of working with large domestic animals is t h a t relatively large quantities of blood can be obtained routinely, limitations in processing samples is usually the problem not the a m o u n t of sample available. Using our system, large numbers of samples can be processed and a second or third vial can be thawed if and when it is needed. In general, the advantages of the system greatly out weigh the minor loss of cells. ACKNOWLEDGEMENTS T h e authors express their sincere t h a n k s to Mr. W.V. Adams for his work with the blood collection from the ponies, to Ms. M.A. Dietrich for the flow c y t o m e t r y work and to Dr. J.J. McClure for the E L A typing antisera. This research study was supported in par t by a grant from the Public Health Service, National Institutes of H e a l t h (A125850).
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