CRYOBIOLOGY
16,
118-124
(1979)
An Improved Method for Preparing Refrozen Rethawed Human Lymphocytes on Plates for Microcytotoxicity Studies PEG The Paul I. Hoxworth and The Departments
A.
SOLLMANN
AND
The use of refrozen rethawed lymphocyte panels on cytotoxicity plates to screen sera for HLA activity has great advantages for the small tissue typing laboratory where only a limited number of fresh cells are available. Recent studies (7, 8) demonstrated that lymphocytes may be subjected to a double freeze and thaw and still be reliably typed. However, two difficulties remain: (a) from the limited ‘data reported for the doubly thawed cell, the average viability was often lower than desired, and (b) elaborate freezing techniques were recommended, such as controlled rate freezes (S), with careful attention to temperature during both freeze and thaw procedures. Therefore, a study was undertaken with the following objectives: to clarify the factors influencing the viabilities of singly and doubly frozenthawed cells and to develop the simplest freeze-thaw techniques possible without sacrificing cell viability as measured in the cytotoxicity assay. Conditions examined for their influence on the viability of singly thawed lymphocytes were: age of the blood sample, effectiveness of the buffer in the freeze-mixture, type of storage tube used, temperature of cells prior to initial freezing, frozen storage Received 1978.
May
30,
1978;
accepted
October
OOll-2240/79/020118-07$02.00/O @ 1979 by Academic Press, Inc. of reproduction in any form reserved.
NATHAN Burns The Ohio
Institute, University 45267
Cincinnati Unit, of Cincinnati,
temperatures of the cells, and need for careful post-thaw washing of the cells. Variables influencing viability of doubly thawed lymphocytes included length of thaw time during plate preparation and enzymatic treatment of lymphocytes after the second thaw. The present work also evaluated the effect of length of storage time on once and twice frozen-thawed lymphocytes. It is now possible to recommend that some steps used in previous initial and second freeze procedures be eliminated, since viabilities produced by the methods described in this report are as good as those obtained with more elaborate techniques. TECHNIQUES
Heparin (100 units/ml) was added to each fresh blood sample (20 cc) obtained from norma human donors. The lymphocytes were separated on a Ficoll-Hypaque gradient (2). Platelets were removed by adding several drops of 0.2% adenosine diphosphate in 0.05 M potassium phosphate buffer at pH 6.8. The tubes were inverted repeatedly for 30 seconds allowing the platelets to clump. The suspension was then centrifuged at 2OOg’s for 6 seconds. The platelet free supernatant was removed and adjusted to a concentration of 4000 lymphocytes/p1 of media. Unless otherwise
30,
118
Copyright All rights
PAUL
Blood Center, The ShTineTs’ Of Physiology and Surgery, College of Medicine, Cincinnati,
METHOD
FOR
REFREEZING
indicated, all lymphocytes were initially frozen the same day the blood was drawn. Initial freexing procedure. The cell suspensions were centrifuged and the supernatants discarded. Cells were not placed on ice during the subsequent procedures. The lymphocytes were resuspended to a concentration of 4000 cells/PI in a freshly thawed cryoprotective mixture. This mixture was kept frozen in 4 cc aliquots at -80°C and was composed of 10% dimethylsulfoxide ( DIMSO), 50% fetal calf serum ( FCS ), 37% N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffered Media 199 (pH 7.4) and 4% glucose. In the buffer experiment described below, the p.H of separate preparations of Media 199 were adjusted with a solution of saturated NaHCO,. Fifty to 400 ~1 of the cell suspensions were then added to 5 x 45 mm microsample tubes (Kew Scientific, Cat. No. 208). The tubes were placed immediately into loo-partition boxes (Crown Paper Box Company, Indianapolis, Indiana) and stored in the vapor phase of a liquid nitrogen (LN2) freezer (-100” to -150°C) or a -80°C mechanical chest freezer which maintained a constant temperature. After frozen storage, cells were thawed within two and a half minutes in air at room temperature. They were then immediately inoculated into normal human serum to test their viability. A three hour incubation at room temperature was followed by staining with eosin and fixation in formalin. The stained cells were observed by phase microscopy to determine their percent viability. All the cells used in the present report were frozen for as long as three months unless otherwise indicated. The preparation of refrozen cells in tissue typing plates. A set of frozen cells with previously determined fresh cell viabilities of at least 90%, were thawed in air at 22°C and added to 72 well typing trays, (Cooke Products, Costar No. 236-72) as three identical 24 cell panels. Tests indicated that satisfactory lymphocyte viability could
RETHAWING
LYMPHOCYTES
119
be maintained if the total time the cells were thawed did not exceed 30 min. The completed plates were immediately placed into prechilled cardboard boxes in either a -80°C mechanical freezer or the vapor phase of a LN, freezer. Placement in or close to the liquid itself will result in a rate of freeze detrimental to viability. Use of plates with refrozen cells. Test plates were removed from the -80°C or LN, freezer and allowed to come to room temperature for IO min in air. Each well can immediately be inoculated with 2 PI of test sera and standard cytotoxicity procedures begun. However, the cell viabilities were usually better if the lymphocytes were first treated with deoxyribonuclease ( DNase, Sigma Chemical Company, St. Louis, Missouri) to eliminate any dead cells. Five microliters of DNase (2.5 mg/ ml) were inoculated into each well. The plate was floated on a layer of water in a 37°C incubator for 20 min. To remove the enzyme mixture and dead cells from the plates, the cells were then washed twice after which sera to be tested were added ta the plate. Cell washing alone, without the addition of DNase, did not raise the average viability above that of an unwashed panel of lymphocytes. Statistical analysis. Analysis of variance was used to determine if differences existed among sample means. Least significant difference (LSD) intervals were generated in all cases where a significant F test was found to locate specific differences. An arcsin transformation for aormality (12) was performed on the data preceding statistical analysis since the data consisted mainly of percentages skewed towards 100~. Conclusions obtained were the same as when the direct data were analyzed and the latter are presented for simplicity. RESULTS
Several vestigated
factors were to determine
individually intheir importance
120
SOLLMANN
AND TABLE
Effect
of Storage
NATHAN 1
of Blood Samples Prior to Freezing Lymphocytes Following a Single Lymphocytes purified and frozen immediately
‘% Viability &SE. single thaw.
following
a
93.1 f 0.6 N = 40
on Subsequent Thaw
Viability
Whole blood stored 24 hr at IOO~ temperature ; lymphocytes frozen next day
I
;
85.2 f 1.5 N = 27 1
in production of once-thawed preparations with viabilities greater than 90%. Such excellent results are necessary for the successful retrieval of useful lymphocyte preparations after the second freeze-thaw. Except for the variable being tested or otherwise indicated, conditions during the various experiments on once-thawed viability of lymphocytes are those noted in Techniques. (a) Buffer used in cryoprotectant mixture. Lymphocytes from 20 normal donors were frozen in NaHC03 buffered cryoprotectant mixture and stored for approximately one week. Similarly, another set of 43 normal lymphocyte preparations were frozen in a mixture buffered with HEPES, Thirty-four of the cells were stored for a similar time period as above and nine were stored for between 6 and 12 weeks. An average viability * standard error of the mean (SEM) of 92.7 * 0.6% was observed after a single freeze-thaw on those cells frozen with the HEPES buffered cryoprotectant compared to an average 85.8 * 0.9% viability on those frozen with NaHC08 buffered medium (P < 0.001). The pH of NaHC03 buffered cryoprotective mixture visibly becomes more alkaline within two weeks at -80°C and this change may adversely affect cell viability. HEPES, which is not affected by the CO2 in the gas phase above the buffer solution maintains a more stable pH. The cryo-
Purified lymphocytes stored 24 hr at romn temperature frozen next day
87.7 f 1.4 N = 33
P < 0.005
of
N.S.
T P < 0.005
protective mixture buffered with HEPES can be prepared in advance, frozen until needed, thawed, mixed with cells, and stored for long periods at -80°C with little change in pH. (b) Type of tube used. Lymphocytes from each of 31 random normal donors were frozen and stored for approximately one week in polypropylene microsample tubes (550 ~1 capacity, Kew Scientific) and polyethylene microsample tubes (400 ~1 capacity, Beckman Part No. 314326). After a single freeze-thaw cycle the average viability observed in the cells frozen in poIypropylene tubes was 88.6 * 1.1% compared to an average of 79.4 t 2.670 for those stored in polyethylene (P < 0.005). The cause of reduced viability in the Beckman tubes was not ascertained. Cells stored in bicarbonate buffer were used in these tests and this accounts for the lower viability in the polypropylene tubes compared to results described above when the cells were stored in HEPES buffer. (c) Freshness of blood samples. Table 1 shows a 5% decrease in viability was obtained following a single thaw if the whole blood was held overnight at room temperature before freezing relative to the results obtained with immediate processing. The standard error of the mean for percentage cell viability in which processing was delayed was more than twice that observed
METHOD
FOR
REFREEZING
RETHAWING TABLE
Percent
Storage
Viability
Conditions LNz Y&PlX
9 to 14 months
90.3 f
Various
Freezing
of storage
LNz vapor and then final 2 mo. -80°C chest
1.30
121
2
of Once-Thawed Lymphocytes Stored Under Conditions for Different Lengths of Time
time
4 to 8 months
LYMPHOCYTES
Upright freezer -80°C
LNz vapor and then final week -20%
N = 10
84.0 f 2.21 N.&a AT = 10
70.0 f 3.55 P < O.Ol* N = 15
26.0 zk 3.23 P < O.Ol* N = 10
85.8 f 2.05 N.Ka !V = 26
75.2 f 5.45 P < 0.01* iv = 10
42.8 f 6.94 P < 0.01* N = 9
24.0 f 5.85 P < O.Ol* N = 10
a N.S., compared to cell stored in LN2 vapor for 4 to 8 months. *P < 0.01, compared to cell stored in LN2 vapor for 4 to 8 months.
with freshly frozen cells. For subsequent use as doubly thawed cells the freshest preparations were preferred since differences obtained after a single thaw became magnified .after the second freeze-thaw. (d) Thawing conditions following a single freeze. Treatment of singly frozen cells, after removal from the freezer, has been simplified. Frozen cells (N = 20 normal lymphocyte donors) thawed in air at room temperature rendered similar results (92 +- 1.2% viable) to cells subjected to a faster thaw in a 37°C water bath (91.5 + 0.9% ), These differences were not significant. Since the diameter of the tubes used for freezing is small, the cells apparently thaw rapidly enough at room temperature (approximately 4O”C/min) to TABLE Effect
of DNase
Age of preparation
1 week
2 B 7 10
to 4 months months months months
n 24 cells/panel.
maintain good viabilities. In addition, post-thaw dilution of the DMSO and cell washing before use on cytotoxicity plates were not found to be necessary. Centrifugation and washing after a single freezethaw of 10 lymphocyte preparations resulted in an average viability of 86.2 * 0.97%. Samples of those same lymphocytes in which post-thaw washing was omitted prior to the cytotoxicity assay resulted in an average viability of 94.0 * 0.69% (P < 0.005). Not only could post-thaw washing be omitted for this test, but higher average viabilities resulted. Temperature and time length of frozen st,orage. The effect of length of storage time on subsequent once-thawed viability varied with the temperature of storage and 3
Treatment After the Second Thaw of Doubly-Thawed Lymphocyte Number prtIL&
2 3 2 1
of
DNase treated: mean viability of panel
89.5 f 0.53 92.0 f 0.79 92.2 f 1.0.5 91.0 f 1.43
on Mean Panelsa
Viability
Number panels
1 4 2 1 1
of
(% f
SE)
No DNase treatment: mean viability of panel
87.7 81.6 56.1 33.5 55.9
f f f f f
1.20 1.58 4.20 4.00 4.10
-
122
SOLLMANN
AND
NATHAN
100 90 -z zso-l 5
P
,”
70-
i I E
60-
s f P
50-
P
40 -
30 -
A
oL 3b
6’0 TIME
do
OF FIRST THAW
FIG. 1. Infiuence of length of first thaw cyte ceil panel after a second freeze-thaw. second freeze-thaw; (B) DNase treatment
(MINUTES)
at either 22” or 4°C on mean viability of 24 lympho(A) No DNase treatment of lymphocytes after the after second freeze-thaw.
its stability. This interaction was significant in most instances studied. TabIe 2 shows that cells fr’ozen with our technique and stored in LN, vapor will retain viabilities of 85 to 90% for at least 14 months. Storage for 2 months in a chest type -80°C freezer will produce similar results. However, the viability significantly decreased if the cells were kept in a -80°C upright freezer in which frequent disturbance resulted in temperature fluctuation. The average viability of cells stored under thlose conditions dropped to 70% within 4 to 8 months and to 42% within a year. Exposure to temperatures of -20°C for 1 week only produced average viabilities of 25%. Doubly thawed cells. Cells used for refreezing had a viability of at least 90% when fresh. All necessary precautions were taken in the initial freezing and storage procedures to maintain that level of viability after the first thaw. In addition, the time length of this thaw during plate preparation was found to be ‘critical. In Fig. l-A, second thaw mean viability of a 24 lymphocyte panel was 84% when it was
subjected to only a 30 min initial thaw period at room temperature during plate preparatio’n. The mean viability of that same panel in which the initial thaw time was extended to 60 min dropped to 66% (P < 0.01). Even when the panel was held on ice during that time, supposedly to reduce DMSO toxicity, the viability was still significantly lower (73% ) than when prepared at room temperature and refrozen within 30 min. Furthermore, when the initial thaw time was 30 min, maintenance of the cells on ice compared to those done at room temperature during this time did not result in better average panel viability after the second thaw. In fact, if the panels were treated with DNase prior to final use ( Fig. 1-B ), room temperature preparations extended as long as an hour resulted in no significant loss of viability following the second freeze-thaw cycle. The majority of refrozen cell panels stored for up to 4 months and used without prior DNase treatment had average viabilities of over 80% after the second thaw (Table 3). However, the viability gradually fell even in LN:, storage and
METHOD
FOR REFREEZING
the need for DNase treatment increased. Use of the enzyme improved preparations 10 months ‘old with an average initial viability of 55% to a final viability of over 90%.
RETHAWING
LYMPHOCYTES
123
perature on various types of tissues and cells (3, 4, 9, 10). While DMSO is undoubtedly toxic in the cases cited, a differential toxicity apparently exists from one cell type to another. Peripheral blood lymphocytes appear to be resistant to DISCUSSION DMSO toxicity at room temperature for at least short periods of time ( 1, 4). The goal of this study was to establish Conditions of frozen storage play a the simplest laboratory conditions needed major role in successful retrieval of viable to produce cytotoxicity test plates conlymphocytes. Storage temperatures must taining viable refrozen rethawed lymphobe stable and maintained at no more than cytes. A detailed study of conditions SUE-80°C. This condition is often difficult to cient for a simplified initial freeze proobtain in a mechanical freezer, however, cedure was undertaken, since cells with because of frequent disturbance, unnoticed excellent viability after a single thaw are voltage drops, frost buildup, and freezer required for successful use in refrozen cell breakdowns. For this reason, vapor phase panels. In the course of that work, the LNZ storage is preferred ( -100” to plastic composition of the storage tube, -150°C) especially for long term storage. buffers in the cryoprotective mixture, freshMaintenance of a brief thaw time during ness of the blood sample at time of freezinoculation of cells onto the microcytoing, and constant low temperature storage toxicity plates is a critical factor. However, conditions were all found to have signifiwe have demonstrated that this operation cant effects on the viability of the onceneed not be performed on ice as previously thawed lymphocytes. suggested (7, 8) provided the period ‘of However, some recommended steps thaw is kept to 30 min. Once exposure noted in the literature for initial lymphotime to DMSO at room temperature or on cyte freezing procedures (5, 6, 11, 13-16) ice exceeds 0.5 hr, viability begins to dewere follnd to be unnecessary under our crease. conditions. For example, a controlled rate The effectiveness ~ofDNase treatment on of freezing was not required for cell presrefrozen rethawed cell plates is impressive. ervation either on the initial freeze, as also Its use is not mandatory in all cases, but observed by Wood et al. ( 16), or on the whenever it is applied, it will improve the second freeze cycle. Also, rather than chillaverage viability of the recovered cells as ing cells prior to freezing (6, 16)) we have well as decrease the viability among obtained excellent results when cells are lymphocytes in the panel. This reagent mixed at room temperature with freshly was originally used to improve fresh cell thawed cryoprotective mixture. In addisuspensions or singly thawed lymphocytes tion, 37°C thaws in water and subsequent with low viability (6 ). To date, we have dilution and washing of the cells prior to made up more than a dozen separate use in cytotoxicity tests did not result in panels of frozen cell plates (N > 300). improved viability. In fact, we observed After DNase treatment the mean percent that centrifugation and washing actually viability 2 SEM was 91.2 * 0.75% even decreased cell viabilities. This effect has for plates almost 1 year old. been previously noted by Bsouroncle (4). The widespread practice of prechilling SUMMARY cells prior to the addition of cryoprotective agents stems from earlier studies showing This report describes simplified methods the toxic effect of DMSO at room tem- for the initial freezing and thawing of hu-
124
SOLLMANN
man lymphocytes and the subsequent use of these cells after refreezing on cytotoxicity plates, storage, and a second thaw. The proposed initial freeze method eliminates some technical inconveniences required previously such as chilling of cells prior to addition of DMSO, preparing cryoprotective mixtures just prior to freezing, controlled rate of freezing and thawing and the washing of cells after thawing. However, pH of the media, blood freshness, type of storage tube used, and constant storage temperature were found to be very important to maintain good cell viability. Most lymphocytes maintain an average viability of 85 to 95% for at least a year when prepared according to the present freezing and thawing technique. When panels of lymphocytes are prepared for refrozen rethawed cytotoxicity test plates, the thaw time between freezes must be brief. Production of test plates on ice, however, was not found to be necessary. As the period of storage of refrozen cells on plates increases, viability of the cells after a second thaw decreases and treatment with DNase to enzymatically remove the dead cells is useful. With this procedure, refrozen rethawed lymphocytes up to a year old can be prepared on microcytotoxicity test plates with average viabilities of 90 * 1%. REFERENCES 1. Birkeland, S. A. The influence of different freezing procedures and cryoprotective agents on the immunological capacity of frozen-stored Iymphocytes, Clyobiology 13, 442-447 ( 1976). 2. Boyum, A. Isolation of leucocytes from human blood-further observations. &and. J. of Clin. Lab. Innest. (Suppl. 97). 20, 31-50 (1968). 3. Bouroncle, B. A. Preservation of living cells at -70°C with dimethylsulfoxide. Proc. of Sot. for Exp. Biol. and Med. 119, 958-961 (1965), U.S.A.
AND
NATHAN
4. Bouroncle, B. A. Preservation of human normal and leukemic cells with dimethylsulfoxide at -80°C. Cryobiology 3, (1967). 5. Chess, L., Bock, G. N., and Mardiney, M. F., Jr. Restoration of reactivity of frozen stored human lymphocytes in the mixed lymphocyte reaction and in response to specific antigens. Transphtation 14, 726733, ( 1972). 6. Fuller, T. C. A simple, inexpensive procedure for freezing, storage and recovery of viable human lymphocytes. In “Manual of Tissue Typing Techniques” (NIH, DHEW Pub. No. 76-545) 190-195 (1976-1977). 7. Nathan, P. Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing, Cryobiology 11, 305-311 ( 1974). 8. Perry, V. P., Martin, J. L., and Droener, C. A. Nutrient medium and frozen storage of human lymphocytes, Cryobiology 12, 386396 ( 1975). 9. Sherman, J. K. Dimethylsulfoxide as a protective agent during freezing and thawing of human spermatozoa. Proc. of Sot. for Erp. Bio. and Med., USA 117, 261-264 (1964). 10. Sherman, J. K. Pretreatment with protective substances as a factor in freeze-thaw survival. Cryobiology 1, 298-300 ( 1965). 11. Simon, J. D., Flinton, L. J., and Albala, M. M. A simple method for the cryopreservation of human lymphocytes at -80°C. Trunsfusion 17, 23-28 ( 1977). 12. Snedecor, G. W., and Cochran, W. G., “Statistical Methods,” 6th ed. Iowa State U. Press, Ames (1967). 13. Stopford, C. R., MacQueen, J. M., Amos, D. B., and Ward, F. E. Some variations in lymphocyte freezing methods which do not affect cell viability. Tissue Antigens 2, 20-26 ( 1972). 14. Strong, D. M., Woody, J. N., Factor, M. S., and Ahmed, A. Immunological responsiveness of frozen-thawed human lymphocytes. Clinical Exp. lmmunol. 21, 442-455 (1976). 15. Thorsby, D., Dubois, R., Bondevik, H., and DuPont, B. Joint report from a mixed lymphocyte culture workshop. Tissue Antigens 4,507-525 ( 1974). 16. Wood, M., Bashir, H., Greally, J., and Amos, D. B. A simple method of freezing and storing live lymphocytes. Tissue Antigens 2, 27-31 (1972).
16,
118-124
(1979)
An Improved Method for Preparing Refrozen Rethawed Human Lymphocytes on Plates for Microcytotoxicity Studies PEG The Paul I. Hoxworth and The Departments
A.
SOLLMANN
AND
The use of refrozen rethawed lymphocyte panels on cytotoxicity plates to screen sera for HLA activity has great advantages for the small tissue typing laboratory where only a limited number of fresh cells are available. Recent studies (7, 8) demonstrated that lymphocytes may be subjected to a double freeze and thaw and still be reliably typed. However, two difficulties remain: (a) from the limited ‘data reported for the doubly thawed cell, the average viability was often lower than desired, and (b) elaborate freezing techniques were recommended, such as controlled rate freezes (S), with careful attention to temperature during both freeze and thaw procedures. Therefore, a study was undertaken with the following objectives: to clarify the factors influencing the viabilities of singly and doubly frozenthawed cells and to develop the simplest freeze-thaw techniques possible without sacrificing cell viability as measured in the cytotoxicity assay. Conditions examined for their influence on the viability of singly thawed lymphocytes were: age of the blood sample, effectiveness of the buffer in the freeze-mixture, type of storage tube used, temperature of cells prior to initial freezing, frozen storage Received 1978.
May
30,
1978;
accepted
October
OOll-2240/79/020118-07$02.00/O @ 1979 by Academic Press, Inc. of reproduction in any form reserved.
NATHAN Burns The Ohio
Institute, University 45267
Cincinnati Unit, of Cincinnati,
temperatures of the cells, and need for careful post-thaw washing of the cells. Variables influencing viability of doubly thawed lymphocytes included length of thaw time during plate preparation and enzymatic treatment of lymphocytes after the second thaw. The present work also evaluated the effect of length of storage time on once and twice frozen-thawed lymphocytes. It is now possible to recommend that some steps used in previous initial and second freeze procedures be eliminated, since viabilities produced by the methods described in this report are as good as those obtained with more elaborate techniques. TECHNIQUES
Heparin (100 units/ml) was added to each fresh blood sample (20 cc) obtained from norma human donors. The lymphocytes were separated on a Ficoll-Hypaque gradient (2). Platelets were removed by adding several drops of 0.2% adenosine diphosphate in 0.05 M potassium phosphate buffer at pH 6.8. The tubes were inverted repeatedly for 30 seconds allowing the platelets to clump. The suspension was then centrifuged at 2OOg’s for 6 seconds. The platelet free supernatant was removed and adjusted to a concentration of 4000 lymphocytes/p1 of media. Unless otherwise
30,
118
Copyright All rights
PAUL
Blood Center, The ShTineTs’ Of Physiology and Surgery, College of Medicine, Cincinnati,
METHOD
FOR
REFREEZING
indicated, all lymphocytes were initially frozen the same day the blood was drawn. Initial freexing procedure. The cell suspensions were centrifuged and the supernatants discarded. Cells were not placed on ice during the subsequent procedures. The lymphocytes were resuspended to a concentration of 4000 cells/PI in a freshly thawed cryoprotective mixture. This mixture was kept frozen in 4 cc aliquots at -80°C and was composed of 10% dimethylsulfoxide ( DIMSO), 50% fetal calf serum ( FCS ), 37% N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffered Media 199 (pH 7.4) and 4% glucose. In the buffer experiment described below, the p.H of separate preparations of Media 199 were adjusted with a solution of saturated NaHCO,. Fifty to 400 ~1 of the cell suspensions were then added to 5 x 45 mm microsample tubes (Kew Scientific, Cat. No. 208). The tubes were placed immediately into loo-partition boxes (Crown Paper Box Company, Indianapolis, Indiana) and stored in the vapor phase of a liquid nitrogen (LN2) freezer (-100” to -150°C) or a -80°C mechanical chest freezer which maintained a constant temperature. After frozen storage, cells were thawed within two and a half minutes in air at room temperature. They were then immediately inoculated into normal human serum to test their viability. A three hour incubation at room temperature was followed by staining with eosin and fixation in formalin. The stained cells were observed by phase microscopy to determine their percent viability. All the cells used in the present report were frozen for as long as three months unless otherwise indicated. The preparation of refrozen cells in tissue typing plates. A set of frozen cells with previously determined fresh cell viabilities of at least 90%, were thawed in air at 22°C and added to 72 well typing trays, (Cooke Products, Costar No. 236-72) as three identical 24 cell panels. Tests indicated that satisfactory lymphocyte viability could
RETHAWING
LYMPHOCYTES
119
be maintained if the total time the cells were thawed did not exceed 30 min. The completed plates were immediately placed into prechilled cardboard boxes in either a -80°C mechanical freezer or the vapor phase of a LN, freezer. Placement in or close to the liquid itself will result in a rate of freeze detrimental to viability. Use of plates with refrozen cells. Test plates were removed from the -80°C or LN, freezer and allowed to come to room temperature for IO min in air. Each well can immediately be inoculated with 2 PI of test sera and standard cytotoxicity procedures begun. However, the cell viabilities were usually better if the lymphocytes were first treated with deoxyribonuclease ( DNase, Sigma Chemical Company, St. Louis, Missouri) to eliminate any dead cells. Five microliters of DNase (2.5 mg/ ml) were inoculated into each well. The plate was floated on a layer of water in a 37°C incubator for 20 min. To remove the enzyme mixture and dead cells from the plates, the cells were then washed twice after which sera to be tested were added ta the plate. Cell washing alone, without the addition of DNase, did not raise the average viability above that of an unwashed panel of lymphocytes. Statistical analysis. Analysis of variance was used to determine if differences existed among sample means. Least significant difference (LSD) intervals were generated in all cases where a significant F test was found to locate specific differences. An arcsin transformation for aormality (12) was performed on the data preceding statistical analysis since the data consisted mainly of percentages skewed towards 100~. Conclusions obtained were the same as when the direct data were analyzed and the latter are presented for simplicity. RESULTS
Several vestigated
factors were to determine
individually intheir importance
120
SOLLMANN
AND TABLE
Effect
of Storage
NATHAN 1
of Blood Samples Prior to Freezing Lymphocytes Following a Single Lymphocytes purified and frozen immediately
‘% Viability &SE. single thaw.
following
a
93.1 f 0.6 N = 40
on Subsequent Thaw
Viability
Whole blood stored 24 hr at IOO~ temperature ; lymphocytes frozen next day
I
;
85.2 f 1.5 N = 27 1
in production of once-thawed preparations with viabilities greater than 90%. Such excellent results are necessary for the successful retrieval of useful lymphocyte preparations after the second freeze-thaw. Except for the variable being tested or otherwise indicated, conditions during the various experiments on once-thawed viability of lymphocytes are those noted in Techniques. (a) Buffer used in cryoprotectant mixture. Lymphocytes from 20 normal donors were frozen in NaHC03 buffered cryoprotectant mixture and stored for approximately one week. Similarly, another set of 43 normal lymphocyte preparations were frozen in a mixture buffered with HEPES, Thirty-four of the cells were stored for a similar time period as above and nine were stored for between 6 and 12 weeks. An average viability * standard error of the mean (SEM) of 92.7 * 0.6% was observed after a single freeze-thaw on those cells frozen with the HEPES buffered cryoprotectant compared to an average 85.8 * 0.9% viability on those frozen with NaHC08 buffered medium (P < 0.001). The pH of NaHC03 buffered cryoprotective mixture visibly becomes more alkaline within two weeks at -80°C and this change may adversely affect cell viability. HEPES, which is not affected by the CO2 in the gas phase above the buffer solution maintains a more stable pH. The cryo-
Purified lymphocytes stored 24 hr at romn temperature frozen next day
87.7 f 1.4 N = 33
P < 0.005
of
N.S.
T P < 0.005
protective mixture buffered with HEPES can be prepared in advance, frozen until needed, thawed, mixed with cells, and stored for long periods at -80°C with little change in pH. (b) Type of tube used. Lymphocytes from each of 31 random normal donors were frozen and stored for approximately one week in polypropylene microsample tubes (550 ~1 capacity, Kew Scientific) and polyethylene microsample tubes (400 ~1 capacity, Beckman Part No. 314326). After a single freeze-thaw cycle the average viability observed in the cells frozen in poIypropylene tubes was 88.6 * 1.1% compared to an average of 79.4 t 2.670 for those stored in polyethylene (P < 0.005). The cause of reduced viability in the Beckman tubes was not ascertained. Cells stored in bicarbonate buffer were used in these tests and this accounts for the lower viability in the polypropylene tubes compared to results described above when the cells were stored in HEPES buffer. (c) Freshness of blood samples. Table 1 shows a 5% decrease in viability was obtained following a single thaw if the whole blood was held overnight at room temperature before freezing relative to the results obtained with immediate processing. The standard error of the mean for percentage cell viability in which processing was delayed was more than twice that observed
METHOD
FOR
REFREEZING
RETHAWING TABLE
Percent
Storage
Viability
Conditions LNz Y&PlX
9 to 14 months
90.3 f
Various
Freezing
of storage
LNz vapor and then final 2 mo. -80°C chest
1.30
121
2
of Once-Thawed Lymphocytes Stored Under Conditions for Different Lengths of Time
time
4 to 8 months
LYMPHOCYTES
Upright freezer -80°C
LNz vapor and then final week -20%
N = 10
84.0 f 2.21 N.&a AT = 10
70.0 f 3.55 P < O.Ol* N = 15
26.0 zk 3.23 P < O.Ol* N = 10
85.8 f 2.05 N.Ka !V = 26
75.2 f 5.45 P < 0.01* iv = 10
42.8 f 6.94 P < 0.01* N = 9
24.0 f 5.85 P < O.Ol* N = 10
a N.S., compared to cell stored in LN2 vapor for 4 to 8 months. *P < 0.01, compared to cell stored in LN2 vapor for 4 to 8 months.
with freshly frozen cells. For subsequent use as doubly thawed cells the freshest preparations were preferred since differences obtained after a single thaw became magnified .after the second freeze-thaw. (d) Thawing conditions following a single freeze. Treatment of singly frozen cells, after removal from the freezer, has been simplified. Frozen cells (N = 20 normal lymphocyte donors) thawed in air at room temperature rendered similar results (92 +- 1.2% viable) to cells subjected to a faster thaw in a 37°C water bath (91.5 + 0.9% ), These differences were not significant. Since the diameter of the tubes used for freezing is small, the cells apparently thaw rapidly enough at room temperature (approximately 4O”C/min) to TABLE Effect
of DNase
Age of preparation
1 week
2 B 7 10
to 4 months months months months
n 24 cells/panel.
maintain good viabilities. In addition, post-thaw dilution of the DMSO and cell washing before use on cytotoxicity plates were not found to be necessary. Centrifugation and washing after a single freezethaw of 10 lymphocyte preparations resulted in an average viability of 86.2 * 0.97%. Samples of those same lymphocytes in which post-thaw washing was omitted prior to the cytotoxicity assay resulted in an average viability of 94.0 * 0.69% (P < 0.005). Not only could post-thaw washing be omitted for this test, but higher average viabilities resulted. Temperature and time length of frozen st,orage. The effect of length of storage time on subsequent once-thawed viability varied with the temperature of storage and 3
Treatment After the Second Thaw of Doubly-Thawed Lymphocyte Number prtIL&
2 3 2 1
of
DNase treated: mean viability of panel
89.5 f 0.53 92.0 f 0.79 92.2 f 1.0.5 91.0 f 1.43
on Mean Panelsa
Viability
Number panels
1 4 2 1 1
of
(% f
SE)
No DNase treatment: mean viability of panel
87.7 81.6 56.1 33.5 55.9
f f f f f
1.20 1.58 4.20 4.00 4.10
-
122
SOLLMANN
AND
NATHAN
100 90 -z zso-l 5
P
,”
70-
i I E
60-
s f P
50-
P
40 -
30 -
A
oL 3b
6’0 TIME
do
OF FIRST THAW
FIG. 1. Infiuence of length of first thaw cyte ceil panel after a second freeze-thaw. second freeze-thaw; (B) DNase treatment
(MINUTES)
at either 22” or 4°C on mean viability of 24 lympho(A) No DNase treatment of lymphocytes after the after second freeze-thaw.
its stability. This interaction was significant in most instances studied. TabIe 2 shows that cells fr’ozen with our technique and stored in LN, vapor will retain viabilities of 85 to 90% for at least 14 months. Storage for 2 months in a chest type -80°C freezer will produce similar results. However, the viability significantly decreased if the cells were kept in a -80°C upright freezer in which frequent disturbance resulted in temperature fluctuation. The average viability of cells stored under thlose conditions dropped to 70% within 4 to 8 months and to 42% within a year. Exposure to temperatures of -20°C for 1 week only produced average viabilities of 25%. Doubly thawed cells. Cells used for refreezing had a viability of at least 90% when fresh. All necessary precautions were taken in the initial freezing and storage procedures to maintain that level of viability after the first thaw. In addition, the time length of this thaw during plate preparation was found to be ‘critical. In Fig. l-A, second thaw mean viability of a 24 lymphocyte panel was 84% when it was
subjected to only a 30 min initial thaw period at room temperature during plate preparatio’n. The mean viability of that same panel in which the initial thaw time was extended to 60 min dropped to 66% (P < 0.01). Even when the panel was held on ice during that time, supposedly to reduce DMSO toxicity, the viability was still significantly lower (73% ) than when prepared at room temperature and refrozen within 30 min. Furthermore, when the initial thaw time was 30 min, maintenance of the cells on ice compared to those done at room temperature during this time did not result in better average panel viability after the second thaw. In fact, if the panels were treated with DNase prior to final use ( Fig. 1-B ), room temperature preparations extended as long as an hour resulted in no significant loss of viability following the second freeze-thaw cycle. The majority of refrozen cell panels stored for up to 4 months and used without prior DNase treatment had average viabilities of over 80% after the second thaw (Table 3). However, the viability gradually fell even in LN:, storage and
METHOD
FOR REFREEZING
the need for DNase treatment increased. Use of the enzyme improved preparations 10 months ‘old with an average initial viability of 55% to a final viability of over 90%.
RETHAWING
LYMPHOCYTES
123
perature on various types of tissues and cells (3, 4, 9, 10). While DMSO is undoubtedly toxic in the cases cited, a differential toxicity apparently exists from one cell type to another. Peripheral blood lymphocytes appear to be resistant to DISCUSSION DMSO toxicity at room temperature for at least short periods of time ( 1, 4). The goal of this study was to establish Conditions of frozen storage play a the simplest laboratory conditions needed major role in successful retrieval of viable to produce cytotoxicity test plates conlymphocytes. Storage temperatures must taining viable refrozen rethawed lymphobe stable and maintained at no more than cytes. A detailed study of conditions SUE-80°C. This condition is often difficult to cient for a simplified initial freeze proobtain in a mechanical freezer, however, cedure was undertaken, since cells with because of frequent disturbance, unnoticed excellent viability after a single thaw are voltage drops, frost buildup, and freezer required for successful use in refrozen cell breakdowns. For this reason, vapor phase panels. In the course of that work, the LNZ storage is preferred ( -100” to plastic composition of the storage tube, -150°C) especially for long term storage. buffers in the cryoprotective mixture, freshMaintenance of a brief thaw time during ness of the blood sample at time of freezinoculation of cells onto the microcytoing, and constant low temperature storage toxicity plates is a critical factor. However, conditions were all found to have signifiwe have demonstrated that this operation cant effects on the viability of the onceneed not be performed on ice as previously thawed lymphocytes. suggested (7, 8) provided the period ‘of However, some recommended steps thaw is kept to 30 min. Once exposure noted in the literature for initial lymphotime to DMSO at room temperature or on cyte freezing procedures (5, 6, 11, 13-16) ice exceeds 0.5 hr, viability begins to dewere follnd to be unnecessary under our crease. conditions. For example, a controlled rate The effectiveness ~ofDNase treatment on of freezing was not required for cell presrefrozen rethawed cell plates is impressive. ervation either on the initial freeze, as also Its use is not mandatory in all cases, but observed by Wood et al. ( 16), or on the whenever it is applied, it will improve the second freeze cycle. Also, rather than chillaverage viability of the recovered cells as ing cells prior to freezing (6, 16)) we have well as decrease the viability among obtained excellent results when cells are lymphocytes in the panel. This reagent mixed at room temperature with freshly was originally used to improve fresh cell thawed cryoprotective mixture. In addisuspensions or singly thawed lymphocytes tion, 37°C thaws in water and subsequent with low viability (6 ). To date, we have dilution and washing of the cells prior to made up more than a dozen separate use in cytotoxicity tests did not result in panels of frozen cell plates (N > 300). improved viability. In fact, we observed After DNase treatment the mean percent that centrifugation and washing actually viability 2 SEM was 91.2 * 0.75% even decreased cell viabilities. This effect has for plates almost 1 year old. been previously noted by Bsouroncle (4). The widespread practice of prechilling SUMMARY cells prior to the addition of cryoprotective agents stems from earlier studies showing This report describes simplified methods the toxic effect of DMSO at room tem- for the initial freezing and thawing of hu-
124
SOLLMANN
man lymphocytes and the subsequent use of these cells after refreezing on cytotoxicity plates, storage, and a second thaw. The proposed initial freeze method eliminates some technical inconveniences required previously such as chilling of cells prior to addition of DMSO, preparing cryoprotective mixtures just prior to freezing, controlled rate of freezing and thawing and the washing of cells after thawing. However, pH of the media, blood freshness, type of storage tube used, and constant storage temperature were found to be very important to maintain good cell viability. Most lymphocytes maintain an average viability of 85 to 95% for at least a year when prepared according to the present freezing and thawing technique. When panels of lymphocytes are prepared for refrozen rethawed cytotoxicity test plates, the thaw time between freezes must be brief. Production of test plates on ice, however, was not found to be necessary. As the period of storage of refrozen cells on plates increases, viability of the cells after a second thaw decreases and treatment with DNase to enzymatically remove the dead cells is useful. With this procedure, refrozen rethawed lymphocytes up to a year old can be prepared on microcytotoxicity test plates with average viabilities of 90 * 1%. REFERENCES 1. Birkeland, S. A. The influence of different freezing procedures and cryoprotective agents on the immunological capacity of frozen-stored Iymphocytes, Clyobiology 13, 442-447 ( 1976). 2. Boyum, A. Isolation of leucocytes from human blood-further observations. &and. J. of Clin. Lab. Innest. (Suppl. 97). 20, 31-50 (1968). 3. Bouroncle, B. A. Preservation of living cells at -70°C with dimethylsulfoxide. Proc. of Sot. for Exp. Biol. and Med. 119, 958-961 (1965), U.S.A.
AND
NATHAN
4. Bouroncle, B. A. Preservation of human normal and leukemic cells with dimethylsulfoxide at -80°C. Cryobiology 3, (1967). 5. Chess, L., Bock, G. N., and Mardiney, M. F., Jr. Restoration of reactivity of frozen stored human lymphocytes in the mixed lymphocyte reaction and in response to specific antigens. Transphtation 14, 726733, ( 1972). 6. Fuller, T. C. A simple, inexpensive procedure for freezing, storage and recovery of viable human lymphocytes. In “Manual of Tissue Typing Techniques” (NIH, DHEW Pub. No. 76-545) 190-195 (1976-1977). 7. Nathan, P. Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing, Cryobiology 11, 305-311 ( 1974). 8. Perry, V. P., Martin, J. L., and Droener, C. A. Nutrient medium and frozen storage of human lymphocytes, Cryobiology 12, 386396 ( 1975). 9. Sherman, J. K. Dimethylsulfoxide as a protective agent during freezing and thawing of human spermatozoa. Proc. of Sot. for Erp. Bio. and Med., USA 117, 261-264 (1964). 10. Sherman, J. K. Pretreatment with protective substances as a factor in freeze-thaw survival. Cryobiology 1, 298-300 ( 1965). 11. Simon, J. D., Flinton, L. J., and Albala, M. M. A simple method for the cryopreservation of human lymphocytes at -80°C. Trunsfusion 17, 23-28 ( 1977). 12. Snedecor, G. W., and Cochran, W. G., “Statistical Methods,” 6th ed. Iowa State U. Press, Ames (1967). 13. Stopford, C. R., MacQueen, J. M., Amos, D. B., and Ward, F. E. Some variations in lymphocyte freezing methods which do not affect cell viability. Tissue Antigens 2, 20-26 ( 1972). 14. Strong, D. M., Woody, J. N., Factor, M. S., and Ahmed, A. Immunological responsiveness of frozen-thawed human lymphocytes. Clinical Exp. lmmunol. 21, 442-455 (1976). 15. Thorsby, D., Dubois, R., Bondevik, H., and DuPont, B. Joint report from a mixed lymphocyte culture workshop. Tissue Antigens 4,507-525 ( 1974). 16. Wood, M., Bashir, H., Greally, J., and Amos, D. B. A simple method of freezing and storing live lymphocytes. Tissue Antigens 2, 27-31 (1972).
