Low-Temperature Preservation of the Biomphalaria glabrata Cell Line

JOURNAL OF INVERTEBRATE PATHOLOGY 29, 332-337 (1977)

Low-Temperature Preservation of the Biomphalaria glabrata Cell Line CHRISTOPHER J.

B A Y N E , J O W E T T C H A O , 1 AND PEGGY SALVATORE

Department of Zoology, Oregon State University, Corvallis, Oregon 97331 Received April 8, 1976

Biomphalaria glabrata embryo (Bge) cells are tolerant of a wide range of conditions for preservation at low temperature. It is recommended that the cells, suspended in complete medium with 10% DMSO, be frozen at a rate of 0.5-2°C/min. If a controlled-rate freezer is not available, successful freezing is achieved by placing the lightly insulated sealed ampoule (Fig. 1) into the freezing compartment of a refrigerator (about - 16°C) for 1 hr, then transferring it directly to an ultradeep freeze at about -86°C for another hour before removing it from the insulation and placing it into liquid nitrogen at -198°C for semipermanent storage. There is minimal lag in growth after the cells are thawed. It is apparent that, if 5 x l0 Bor more cells are used for freezing, a certain and quick recovery of the culture after thawing is assured. Using this technique, we have accumulated a bank of 38 frozen ampoules in which cells are preserved with the genetic constitution inherent to this cell line at the fifty-sixth to sixty-third passage.

INTRODUCTION The recent establishment of a cell line from the schistosome intermediate host snail Biomphalaria glabrata (Hansen, 1976) has led to the possibility of answering numerous previously unapproachable questions relating to host-parasite interactions and basic cell biology of the host snail. It is unrealistic, however, to presume that the cell line will be rapidly incorporated into the research frameworks of many laboratories. So long as such a valuable tool is in the custody of only a few laboratories, it remains an essential safeguard to maintain a bank of cells available for shipment to interested investigators. This, then, is one reason why we have experimented with procedures for freeze preservation of the B. glabrata embryo (Bge) cell line. An equally important reason is that the line, currently in its seventy-eighth passage (July, 1976), is susceptible, as is any cell line, to genetic alteration as it is continued. By cryopreservation of a bank of cells in relatively early passages, we insure the retention of genetic characteristics (Bayne, Allen, Owczarzak, and Salvatore, unpubl.) possessed by the newly established cell line. Presently there i Present address: Department of Biology, University of California, Los Angeles, California 90024.

is no indication that further lines can be developed at will from snail tissues (Bayne et al., 1975). An essential element of responsible management of frozen cell banks is that not all of the frozen cells be kept at the same location (Rinfret, 1975). With these considerations in mind, we have sought to find the simplest method which can, with a wide margin of safety, be used for long-term preservation of the snail cells. Since the completion of this work, DiConza and Basch (1976) have published a note on freeze-preservation of this same cell line. It is hoped that the present report of a simple method for freeze-preservation will lead other investigators to put aside populations of these cells for future research.

MATERIALS AND METHODS The Bge cells are grown at 26°C in Falcon T-30 plastic flasks, each with 3 ml of culture medium,pH 7.4, prepared as follows for 100 ml: 22 ml of Schneider' s Drosophila medium (GIBCO No. 172), l0 ml of fetal calf serum 2 2 Variability in the growth of the snail cell line has frequently been shown to be due to the variable properties of different batches of fetal calf serum. The investigators should screen several batches before selecting one to buy in quantity. 332

Copyright © 1977by AcademicPress, Inc. All rightsof reproductionin any formreserved.

ISSN 0022-2011

333

CRYOPRESERVATION OF SNAIL CELLS

(FCS) heat inactivated at 56°C for 30 min, 130 mg of galactose, 450 mg of lactalbumin hydrolysate, 100 t~g/ml of gentamicin, and water to 100 ml. If necessary, the p H can be adjusted with 0.1 N HC1 or 0.1 NaOH. The complete medium is sterilized by filtration through a 0.45-/zm Miilipore filter. Cells can be harvested by use of 0.5 mM EDTA in Hansen's buffered saline (Hansen 1976), or, preferably, by the use of 0.05% trypsin followed by flushing with complete medium and suspension in this medium, or, due to the weak adherent properties of the cells, by simple flushing of complete medium with a bent-tip Pasteur pipet. Cells to be frozen were obtained from cultures which were fed with new medium 2 days previously. The medium was removed from the flask and replaced by 1 ml of 0.05% trypsin in Hansen's buffered saline. After only 15-20 sec at room temperature, the flask was tilted, and the enzyme solution was removed by pipet. One milliliter of complete medium was then introduced into the flask by a bent-tip Pasteur pipet. The medium, which, due to the FCS present, inactivated the trypsin, was pulled in and out of the pipet directed at the monolayer to wash the cells off the plastic culture surface. Each T-30 flask (25-cm 2 growth area) contains 1-5 x 106 cells in a culture which is approaching the end of log-phase growth.

~

l

snap cap

, ....... / , , , J, ~ , ~..~

~

',': 1 ~l'~fl,

Approximately 1 x 106 cells in 1 ml of complete medium were then dispensed into 1 ml of precooled (10°C) 20% Millipore-filtered glycerol (Baker Analyzed reagent) or DMSO (dimethyl sulfoxide, Matheson, Coleman, and Bell, reagent grade) in a freezing ampoule or 2-ml vial. After 30 rain, during which time the cryoprotectant is presumed to come to equilibrium within the cell cytoplasm, the ampoules were sealed in a gas-air flame. To facilitate later identification and recovery from the liquid nitrogen container, a labeled string was tied to each ampoule. The basic procedure for cryopreservation of cells was similar to that given by LaSalle (1975). One experiment was done with an electronically controlled freezing apparatus set at a cooling rate of 1.4°C/rain. Slow cooling in other experiments, 14 in total, was achieved by placing ampoules in insulated 75 x 25-mm vials and holding them for some time in each of several stepwise lower temperatures. For example, an insulated ampoule (Fig. IA) was first placed in the freezing compartment at - 16°C for 1 hr followed by 1 hr in an uitradeep freezer at -86°C, and, finally, the ampoule was removed and placed into liquid nitrogen at -198°C for long-term preservation. The protocol just described proved optimal after several variations were tested for simplification and improvement of the procedure. A successful variation in the procedure ,______-- c ap

7 p l a s t i c ,vial

v ~ r a p p e r , for iqsdJc]tlO n dbsoiute e~hanol

ampule COP!a r/,rlg

95ml

(el'S in

' ~

~,

@

crLjoprotectd

®

~ampu'e a " t h ce',',s ,r/ c r , j o p r o + e ~ t a n T

FIG. 1. A m p o u l e s within the two alternative insulators used: (A) in a 75 × 25-mm plastic snap-cap vial; (B) in a 100 ml s e r u m bottle filled with absolute ethanol.

334

BAYNE, CHAO, ANDSALVATORE ]4

A!

,o_S// a.

/11 TRYPSIN TRYPSIN EOTA MECHANICAL

6

c~

I

~ w o

EDTA

2

-30

' -2@

-EO

0

HOURS RELATIVE

I0 TO

20

50

40

50

PASSAGING

FIG. 2. Cell numbers in flasks prior to and after passaging by each of four methods. Cells harvested by (0) mechanical flushing with medium only; (4) loosened with 0.5 mM EDTA in HBS before flushing with medium; (11) loosened by exposure to 0.05% trypsin in HBS for 30 sec before flushing with complete medium; and (&) loosened with 0.05 mM EDTA, 0.05% trypsin in HBS for 30 sec before flushing with complete medium. involved the use of 100-ml serum bottles (GIBCO) in place of the 75 × 25-mm plastic vials for insulation (Fig. 1B). Each bottle containing an ampoule with cell suspension was filled with 100 ml of absolute ethanol at room temperature and was then transferred from one temperature to the next lower one at 60-min intervals, until the ampoule was ready for thawing or for being transferred to liquid nitrogen. Thawing was completed in less than 60 sec by placing the frozen ampoule directly into water at room temperature (25°C). When thawed, the sample was transferred into a centrifuge tube and was mixed with 1, 2, and 4 ml of fresh medium, at 15-min invervals to gradually dilute the cryoprotectant. Fifteen minutes after the last dilution, the tube was centrifuged at 170g for 5 rain, and the supernatant was withdrawn by pipet. The cell pellet was loosened in 3 ml of culture medium by pipetting, and cells were transferred to a T-30 flask. T w e n t y points were premarked on the bottom of the flask with a needle for exact fields of cell counts. At 100× it was, with practice, easy to distinguish live from moribund and dead cells, on the

basis of whether or not they were attached to the substrate, by the degree o f refractility, and by cell shape. Trypan blue is not an effective agent for determining viability of these molluscan cells. The total number of live cells in each flask was calculated from the numbers in the 20 fields at 100×. The controls were treated identically except that there was no freezing step; the prefreezing and cryoprotectant dilution steps were the same as those of the freezing experiments.

RESULTS AND DISCUSSION Mortality occurs at several stages in the transfer and cryopreservation procedure, and we have determined this quantitatively as follows. By making counts of cells in culture flasks 25-30 hr before and shortly prior to passaging and at 17-21 and 4 2 - 4 6 hr after passaging, it was shown that trypsinization was the method of choice to loosen the cells (Fig. 2); mortality was lowest (24%) when 0.05% trypsin was used and highest (65%) when 0.5 mM EDTA was used. Hydraulic flushing of cells without the aid of e n z y m e or chelating agent is also

CRYOPRESERVATION

OF

TABLE

SNAIL

335

CELLS

1

R E C O V E R Y OF C E L L S A F T E R V A R I O U S F R E E Z I N G P R O C E D U R E S Number of cells recovered Experiment Mc Mt M2 M3

Treatment

Percentage

Earliest count

Extrapolated value on thaw + 1 day

of recovery

5.4 x 105 4.1 × 105 0.76 x 104 2.1 x 104 1.3 x 105

Counted thaw + 1 day Counted thaw + 1 day 0.6 x 104 1.45 x 104 Counted thaw + 1 day

100 76 I1 27 24

M4

Control" 24 Hr, at 86°C 24 Hr, at 86°C and 5 days at 6Days at -86°C 12 Days at -86°C

Nt Nc N

Simple transfer (trypsin) ControF' 24 Hr at - g 6 ° c

3.7 x 105 2.4 × 1@ 2.5 x l0 s

Counted thaw + 1 day Counted thaw + 1 day Counted thaw + 1 day

100 65 68

Kc K~ K2 K3 K4

Control" 3 Days at - 16°C, 104)~,DMSO 3 Days at -86°C, 10% DMSO 3 Days at 16°C, 10% glycerol 3 Days at 86°C, 10% glycerol

12.6 x 103 0.4 x 10~ 4.6 × 105 3.5 x 104 3.7 x 104

Counted Counted Counted Counted Counted

1 day 1 day 1 day 1 day 1 day

100 3 37 28 29

Gc G~ G2 G3 G~

Control" 3°C (1.5 hr), 16°C(1 hr), 86°C (0.5 hr), 198°C(10 days) 16°C (1.5 hr), - g 6 ° c (0.5 hr), -198°C (10 days) 16°C (I.5 hr), -198°C (10 days) 3°C (1.5 hr), -16°C (I hr), -198°C (10 days)

7.6 3.0 4.2 1.0

Counted thaw + 1 day 2.8 x 104 4.0 x 104 0.g x 104 No living cells

100 37 53 11 0

198°C

× x x x

10~ 104 10~ 104

thaw thaw thaw thaw thaw

+ + + + +

" Control: cells suspended in 10% DMSO, not frozen, but then removed by dilution and centrifugation.

satisfactory for passaging with an accept- DMSO, in our laboratory, Bge cells slowly able cell mortality (41%). return to normal growth rates after 5 days The superior protection provided by in 7.5% DMSO at 26°C; they do not survive DMSO was evident from an experiment in 5 days in 7.5% glycerol at 26°C. Some mortality is also caused by the which cells were frozen and stored for 11 days at -198°C in either 10% glycerol or actual freezing and thawing of the cells, 10% DMSO; survival was 12 and 42% of and it was the main purpose of our expericontrol values, respectively. Another ex- ments to determine which procedure would periment showed that 10% DMSO was lead to the least mortality from this cause. better than 7.5 and 5% DMSO, for which Variables included the chemical nature of survival values were 63 and 16% of the 10% the cryopreservative (glycerol, DMSO, DMSO value 10 days after thawing. In a sucrose), the rate of cooling, the lowest third experiment in which the cells were not temperature achieved, and the time spent at frozen, a measure was obtained of cell this lowest temperature (Table 1). Conclumortality due to the addition of DMSO and sions which can be drawn include the folits later removal by gradual dilution and lowing. The death rate of cells at -86°C centrifugation. Expressed as a percentage of (Expt M, Fig. 3) is marked during the first those surviving direct passaging, survival several days of storage, indicating the need with this (control) procedure was 65%. We to store cells at liquid nitrogen temperature have found that, at normal growth tempera- (-198°C). However, in another experiture (26°C), primary cultures of adult cells ment, some survival was observed even (Bayne, et al., 1975) do not suffer from any after 3 days at - 16°C so long as a cryoprotoxicity, even when maintained in the tectant was present; all cells died in 20 hr presence of 7.5% DMSO for 5 days. In at this temperature without cyroprotectant. marked contrast to DiConza and Basch's Although percentages of survival cannot (1976) experience of marked toxicity of be confidently compared between experi-

336

BAYNE, CHAO, AND SALVATORE

ments due to variation, it is apparent that passage to liquid nitrogen temperatures is best done within an hour or so of reaching G1 -86°C. This is evident from a comparison of Ms with M3 (Fig. 3) (8.2 and 23% survival vs control from cell counts 4 days after t.~ -M4 . thawing) and Exp G in which survival was iii 53% when a cell suspension (G2) in 10% DMSO was cooled at - 16°C for 1.5 hr and -86°C for only 0.5 hr before transfer to ." a liquid nitrogen. P a s s a g e from f r e e z e r t e m p e r a t u r e (-16°C) directly to liquid nitrogen is considerably less damaging than a protocol with 1.5 hr at 3°C before the - 16°C step; survival U x / / values were as follows: -16°C (1.5 hr), / -198°C (10 d a y s ) = 11%; 3°C (1.5 hr), / -16°C (1 hr), -198°C (10 days) = 0%. In the electronically controlled freezing rate 0 2 4 I0 12 experiment, mortality was high in all experiDAYS AFTER THAW mental ampoules and in the control, making FIG. 3. Growth rates of Bge cells after freezeinterpretation difficult. The rate used was preservation. Experiment G: Gc = control. G~ = 4°C, 1.4°C/min from 25 to -60°C, then trans- 1.5 hr; then -16°C, 1.5 hr; then -86°C, 0.5 hr; then fer to liquid nitrogen. Each frozen culture, -198°C, 10 days. Gz = - 1 6 ° C , 1.5 hr; then -86°C, thawed after 10 days at - 198°C, was equally 0.5 hr; then -198°C, 10 days. G3 = -16°C, 1.5 hr; -198°C, 10 days. G4 (not plotted: no live cells) as viable as the control (no freezing), irre- = 3°C, 1.5 hr; then -16°C, 1.0 hr; then -198°C, 10 spective of the nature of the cryoprotectant: days. Experiment M: Mc = control. M1 = - 8 6 ° C , 1 10% DMSO, 15% DMSO, or 10% glycerol. day. Mz = -86°C, 1 day; then -198°C, 5 days. M3 From these data and from measurements = -86°C, 6 days. M4 = -86°C, 12 days. taken of cooling rates in the stepwise procedure used for other experiments, it Sucrose at 10 or 5% provided no protection is evident that a steady rate of cooling at against freezing damage. about 0.5-2°C/min is optimal for these The Bge cells returned to active proliferacells. This rate is within the range normally tion surprisingly rapidly after thawing (Figs. used for cryopreservation of animal cells 2, 3). This is true of cultures after being (Rinfret, 1975). frozen 119 days, the longest period yet Some cells revived when returned to 26°C tested, and there is no indication that "inafter 4-12 days at 3°C without cryoprotec- definite" storage will be any problem. The tant, but they were dead after being exposed average growth rate during active proliferato - 16°C for 20 hr. The addition of either tion is calculated to give a population DMSO or glycerol to 10% prior to cooling doubling time of 3.4 days. The resilience to these two temperatures led to survival. In of these cells is also evident from the findsome experiments (-16°C, 3 days), glycerol ing that as few as 1 × 104 or even 1 × 103 was the superior cryoprotectant, whereas, cells proved sufficient for initiating a new in others (-86°C, 3 days), DMSO proved culture in 2 ml of medium in a T-30 flask. superior. The more important consideration Where possible, of course, more cells, e.g., in selecting a cryoprotectant is considered 5 × 105, should be used. to be freshness; old stocks apparently beFarrant et al. (1974) have indicated that, come increasingly toxic (Shannon, 1972). at least for certain mammalian cell cultures,

/.Me

:¢;/

CRYOPRESERVATION OF SNAIL CELLS

protection from temperature change d a m a g e is conferred by holding the cells at a specific subzero t e m p e r a t u r e for a period in excess of 10 min before continued cooling to storage temperature. F o r m a m m a l i a n cells, the critical c r y o p r o t e c t i v e t e m p e r a t u r e was - 2 6 ° C (variation dep e n d e d upon cell line). I f this p h e n o m e n o n is found to be m o r e widespread, it will p r o b a b l y be the key in understanding the nature of c r y o p r e s e r v a t i o n . While we h a v e not specifically u n d e r t a k e n to determine whether or not a similar p h e n o m e n o n occurs in snail cell cultures, some of our data indicate that it p r o b a b l y does. Sudden cooling to - 1 9 8 ° C from - 1 6 ° C is very damaging, whereas such damage is not o b s e r v e d if the cell suspension is placed at - 8 6 ° C e v e n for only 30 min before transfer to liquid nitrogen. Cooling at - 8 6 ° C for longer than necessary (30 min to 2 hr) should be avoided, since cell viability is not maintained well at this temperature. The possibility of an " o p t i m a l protective t e m p e r a t u r e " and time (Farrant et al., 1974) prior to the drastic cooling to c r y o p r e s e r v a t i v e t e m p e r a t u r e s is certainly worthy of further investigation.

ACKNOWLEDGMENTS Funds were provided by National Institutes of

Health, NIAID contract, NIH-NIAID-22-527.The orig-

337

inal manuscript was improved after critical reading by our colleagues A. Owczarzak and J. Allen, to whom we express our thanks.

REFERENCES BAYNE, C. J., OWCZARZAK,A., AND NOONAN, W. E. 1975. In vitro cultivation of cells and a microsporidian parasite of Biomphalaria glabrata. Ann. N. Y. Acad. Sci., 266, 513-529. DICONZA, J. J., AND BASCH,P. F. 1976. Cryopreservation of Biomphalaria glabrata (Mollusca) cells. J. Invertebr. Pathol., 27, 273-274. FARRANT, J., KNIGHT, S. C., MCGANN, L. E., AND J. O'BRIEN. 1974. Optimal recovery of lymphocytes

and tissue culture cells following rapid cooling. Nature (London) 249, 452-453.

HANSEN, E. L. 1976. A cell line from embryos of Biomphalaria glabrata (Pulmonata): Establishment and characteristics. In "Invertebrate Tissue Culture: Research Applications" (K. Maramorosch, ed.). Academic Press, New York. LASALLE, B. 1975. Introduction. In "Round Table Conference on the Cryogenic Preservation of Cell Cultures" (A. P. Rinfret and B. LaSalle, eds.). National Academy of Sciences, Washington, D.C. RINFRET, A. P. 1975. Comments on various aspects of the cryogenic preservation of cell cultures. In "Round Table Conference on the Cryogenic Preservation of Cell Cultures" (A. P. Rinfret and B. LaSalle, eds.). National Academy of Sciences, Washington, D.C. SHANNON,J. E. 1972. Recommendations on the freezing of animal cell lines. In "Registry of Animal Cell Lines." American Type Culture Collection, Rockville, Md.