PROCEDURE NO. 76180

A TWO-STEP TECHNIQUE FOR THE CRYOPRESERVATION OF NORMAL MOUSE M A M M A R Y TISSUE

Michael Rosenberg, Lisa Larson, and Satyabrata Nandi

Cancer Research Laboratory and Department of Zoology University of California

Berkeley, California 94720

SUMMARY: Dimethylsulfoxide is used as a cryoprotectant for the preservation of mammary tissue to be used in primary cell culture. The method involves a two-step process that reduces the sample temperature to -20 ° C for a short-time, which is followed by storage at -90 ° C or immersion into liquid nitrogen. Comparison between fresh and frozen-thawed tissue was made by the cells' ability to grow inside collagen gel in response to various hormones and growth factors. Growth of frozen-thawed and collagenase-digested tissue achieved 70 to 90% the DNA values of their nonfrozen counterparts.

Key words: two-step; cryopreservation; mammary tissue; DMSO.

I. INTRODUCTION

We have developed a procedure for the cryo- preservation of mouse mammary tissue, bor- rowing from descriptions for a two-step method in earlier publications {1-4). The purpose of holding cells initially at a subzero temperature is to protect against the formation of intracellu- lar ice and, ultimately, membrane damage upon thawing. The rate at which the temperature is lowered affects cell shrinkage and the build up of toxic solute concentrations. This method does not, however, require a temperature- controlled unit to produce a cooling rate com- patible for mammary tissue (mouse or human) and is applicable to the culture of enzymati- cally dissociated tissue, as well as that from cell lines.

II. MATERIALS

A. Mechanical dissociation equipment

Teflon chopping block; 5 x 5 cm, 7-mm thick Polystyrene petri dishes, 100-mm diam x

15 mm, D-1906, American Scientific Products '

Single-edge razor blade and paint-scraping adapter (for easy handling)

Forceps, D-2568-1'

B. Cryogenic equipment

Tissue Tek, disposoplastic grids; rectangular dimensions 2.5 x0.8 cm (have to be cut specifically to fit inside cryotube). Immerse

grids in 70% ETOH to sterilize after cutting. Cryotubes, 2-ml capacity with screw cap and

writing area, 1073-1, GIBCO ~ India ink and pen or other labeling system that

is semipermanent on the tube and resists brief exposure to lab solvents and immersion in liquid nitrogen

Freezer, to -20 ° C Freezer, to -90 ° C, Revco Inc., Ultralow 3 Liquid nitrogen tank, type XR-16 (25 liters),

Linde Co. 4 Aluminum canes, tube adaptable for organiza-

tion into liquid nitrogen tank canisters, C- 100, Vangard Intl., Inc. »

Aluminum canisters, to hold aluminum canes for immersion into liquid nitrogen, V-255

C. Cryoprotective medium

Medium 199,320-11502 Supplementation of Medium 199 with 30 ~g/ml penicillin-G, PEN-NA, Sigma6; 50 ~g/ml streptomycin sulfate, S-65016; 50 ~g/ml gentamycin sulfate, G-36326; and 2.5 gg/ml amphotericin B, A548886 is later referred to as the Basal Medium 199.

DMSO, D58796 Serum, porcine, Sterile Systems, Inc. 7 Insulin, bovine, solubilized in 0.05 N HCL at a

concentration of 10 mg/ml, sterilized through a 0.22-~m filter, 155006

D. Miscellaneous equipment

Millex-GV sterilizing filter unit, SLGV025LS, Millipore Corp. 8

Journal of Tissue Culture Methods Vol. 8, No. 1 ,1983 37 « 1984 Tissue Culture Association, Inc.

ROSENBERG, LARSON, AND NANDI -- PROCEDURE NO. 76180

Corning conical eentrifuge tubes, Corning 9

Pipettes, serologieal, 53221-030, VWR '°

25330, 4. The tissue is now ready for enzymatic dis- sociation (6,7).

III. PROCEDURE

A. Work with t issue under laminar flow hood with instruments appropriately sterilized.

B. Prepare cryoprotective medium. For a 50-ml vol combine 40 ml Basal Medium 199; 5 ml DMSO (10% vol/vol); 5 m l serum (10% vol/vol); 10/ag/ml insulin.

C. Prepare tissue for freezing.

1. Remove mammary tissue from miee by the technique deseribed in the TCA Manual (5).

2. Collect glands into a 50-ml tes t tube that is partially filled with Medium 199 to bathe the t issue temporarily.

3. Transfer t issue onto a Teflon ehopping block with foreeps and minee the t issue finely using a razor blade, which has been inserted onto a paint seraper for ease in holding, at a 90 ° angle.

4. Remove plastie grid from ethanol and dry aseptieally.

5. Using foreeps, eover both sides of the grid with t issue (approx. 0.5 g).

6. Place t issue-supporting grid into the eryo- tube and add 1.5 ml eryoproteetive medium so that the t issue is eompletely eovered.

7. Cap tightly.

8. Set the tube on iee for 15 min.

D. Cool the t i ssue .

1. Snap cryotube onto an aluminum eane.

2. Transfer eane to -20 ° C freezer for 30 min.

3. Transfer eane to - 90 ° C freezer for 30 min.

4. Submerge eane slowly into liquid nitrogen tank for indefinite storage.

CAUTION: liquid nitrogen will boil over from too rapid an immersion of cane into tank. Wear gloves and a face shield.

E. Thaw

1. Remove aluminum cane from liquid nitrogen tank.

2. Plunge tube into 3 7 ° C water bath and agitate gently for rapid thawing.

3. Dilute DMSO from tissue by removing tissue from grid and placing in a fluted Erlenmeyer flask at a ratio of approximately 1 g tissue/10 ml Medium 199.

IV. DISCUSSION

Successful cryopreservation depends not only on tailoring the variables of freeze~thaw conditions to fit a specific cell type but also on the post- thaw conditions by which cells are ult imately judged to be functional. Our indica- tor of damage suffered during the freeze-thaw procedure was measured by growth of collagenase-dissociated mouse mammary epi- thelial cells inside collagen gel with serum-free and serum-containing medium supplemented with various hormones and growth factors. The details of the dissociation and culture pro- cedure are described elsewhere (6 and Richards et al., submit ted for publication). Through re- peated application of this growth test to numerous freezing protocols we found that thawed mammary cells achieved 80% growth of their nonfrozen counterparts over equivalent culture periods, as judged by DNA assay (7).

Ten percent DMSO was found to serve as a more effective cryoprotectant than did a 5% DMSO/5% glycerol mixture. Also, mammary tissue that was first subj ected to collagenase or pronase treatment, or both, did not exhibit as high a level of viability upon thawing as did nondissociated, frozen tissue from the same source. Holding tissue at -20 ° C for prolonged periods up to 1.5 h decreased the tissues' viability in culture, presumably by prolonging the cooling rate. No experiment was under- taken to examine the preservation of mammary tissue in liquid nitrogen for a time greater than 1 mo.

Finally, this procedure may be suitable for the frozen storage of normal and neoplastic mammary tissue from mouse, rat, and human origins: both primary and cell line derived. As such, the method may provide a means of bank- ing t issues for those working in breast cancer research who need to store a supply of t issue for future experimentation.

V. REFERENCES

1. Ashina, E. Prefreezing as a method enabling animals to sur- vive freezing a t an extremely low temperature. Nature 184: 1003-1004; 1959.

2. Walter, C.A.; Knight , S.C.; Far rant , J. Ul t ras t ruc tura l appearance of freeze-subst i tuted lymphocytes frozen by in te r rupt ing rapid cooling with a period a t - 26 ° C. Cryobiology 12: 103-109; 1975.

3. McGann, L. E.; Far rant , J. Survival of t issue culture ceUs frozen by a two-step procedure to -196 ° C. Cryobiology 13: 261-268; 1976.

38 Journa l of Tissue Culture Methods Vol. 8, No. 1, 1983

ROSENBERG, LARSON, AND NANDI -- PROCEDURE NO. 76180

4. Farrant, J.; Walter, C. A.; Lee, H.; McGann, L. E. Use of two-step cooling procedures to examine factors influenc- ing cell survival foUowing freezing and thawing. Cryo- biology 14: 273-286; 1977.

5. Foster, R. C,; Feldman, M. K. Techniques for mammary epi- thelial cell isolation and cultivation. TCA Manual 1: 27-30; 1975.

6. Imagawa, W.; Tomooka, Y.; Yang, J.; Guzman, R.; Richards, J.; Nandi, S. The isolation and serum-free cultivation of

mammary epithelial cells within a coUagen gel matrix. Sato, G.; Sirbasku, D.; Barnes, D., eds. Methods for preparation of serum-free culture media and methods for growth of cells in serum-free culture. New York: Alan R. Liss, Inc. In press.

7. Hindegardner, R.T. An improved fluorimetric assay for DNA. Anal. Biochem. 30: 197-201; 1971.

1 American Scientific Products, Sunnyvale, CA. 2 GIBCO, Santa Clara, CA.

Revco, Inc. West Columbia, SC. 4 Linde Co., San Francisco, CA. » Vangard International, Inc., Neptune, NJ.

6 Sigma Chemical Co., Saint Louis, MO. 7 Sterile Systems, Inc., Logan, UT. 8 Millipore Corp., Bedford, MA. 9 Corning Glass Works, Corning, NY.

,o Van Waters & Rogers, San Francisco, CA.

This investigation was supported by PHS Grants CA05388 and CA09041 awarded by the National Cancer Institute, DHHS, and in part from funds from the Committee on Research, University of California, Berkeley.

Journal of Tissue Culture Methods Vol. 8, No. 1, 1983 39