CRYO-CONSERVATION MEDIUM FOR IN-VITRO CULTURED CELLS

FIELD OF THE INVENTION

The present invention relates to a serum-free cryo-conservation medium for in-vitro cultured cell lines.

BACKGROUND OF THE INVENTION

Long term storage of living cells is a central issue in cell biology and in medicine in general. A living eukaryotic cell is a rather fragile subject and needs special precautions to be taken prior cryo-conservation at very low temperatures (Aswood- Smith, M. J., 1980. Low Temperature Preservation of Cells, Tissues and Organs, p. 19-44. In Low Temperature Preservation in Medicine and Biology. M. J. Aswood-Smith and J. Farrant, Eds. (Pitman Medical Limited, Kent, England)). Already in 1949, the effect of Glycerol on cells upon their freezing has been discovered. Since then, it is well established that a cryo-conservation medium has to contain an anti-freezing agent to prevent ice crystal formation which disrupts the cell membrane (Polge, C, A. U. Smith, and A. S. Parkes, 1949. Revival of Spermatozoa after Vitrification and Dehydration at Low Temperatures. Nature 164: 666). Often Dimethylsulfoxide (DMSO) is used since this compound is, at low concentrations and low temperatures non-toxic for the cells. Additionally, it can easily penetrate the cell wall and act in the cytoplasm where its action is needed. Fetal bovine serum (FBS) is routinely added to freezing media for the conservation of serum dependent cell lines. FBS with its high protein content (bovine serum albumin and many unknown growth factors, enzyme inhibitors and more) prevents the cells against shear forces and gives the medium the desired osmotic environment with a physiological viscosity. For ethical and other reasons, however, there is the tendency to get away from FBS containing cultivation media. Serum- and protein-free cultured cells, however, must not be stored

in a serum-containing freezing medium.

Today, different serum-free freezing media with proprietary composition are commercially available; many home made mixtures as simple as conditioned medium + DMSO are used by researchers (Waymouth, C. and Varnum, D., Simple freezing procedure for storage in serum-free media of cultured and tumor cells of mouse. TCA Manual, (1976) 2(1), 311-313). However, surviving of the cells in such media ranges from 30 to 50% only compared to about 75% in FBS containing freezing media.

DESCRIPTION OF THE INVENTION The technical problem to be solved was to get a serum- and protein-free cryo- conservation medium with cell surviving rates equal or better than with serum- containing media. Such medium should be usable for freezing any eukaryotic cells, preferably mammalian cells. The problem is solved by a cryo-conservation medium according to claim 1. Further preferred embodiments are subject of claims 2 to 9.

Addition of an anti-freezing agent and an ethylene oxide/propylene oxide block copolymer to any commercially available basal culture medium (e.g. Iscove's Modified Dulbecco's Medium, RPMI-1640 Medium) or even to a physiological NaCI solution (Phosphate buffered saline) in any concentration has an outstanding surviving effect on cells which have to be cryo-conserved. The viability and surviving rates of cells and the growth behaviour of cells after freezing and thawing is even better than with traditional FBS containing media. Moreover, the new cryo-conservation medium is very easy to produce and the production costs are lower compared to other freezing media. A very important point is that it circumvents the ethical problems caused by the use of fetal bovine serum.

In order to prevent crystal formation, any anti-freezing agent has to be added to the cryo-conservation medium. Anti-freezing agents are agents that prevent freezing of liquids and therefore prevent crystal formation which would destroy the cells. Preferred anti-freezing agents are selected from the group consisting of Methanol [CH3OH], Ethanol [C2H5OH], Ethyleneglycol [C2H4(0H)2], 1-2 Isopropanediol [C3H6(0H)2],

Glycerol [C3H5(OH)3], Dimethylsulfoxide [(CH3)SO], Polyvinylpyrrolidone (PVP),

Methylcellulose, and Hydroxyethyl Starch or mixtures thereof. The most preferred anti- freezing agent is Dimethylsulfoxide (DMSO). A concentration of 0.5 to 10% (vol%), preferably 5 to 10% protects well.

Ethylene oxide/propylene oxide block copolymers are block copolymers of the general formula EO(n1)-PO(n2)-EO(n1), whereby EO stands for Ethylene Oxide and PO for Propylene Oxide and n1, n2 are average numbers of units of Ethylene Oxide and Propylene Oxide, respectively.

Preferred ethylene oxide/propylene oxide block copolymers have the formula of EO(50 to 20O)-PO(IO to 80)-EO(50 to 200), even preferred of EO(70 to 140)-PO(25 to 55)- EO(70 to 140)

More preferred ethylene oxide/propylene oxide block copolymers are selected from the group consisting of EO(76)-PO(30)-EO(76) (Pluronic F68); EO(104)-PO(39)- E(104) (Pluronic F88); EO(123)-PO(47)-EO(123) (Pluronic F98); and EO(132)- PO(50)-EO(132) (Pluronic F108) or mixtures thereof.

The most preferred embodiment of the invention is EO(76)-PO(30)-EO(76) (Pluronic F68). The synthetic Pluronic F68 is a difunctional block copolymer surfactant terminating in primary hydroxyl groups, a non-ionic surfactant which is known to stabilize cell membranes of eukaryotic cells. In combination with an anti-freezing agent, most preferably with DMSO, it is perfectly suited for the use in freezing media for low temperature cryo-preservation.

Optimal concentrations of the ethylene oxide/propylene oxide block copolymer range from 0.1 to 10% (weight%), even preferred concentrations are 0.1 to 1%, the most preferred concentration is about 1%. The very best results have been obtained with cryo-conserving media consisting of about 89% basal culture medium, about 10% DMSO and about 1% Pluronic F68. At this concentration of Pluronic F68, the recovery of cells after cryo-conservation exhibits its maximum, while maintaining a viscosity which allows the operator a good handling. ,

The cryo-conservation medium may be provided as a concentrate or as solution ready for use. All the concentrations described are for the ready for use medium and have to be adapted accordingly in case of a concentrate.

Outstanding results have been obtained with the use of the cryo-conservation medium according to the present invention for freezing of mammalian cells.

FIGURES Figure 1 shows a selection of the tested standard cryo-conservation media compared to the cryo-conserving media containing Pluronic F68. Surviving of cells 24 hours after thawing is significantly higher in media containing Pluronic F68 as compared to the corresponding media without Pluronic F68 or standard FBS containing media. Values higher than 100% indicate cell proliferation within the first 24 hours after thawing. Such values were never observed if cells were frozen in absence of Pluronic F68. The following legend for Figure 1 describes the composition of the tested media:

1. FBS 10% DMSO

2. Commercial Medium

3. 45% Fresh Medium/45% Conditioned Medium/10% DMSO 4. 48.7% Fresh Medium/48.7% Conditioned Medium/2.5% DMSO

5. 47.5% Fresh Medium/47.5% Conditioned Medium/5% DMSO

6. Hypothermosol 1.5% DMSO

7. Hypothermosol 2.5% DMSO

8. Hypothermosol 5% DMSO 9. Hypothermosol 10% DMSO

10. Hypothermosol 2.5% DMSO+0.1% Methylcellulose

11. Hypothermosol 2.5% DMSO+3% Polyvinylpyrrolidone

12. Hypothermosol 10% DMSO+0.1% Methylcellulose

13. Hypothermosol 10% DMSO+3% Polyvinylpyrrolidone 14. Hypothermosol 2.5% DMSO+0.1% Pluronic F68

15. Hypothermosol 2.5% DMSO+1% Pluronic F68

16. Hypothermosol 10% DMSO+0.1% Pluronic F68

17. Hypothermosol 10% DMSO+1% Pluronic F68

18. 47.5% Fresh Medium/47.5% Conditioned medium/5% DMSO+0.1% Pluronic F68 19. 47.5% Fresh Medium/47.5% Conditioned Medium/5% DMSO+1% Pluronic F68

20. 45 % Fresh Medium/45% Conditioned Medium/10% DMSO+0.01% Pluronic F68 21. 45% Fresh Medium/45% Conditioned Medium/10% DMSO+0.1% Pluronic F68

22. 45% Fresh Medium /45% Conditioned Medium/10% DMSO+1% Pluronic F68 23. 45% Fresh Medium/45% Conditioned Medium/10% DMSO+10% Pluronic F68

24. PBS 10% DMSO+0.1% Pluronic F68

25. PBS 10% DMSO+1% Pluronic F68 26. PBS 10% DMSO+5% Pluronic F68

27. Fresh Medium 10% DMSO+0.01% Pluronic F68

28. Fresh Medium 10% DMSO+0.1 % Pluronic F68

29. Fresh Medium 10% DMSO+1% Pluronic F68

30. Fresh Medium 10% DMSO+5% Pluronic F68 31. Fresh Medium 10% DMSO+10% Pluronic F68

32. Conditioned Medium 10% DMSO+0.01% Pluronic F68

33. Conditioned Medium 10% DMSO+0.1% Pluronic F68

34. Conditioned Medium 10% DMSO+1% Pluronic F68

35. Conditioned Medium 10% DMSO+5% Pluronic F68 36. Conditioned Medium 10% DMSO+10% Pluronic F68

37. Fresh Medium 0.5% DMSO+1% Pluronic F68

38. Fresh Medium 1.5% DMSO+1% Pluronic F68

39. Fresh Medium 2.5% DMSO+1% Pluronic F68

40. Fresh Medium 5% DMSO+1% Pluronic F68

EXAMPLES Example 1

As basal culture medium, the following components have been used either as single ingredients or in combinations:

PBS (Phosphate buffered saline);

HTS (Hypothermosol according: Lakey, J. R., Rajotte, R. V., Fedorow, C. A. and Taylor, M. J. (2001). Cell Transplantation 10, 583-589.);

Fresh, chemically fully defined medium, (HEKTOR, In Vitrus, Turbodoma Messi Cell Culture Technologies, Gravesano, Switzerland);

Conditioned medium (conditioned medium is an originally chemically fully defined medium that already has been used and therefore contains several components that have been produced and secreted by the cells such as cytokines, growth factors and others);

Any standard serum containing cell culture medium, e.g. Iscove's modified Dulbeccos medium.

Additional components of the cryo-conservation medium:

FBS (Fetal Bovine Serum; Sigma, Buchs, Switzerland);

DMSO (Fluka, Buchs, Switzerland) at a concentration range of 0.5 to 10% (final concentration);

Pluronic F68 (Sigma, Buchs, Switzerland) at a concentration range of 0.01 to 20% (final concentration);

Methylcellulose (Fluka, Buchs, Switzerland) at a concentration of about 0.1% (final concentration);

PVP (Polyvinylpyrrolidone Fluka, Buchs, Switzerland) at a final concentration of about 3%.

Cell lines:

Murine myeloma cell line P3X63Ag8.653 and an antibody secreting Hybrydoma cell line, HEP-1 , VERO-cells, HEK-293, COS-1 and COS-7 cells.

2x10E6 Cells were frozen in 1 ml of freezing medium in Nalgene cryo-vials at controlled cooling rates ranging from 0.5 to 5 °C/min starting from room temperature down to -80 ⁰ C. The samples were subsequently transferred to liquid nitrogen. Thawing was achieved by melting the samples for exactly 3 min. in a 37 ⁰ C water bath followed by a transfer into a 15 ml sterile test tube (TPP, Switzerland) containing 10 ml of fresh medium. After a 5 min centrifugation at 350 g the supernatant was discarded and the cell pellet resuspended in 3 ml of fresh medium. Efficiency was determined after 24 hours by counting the cells in a CASY ® Cell counter. The growth behaviour

was monitored after adjusting the cell density to 5x10E4 cells/ml cells were counted in 24 hour intervals.

The results and surviving rates are shown in Figure 1.