CH698250B1 - Cryopreservation In vitro cultured cells. - Google Patents

Abstract

The present invention is a serum-free cryopreservation medium for in vitro cultured cells which contains an antifreeze agent and an ethylene oxide / propylene oxide block copolymer.

Description

Technical area

The present invention is a serum-free cryopreservation medium for in vitro cultured cells.

State of the art

The long-term storage of living cells is a major concern in cell biology and medicine. A living eukaryotic cell is a fragile biological complex that requires special protective measures so that it can be cryopreserved at very low temperatures. (Aswood-Smith, MJ, 1980. Low Temperature Preservation of Cells, Tissues and Organs, pp. 19-44, in Low Temperature Preservation in Medicine and Biology, MJ Aswood-Smith, and J. Farrant, Eds. (Pitman Medical Limited). Kent, England)).

As early as 1949, the positive effect of glycerol on cells was discovered during their freezing. Since then, it has been known that every cryogenic medium must contain a cryoprotectant which prevents the formation of ice crystals and the associated cell damage. (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 as this reagent is non-toxic to the cells at low concentrations and low temperatures. In addition, DMSO can easily penetrate into the cell interior where its action is needed. Fetal calf serum (FCS) is added by default to the freezing media used to preserve serum-dependent cell lines. FKS with its high protein content (bovine serum albumin, many unidentified growth factors, enzyme inhibitors, etc.) protects the cells from shear forces and gives the medium the desired osmolarity and physiological viscosity.

There are, among other ethical reasons, which initiated the trend away from serum-containing culture media. However, serum and protein-free cultured cells should not be frozen in cryomials containing serum. At present, various serum-free media whose compositions are not disclosed by the manufacturers are commercially available. Researcher's own simple mixtures such as conditioned medium + DMSO are also used. (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, the survival of cells frozen in these media only ranges between 30 and 50% compared to about 75% when using a serum-containing medium.

Description of the invention

The technical problem that had to be solved was the development of a serum and protein free cryopreservation medium with cell survival rates equal to or better than those achieved with serum containing cryomedia. The cryomedium should be suitable for the preservation of all eukaryotic cells, in particular mammalian cells.

The problem is solved by the cryogenic medium according to claim 1. Further preferred embodiments are described in claims 2 to 9. The addition of a cryoprotectant and an ethylene oxide / propylene oxide block copolymer to any commercially available basal medium (eg, Iscove's modified Dulbecco's medium, RPMI-1640 medium) or even to only physiological saline (Phosphate Buffered Saline, PBS) at any concentration, has an excellent effect on the Survival of frozen cells. The viability and survival of cells after freezing and thawing are even better than those of cells cryopreserved in FKS-containing media. In addition, the new cryomedium is very simple in its manufacture; This results in lower production costs compared to other cryomedias. Another important point is that with the serum-free cryomedium also the ethical concerns become obsolete.

In order to prevent the formation of ice crystals, any cryoprotectant must be added to the cryomedium. Such antifreezes prevent the freezing of liquids and thus prevent the formation of ice crystals, which would destroy the cell membranes and thus the cells. Preferred antifreezes are selected from the group of methanol [CH30H], ethanol [C2H50H], ethylene glycol [C2H4 (OH) 2], 1-2 isopropanediol [C3H6 (OH) 2], glycerol [C3H5 (OH) 3], dimethylsulfoxide [ (CH3) SO], polyvinylpyrrolidone (PVP), methylcellulose or hydroxyethyl starch or mixtures thereof. The most preferred antifreeze is dimethyl sulfoxide (DMSO). Concentrations of from 0.5 to 10% (% by volume), preferably from 5 to 10%, show a good protective effect.

Ethylene oxide / propylene oxide block copolymers are block copolymers having the general formula EO (n1) -PO (n 2) -EO (n1), where EO is "ethylene oxide" and PO is "propylene oxide". n1, n2 denote the average number of ethylene oxide and propylene oxide molecules.

Preferred ethylene oxide / propylene oxide block copolymers have the formula EO (50 to 200) -PO (10 to 80) -EO (50 to 200), more preferred are those having the formula EO (70 to 140) -PO (25 to 55) -EO (70 to 140).

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

The most preferred block copolymer is EO (76) -PO (30) -EO (76) (Pluronic F68). The synthetic Pluronic F68 is a bifunctional primary hydroxyl functional block copolymer, a nonionic surfactant known to stabilize cell membranes of eukaryotic cells. In combination with an antifreeze, preferably DMSO, this shows an optimal effect in freezing media for cryopreservation at low temperatures.

The optimum concentration range of the ethylene oxide / propylene oxide block copolymer is between 0.1 and 10% (weight%). Preferred concentrations are between 0.1 and 1%, the most preferred concentration is about 1%. The best results were obtained with a cryogenic medium having the following composition: about 89% basal medium, about 10% DMSO and about 1% Pluronic F68. At this concentration of Pluronic F68, the yield of surviving cells was greatest and the viscosity allowed optimal handling of the cryomedium.

The cryogenic medium can be prepared as a concentrate or as a ready-to-use solution. The concentrations given refer to the ready-to-use medium; Concentrate concentrations must be adjusted accordingly. Mammalian cells were frozen and thawed with the cryopreservation medium described in this invention; excellent results were achieved.

pictures

Figure 1 shows a selection of standard cryopreservation media tested compared to cryomials containing Pluronic F68. Cell survival 24 hours after thawing in media containing Pluronic F68 was significantly higher than in the corresponding media without Pluronic F68 or in serum-containing standard media. Survival rates greater than 100% indicate that cells proliferate as early as the first 24 hours. Such values were never observed in media without Pluronic F68.

In the legend to Figure 1, the compositions of the media tested are described: <tb> 1. <sep> FCS 10% DMSO <tb> 2. <sep> Commercial medium <tb> 3. <sep> 45% fresh medium / 45% conditioned medium / 10% DMSO <tb> 4. <sep> 48.7% fresh medium / 48.7% conditioned medium / 2.5% DMSO <tb> 5. <sep> 47.5% fresh medium / 47.5% conditioned medium / 5% DMSO <tb> 6. <sep> Hypothermosol 1.5% DMSO <tb> 7. <sep> Hypothermosol 2.5% DMSO <tb> 8. <sep> Hypothermosol 5% DMSO <tb> 9. <sep> Hypothermosol 10% DMSO <tb> 10. <sep> Hypothermosol 2.5% DMSO + 0.1% methylcellulose <tb> 11. <sep> hypothermosol 2.5% DMSO + 3% polyvinylpyrrolidone <tb> 12. <sep> hypothermosol 10% DMSO + 0.1% methylcellulose <tb> 13. <sep> hypothermosol 10% DMSO + 3% polyvinylpyrrolidone <tb> 14. <sep> Hypothermosol 2.5% DMSO + 0.1% Pluronic F68 <tb> 15. <sep> Hypothermosol 2.5% DMSO + 1% Pluronic F68 <tb> 16. <sep> Hypothermosol 10% DMSO + 0.1% Pluronic F68 <tb> 17. <sep> Hypothermosol 10% DMSO + 1% Pluronic F68 <tb> 18. <sep> 47.5% fresh medium / 47.5% conditioned medium / 5% DMSO + 0.1% Pluronic <Tb> <sep> F68 <tb> 19. <sep> 47.5% fresh medium / 47.5% conditioned medium / 5% DMSO + 1% Pluronic <Tb> <sep> F68 <tb> 20. <sep> 45% fresh medium / 45% conditioned medium / 10% DMSO + 0.01% Pluronic F68 <tb> 21. <sep> 45% fresh medium / 45% conditioned medium / 10% DMSO + 0.1% Pluronic F68 <tb> 22. <sep> 45% fresh medium / 45% conditioned medium / 10% DMSO + 1% Pluronic F68 <tb> 23. <sep> 45% fresh medium / 45% conditioned medium / 10% DMSO + 10% Pluronic F68 <tb> 24. <sep> PBS 10% DMSO + 0.1% Pluronic F68 <tb> 25. <sep> PBS 10% DMSO + 1% Pluronic F68 <tb> 26. <sep> PBS 10% DMSO + 5% Pluronic F68 <tb> 27. <sep> Fresh medium 10% DMSO + 0.01% Pluronic F68 <tb> 28. <sep> Fresh medium 10% DMSO + 0.1% Pluronic F68 <tb> 29. <sep> Fresh medium 10% DMSO + 1% Pluronic F68 <tb> 30. <sep> Fresh medium 10% DMSO + 5% Pluronic F68 <tb> 31. <sep> Fresh medium 10% DMSO + 10% Pluronic F68 <tb> 32. <sep> Conditioned medium 10% DMSO + 0.01% Pluronic F68 <tb> 33. <sep> Conditioned medium 10% DMSO + 0.1% Pluronic F68 <tb> 34. <sep> Conditioned medium 10% DMSO + 1% Pluronic F68 <tb> 35. <sep> Conditioned medium 10% DMSO + 5% Pluronic F68 <tb> 36. <sep> Conditioned medium 10% DMSO + 10% Pluronic F68 <tb> 37. <sep> Fresh Medium 0.5% DMSO + 1% Pluronic F68 <tb> 38. <sep> Fresh Medium 1.5% DMSO + 1% Pluronic F68 <tb> 39. <sep> Fresh Medium 2.5% DMSO + 1% Pluronic F68 <tb> 40. <sep> Fresh medium 5% DMSO + 1% Pluronic F68

Examples

example 1

The following basal media were used either individually or in combination: PBS (Phosphate buffered saline); HTS (hypothermosol according to: Lakey, J.R., Rajotte, R.V., Fedorow, C.A. and Taylor, M.J. (2001) Cell Transplantation 10, 583-589.); Fresh, chemically-defined medium, (HEKTOR, In-Vitrus, Turbodoma Messi Cell Culture Technologies, Gravesano, Switzerland);

Conditioned medium (a conditioned medium is an originally chemically fully defined medium in which cells have been cultured and therefore various cell products such as cytokines, growth factors and others are included); Various standard cell culture media such as e.g. Iscove's modified Dulbeccos medium.

Further components in the cryopreservation medium: FKS (fetal calf serum, Sigma, Buchs, Switzerland); DMSO (Fluka, Buchs, Switzerland) at a concentration of 0.5 to 10% (final concentration); Pluronic F68 (Sigma, Buchs, Switzerland) at a concentration 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 concentration of about 3%.

Cell lines:

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

2x10E6 cells were frozen in 1 ml of cryomedium in a Nalgene cryotube at controlled cooling rates of 0.5 to 5 ° C / min starting at room temperature down to -80 ° C. The samples were then transferred to liquid nitrogen. For thawing, the cryotubes were warmed for exactly 3 minutes in a 37 ° C water bath. The contents were then pipetted into 10 ml of fresh cell culture medium in a sterile 15 ml test tube (TPP, Switzerland). After centrifugation (5 min at 350 g), the supernatant was discarded and the cells resuspended in 3 ml of fresh medium. The survival rate was determined 24 hours after thawing by cell counting (Casy <>> Cell Counter). The growth behavior of the cells was observed after cell seeding (cell density 5x10E4 / ml) by cell counting at 24 hour intervals.

The survival rates of the various media are shown in Figure1.

Claims (10)

1. A cryopreservation medium containing an antifreeze and an ethylene oxide / propylene oxide block copolymer. 2. The cryopreservation medium according to claim 1, wherein the antifreeze agent is selected from the group consisting of methanol, ethanol, ethylene glycol, 1-2 isopropanediol, glycerol, dimethylsulfoxide, polyvinylpyrrolidone, methylcellulose or hydroxyethyl starch or mixtures thereof. 3. The cryopreservation medium according to claim 1 or 2, wherein the ethylene oxide / propylene oxide block copolymer has the formula EO (50 to 200) -PO (10 to 80) -EO (50 to 200), preferably the formula EO (70 to 140) - PO (25 to 55) EO (70 to 140). 4. The cryopreservation medium of any one of claims 1 to 3, wherein the ethylene oxide / propylene oxide block copolymer is selected from the group consisting of EO (76) -PO (30) -EO (76), EO (104) -PO (39) - E (104), EO (123) -PO (47) -EO (123) or EO (132) -PO (50) -EO (132) or mixtures thereof. The cryopreservation medium of any one of claims 1 to 4, wherein the antifreeze is dimethylsulfoxide and the ethylene oxide / propylene oxide block copolymer is EO (76) -PO (30) -EO (76). 6. The cryopreservation medium according to any one of claims 1 to 5, wherein the concentration of the ethylene oxide / propylene oxide block copolymer is between 0.1 and 10%, preferably between 0.1 and 1%, more preferably about 1%. 7. The Kryokonservierungsmedium according to any one of claims 1 to 6, wherein the concentration of the cryoprotectant is between 0.5 and 10%, preferably between 5 and 10%. The cryopreservation medium of any one of claims 1 to 7 containing about 89% basal medium, about 10% dimethyl sulfoxide, and about 1% EO (76) -PO (30) -EO (76). 9. The use of an ethylene oxide / propylene oxide block copolymer as constituent of a cryopreservation medium, according to any one of claims 1 to 8. 10. The use of the cryopreservation medium according to any one of claims 1 to 8 for the freezing of mammalian cells.