Method of protecting biological materials from destructive reactions in the dry state

• US 5,290,765 A
• Filed: 02/11/1992
• Issued: 03/01/1994
• Est. Priority Date: 09/14/1990
• Status: Expired due to Term

• Current Assignee: Boyce Thompson Institute for Plant Research, Inc.
• Original Assignee: Boyce Thompson Institute for Plant Research, Inc.
• Inventors: Leopold, Aldo C., Wettlaufer, Scott H.
• Primary Examiner(s): Rollins, John W.
• Application Number:
  US07/833,735
• Time in Patent Office: 749 Days
• Field of Search: 514/53, 514/23, 514/78, 424/195.1, 427/3, 427/4
• US Class Current: 514/23
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A01N 3/00 Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions preservation or chemical ripening of fruit or vegetables A23B7/00; Grafting wax
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 31/7004 Monosaccharides having only carbon, hydrogen and oxygen atoms

Abstract

A method is provided for protection of biological materials from the stresses of air-drying and also from destructive reactions, such as oxidation and free-radical attack, which degrade the materials during long-term storage. The method involves drying the materials, which may contain potentially destructive agents such as free-radical generators or reducing sugars, in the presence of a vitrifying substance, and under conditions which allow the protective substance to become vitrified.

Claims

1. A method of protecting biological materials selected from the group consisting of tissues, cells, organelles, and biologically active compounds from drying and from destructive reactions which take place during storage, comprising the steps of:

(a) selecting a mixture including cells such as living rhizobia cells;

(b) combining said mixture with sufficient quantity of one or more vitrifying solutes to protect said mixture during drying and to inhibit said destructive reactions,

(c) drying said combination, by exposing said combination to a desiccant, at a temperature above that at which said combination will freeze and below that at which said vitrifying solutes achieve the vitrified state, at approximately normal atmospheric pressure, until said combination is substantially dry, and

(d) storing said combination in a dry state.

2. The method of claim 1, wherein said mixture further comprises one or more destructive substances which would, in the absence of a protective effect, initiate said destructive reactions.

3. The method of claim 2, wherein said destructive substances include a reducing sugar.

4. The method of claim 3, wherein said reducing sugar is glucose.

5. The method of claim 2, wherein said destructive substances include a free-radical generating substance.

6. The method of claim 5, wherein said free-radical generating substance is riboflavin.

7. The method of claim 2, wherein said vitrifying solutes include at least one non-reducing sugar.

8. The method of claim 1, wherein said vitrifying solutes include at least one non-reducing sugar.

9. The method of claim 8, wherein said non-reducing sugars include sucrose.

10. The method of claim 9, wherein said non-reducing sugars includes an oligosaccharide.

11. The method of claim 10, wherein said oligosaccharide is raffinose.

12. The method of claim 11, wherein said sucrose and raffinose are in present in a 3:

1 mass ratio.

13. The method of claim 8, wherein said non-reducing sugars include raffinose.

14. A composition produced by a method of preparing materials to be stored in a dry state comprising the steps of:

a. selecting a mixture including cells such as living rhizobia cells,

b. combining said mixture with sufficient quantity of one or more vitrifying solutes to protect said mixture during drying and to inhibit said destructive reactions, and

c. drying said combination, by exposing said combination to a desiccant, at a temperature above that which said combination will freeze and below that at which said vitrifying solutes achieve the vitrified state, at approximately normal atmospheric pressure, until said combination is substantially dry.

15. A composition of protected biological materials in the dry state, comprising a combination of biological materials including cells such as living rhizobia cells, and one or more vitrified solutes, wherein said vitrified solutes are present in sufficient quantity to protect said biological materials from destructive reactions which render said materials less biochemically active over time.

16. The composition of claim 15, further comprising one or more destructive substances which would, in the absence of a protective effect, initiate said destructive reactions.

17. The composition of claim 16, wherein said destructive substances include a reducing sugar.

18. The composition of claim 17, wherein said reducing sugar is glucose.

19. The composition of claim 16, wherein said destructive substances include a free-radical generating substance.

20. The composition of claim 19, wherein said free-radical generating substance is riboflavin.

21. The composition of claim 15, wherein said vitrified substances include at least one non-reducing sugar.

22. The composition of claim 21, wherein said non-reducing sugars include sucrose.

23. The composition of claim 22, wherein said non-reducing sugars includes an oligosaccharide.

24. The composition of claim 23, wherein said oligosaccharide is raffinose.

25. The composition of claim 24, wherein said sucrose and raffinose are in present in a 3:

1 mass ratio.

26. The composition of claim 21, wherein said non-reducing sugars include raffinose.

27. A composition comprising the parts of:

a. one or more vitrifying solutes capable of protecting cells such as living rhizobia cells during drying and from destructive reactions which take place during storage of said cells, and

b. a desiccant, used to dry said vitrifying solutes and said cells at a temperature above that at which said vitrifying solutes and said cells will freeze and below that at which said vitrifying solutes achieve the vitrified state, at approximately normal atmospheric pressure, until said vitrified solutes and said cells are substantially dry.