Controlled reversible poration for preservation of biological materials

• US 6,127,177 A
• Filed: 09/11/1998
• Issued: 10/03/2000
• Est. Priority Date: 09/11/1998
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology, The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Massachusetts Institute of Technology, The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Russo, Michael, Bieganski, Robert, Toner, Mehmet
• Primary Examiner(s): Saucier, Sandra E.
• Assistant Examiner(s): Afremova, Vera
• Application Number:
  US09/151,821
• Time in Patent Office: 753 Days
• Field of Search: 435/374, 435/1.3, 435/2
• US Class Current: 435/374
• 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: Y10S 977/902 Specified use of nanostructure
• CPC Class Codes: Y10S 977/92 Detection of biochemical
• CPC Class Codes: Y10S 977/944 Biochemical memory

Abstract

A preservation method for biological material having cell membranes includes reversibly porating the cell membranes; loading a bio-protective agent having bio-preservation properties to a predetermined intracellular concentration; preparing the bio-protective agent loaded biological material for storage; storing the biological material; recovering the stored biological material from storage; and reversing the cell membrane poration. H5 α-toxin, a genetically engineered mutant of Staphylococcus aureus α-hemolysin, may be used as a porating agent. Non-permeating sugars such as trehalose and sucrose may be used as the bio-protective agent.

Claims

1. A method for dry storing living nucleated mammalian cells having cell membranes, comprising:

a. Applying a membrane toxin to reversibly porate the cell membranes of the nucleated cells;

b. Loading an agent having bio-preservation properties to a predetermined intracellular concentration sufficient for preserving the cellular material, the predetermined intracellular concentration being less than or equal to about 1.0 M, the bio-preservation agent comprising a sugar;

c. Drying the sugar loaded cells to a level sufficient to permit dry storage;

d. Placing the dried cells in dry storage;

e. Rehydrating the dried cells to a level sufficient for cell viability; and

f. Reversing the cell membrane poration to an extent sufficient to permit survival and growth of the cells;

wherein sugar is the only bio-protective agent employed.

2. The method of claim 1, wherein the cell membranes are reversibly porated using H5 α

-toxin.

3. The method of claim 1, wherein the sugar having bio-preservation properties is selected from a group consisting of trehalose, sucrose, glucose and maltose.

4. The method of claim 3, wherein the membrane toxin forms pores of at least about 2.0 nanometers in the membrane.

5. The method of claim 3, wherein the biological material is loaded with an intracellular concentration of sugar less than or equal to about 0.4 M.

6. The method of claim 3, wherein the drying is accomplished by freeze drying.

7. The method of claim 6, wherein the sugar loaded cells are plunge frozen to a cryogenic temperature.

8. The method of claim 3, wherein the drying is a vacuum or air drying.

9. The method of claim 8, wherein the drying is performed at a non-cryogenic temperature.

10. A method for cryopreserved living nucleated mammalian cells having cell membranes, consisting essentially of:

a. Applying a membrane toxin to reversibly porate the cell membranes of the nucleated cells using H5 α

-toxin;

b. Loading the porated cells with a bio-preservation agent consisting essentially of a sugar to a predetermined intracellular concentration sufficient for preserving the nucleated cells, the predetermined intracellular concentration being less than or equal to about 1.0 M;

c. Freezing the sugar loaded cells to a cryopreservation temperature;

d. Storing the frozen biological material at a cryo-storage temperature;

e. Thawing the cryo-stored biological material to a viable state; and

f. Reversing the cell membrane poration to an extent sufficient to permit survival and growth of the cells.

11. The method of claim 10, wherein the sugar having bio-preservation properties is selected from a group consisting of trehalose, sucrose, glucose and maltose.

12. The method of claim 11, wherein the intracellular concentration of the sugar is less than or equal to about 0.4 M.

13. The method of claim 11, wherein the cells are plunge frozen.