Controlled reversible poration for preservation of biological materials
AssigneesMassachusetts Institute of Technology, The General Hospital Corporation
InventorsMehmet Toner, Michael Russo, Robert Bieganski
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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 .alpha.-toxin, a genetically engineered mutant of Staphylococcus aureus .alpha.-hemolysin, may be used as a porating agent. Non-permeating sugars such as trehalose and sucrose may be used as the bio-protective agent.
Claims13
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.
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 .alpha.-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.
Dependencies are inferred from the claim text.