Microinjection of cryoprotectants for preservation of cells

Abstract

A preservation method for biological material having cell membranes includes microinjecting the cell membranes with sugar; preparing the cells for storage; storing the biological material; and recovering the stored biological material from storage. Carbohydrate sugars such as trehalose, sucrose, fructose, dextran, and raffinose, may be used as bio-protective agents.

What is claimed is:  
     
         1 . A method for treating a living cell, said method comprising the steps of: 
 (a) microinjecting into the cytoplasm of said cell a protective agent which (i) comprises a sugar, (ii) is substantially non-permeating with respect to mammalian cell membranes, and (iii) maintains the viability of said cell such that it can be stored in a temporarily dormant state and substantially restored to an active state;    (b) treating said cell to cause it to enter said dormant state; and    (c) storing said cell in said dormant state.    
     
     
         2 . The method of  claim 1 , further comprising the step of (d) treating said cell to restore it to an active state.  
     
     
         3 . The method of  claim 1 , wherein said cell is a mammalian cell.  
     
     
         4 . The method of  claim 3 , wherein said cell is an oocyte.  
     
     
         5 . The method of  claim 3 , wherein said cell is an epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, B-cell, T-cell, erythrocyte, macrophage, monocyte, fibroblast, muscle cell, embryonic stem cell, or adult stem cell.  
     
     
         6 . The method of  claim 1 , further comprising contacting said cell with an extracellular protective agent that is substantially non-permeating with respect to mammalian cell membranes and that stabilizes the cell membrane of said cell.  
     
     
         7 . The method of  claim 1 , wherein said protective agent comprises at least one sugar selected from the group consisting of sucrose, trehalose, fructose, dextran, and raffinose.  
     
     
         8 . The method of  claim 1 , wherein said protective agent comprises at least one sugar selected from the group consisting of glucose, sorbitol, mannitol, lactose, maltose, and stachyose.  
     
     
         9 . The method of  claim 1 , wherein said protective agent comprises at least one sugar with a glass transition temperature greater than −50° C.  
     
     
         10 . The method of  claim 9 , wherein said protective agent comprises at least one sugar with a glass transition temperature greater than −30o C.  
     
     
         11 . The method of  claim 1 , wherein said protective agent comprises at least one sugar with a molecular weight greater than 120 daltons.  
     
     
         12 . The method of  claim 1 , wherein said protective agent comprises at least one sugar with a glass transition temperature greater than −30° C. and a molecular weight greater than 120 daltons.  
     
     
         13 . The method of  claim 1 , wherein said protective agent comprises a glycolipid or a glycoprotein that comprises at least one sugar moiety derived from a sugar with a glass transition temperature greater than −50° C.  
     
     
         14 . The method of  claim 1 , wherein the cytoplasmic concentration of said sugar is less than or equal to about 1.0 M following step (a) and prior to step (b).  
     
     
         15 . The method of  claim 14 , wherein the cytoplasmic concentration of said sugar is less than or equal to about 0.2 M following step (a) and prior to step (b).  
     
     
         16 . The method of  claim 6 , wherein said extracellular protective agent comprises an extracellular sugar.  
     
     
         17 . The method of  claim 16 , wherein said cell is maintained in a liquid medium, and wherein the extracellular concentration of said extracellular sugar is less than or equal to about 1.0 M following dilution into said liquid medium.  
     
     
         18 . The method of  claim 17 , wherein the extracellular concentration of said extracellular sugar is less than or equal to about 0.2 M following dilution into said liquid medium.  
     
     
         19 . The method of  claim 16 , wherein said cell is maintained on a solid medium, and wherein the concentration of said extracellular sugar is less than or equal to about 1.0 M following administration to said cell.  
     
     
         20 . The method of  claim 19 , wherein the concentration of said extracellular sugar is less than or equal to about 0.2 M following administration to said cell.  
     
     
         21 . The method of  claim 1 , wherein step (b) comprises freezing said cell to a cryogenic temperature.  
     
     
         22 . The method of  claim 21 , wherein said cell is plunge frozen.  
     
     
         23 . The method of  claim 21 , wherein step (d) comprises thawing said cell.  
     
     
         24 . The method of  claim 1 , wherein step (b) comprises drying said cell to a level sufficient to permit dry storage.  
     
     
         25 . The method of  claim 24 , wherein step (b) comprises freeze drying said cell.  
     
     
         26 . The method of  claim 24 , wherein step (b) comprises vacuum or convective drying said cell.  
     
     
         27 . The method of  claim 24 , wherein step (d) comprises rehydrating said cell.  
     
     
         28 . The method of  claim 1 , wherein only said protective agent is employed.  
     
     
         29 . The method of  claim 1 , wherein prior to step (a), said cell is maintained in a hypertonic medium having an osmolarity greater than 300 mosm.  
     
     
         30 . The method of  claim 2 , wherein following step (d), said cell is cultured in a hypertonic medium having an osmolarity greater than 300 mosm.  
     
     
         31 . The method of  claim 1 , wherein a penetrating cryoprotectant mixture is added to said preservation agent.  
     
     
         32 . A method of culturing a cell in vitro, comprising incubating said cell in a hypertonic medium having an osmolarity greater than 300 mosm.  
     
     
         33 . The method of  claim 32 , wherein the osmolarity of said medium is greater than 320 mosm.