Methods and gel compositions for encapsulating living cells and organic molecules

Abstract

A method for encapsulating biologics within a hydrogel by using an aqueous solution of an isocyanate-functional polyurethane prepolymer which is mixed with an amount of biologics and an aqueous solution containing a dithiol crosslinking agent under physiological pH conditions to create a hydrogel. An additional bidentate crosslinking agent is optionally included. The product of such method may be a bioreactor or an assay device having a plurality of different biologics encapsulated at predetermined locations in a substrate.

1 . A method of encapsulating biologics within a hydrogel comprising: 
 (a) providing an isocyanate-functional hydrogel prepolymer;    (b) providing an amount of biologics;    (c) providing crosslinkers having at least two thiol groups;    (d) mixing said prepolymer, said crosslinkers, and said biologics in aqueous solution to initiate polymerization under physiological pH and osmolality and create a polymerizing hydrogel; and    (e) dispensing said polymerizing hydrogel into a desired physical form.    
     
     
         2 . The method of  claim 1  wherein said amount of biologics is mixed in said aqueous solution with said thiol crosslinker prior to mixing with said prepolymer.  
     
     
         3 . The method of  claim 1  wherein said thiol crosslinkers comprise a crosslinking agent having a backbone of polyethylene glycol, polypropylene glycol, or a copolymer thereof and a molecular weight between about 2,000 and about 6,000.  
     
     
         4 . The method of  claim 1  wherein said thiol crosslinkers include a crosslinking agent having two thiol groups and a bidentate crosslinking agent having one thiol group and another different isocyanate-reactive group.  
     
     
         5 . The method of  claim 4  wherein the bidentate crosslinking agent comprises cysteine or homocysteine.  
     
     
         6 . The method of  claim 1  wherein the polymerizing hydrogel is dispensed onto a solid substrate so as to result in attachment thereto of the hydrogel-encapsulated biologics.  
     
     
         7 . The method of  claim 1  wherein the polymerizing hydrogel is repeatedly dispensed onto multiple, pre-identified areas of a substrate to form discrete spots arranged as an array.  
     
     
         8 . The method of  claim 7  wherein different of said spots at known locations contain at least two different biologics.  
     
     
         9 . The method of  claim 1  wherein said biologic has a molecular weight not greater than about 500 and contains a thiol-reactive group at a location that will not interfere with its spatial configuration for linking to an isocyanate group of the polymerizing hydrogel.  
     
     
         10 . The method of  claim 1  wherein said mixture is maintained at a pH of about 6.6 to about 7.1.  
     
     
         11 . The method of  claim 1  wherein said mixture is maintained at an osmolality of about 250 to about 400 mOsm/kg.  
     
     
         12 . A method of preparing a hydrogel composition having a biologic encapsulated therein, which method comprises the steps of: 
 (a) providing an isocyanate-functional hydrogel prepolymer, a biologic, and crosslinkers having at least two thiol groups that will react with said isocyanate-functional hydrogel prepolymer,    (b) mixing said isocyanate-functional hydrogel prepolymer, said biologic and said crosslinkers in an aqueous solution at neutral pH, to provide a solution containing about 3 to about 5 weight % prepolymer, and    (c) maintaining said mixture of step (b) at neutral pH, so as to cause said biologic to be encapsulated within a polymerized hydrogel wherein said biologic remains bioactive.    
     
     
         13 . The method according to  claim 12  wherein said mixture is maintained at a pH of between about 6.5 and about 7.5.  
     
     
         14 . The method according to  claim 12  wherein said aqueous solution also contains a water-soluble bidentate crosslinking agent having two different isocyanate-reactive nucleophilic groups.  
     
     
         15 . A hydrogel composition having a bioactive biologic encapsulated therein, which composition comprises: 
 the reaction product of an isocyanate-functional prepolymer, a biologic, and a crosslinker having at least two thiol groups,    said isocyanate-functional prepolymer having been crosslinked in the presence of said biologic by reaction with said crosslinker in an aqueous solution at physiological conditions and, as a result of said crosslinking, said biologic is encapsulated within a stable polymerized hydrogel in a manner so that said biologic remains bioactive.    
     
     
         16 . The hydrogel composition of  claim 15  wherein said prepolymer is a polyurethane prepolymer having a backbone of polyoxyalkylene diols or polyols having a molecular weight between about 5000 and about 30,000 which were reacted with difunctional or polyfunctional isocyanates.  
     
     
         17 . The hydrogel composition of  claim 15  wherein said crosslinker comprises a thiol-functional crosslinking agent having two thiol groups and a bidentate crosslinking agent having one thiol group and another different isocyanate-reactive group.  
     
     
         18 . The hydrogel composition of  claim 17  wherein the bidentate crosslinking agent comprises cysteine.  
     
     
         19 . The hydrogel composition of  claim 15  wherein said biologic is selected from the group consisting of proteins, nucleic acids, living cells, and organic molecules having a molecular weight between about 100 and about 2000.  
     
     
         20 . The hydrogel composition of  claim 15  wherein said thiol-functional crosslinker comprises a backbone of polyethylene glycol, polypropylene glycol, or a copolymer thereof and has a molecular weight between about 2,000 and about 6,000.