Arrays of discrete cell culture microenvironments, method of making such arrays and uses thereof

Abstract

The invention pertains to a combinatorial method of identifying the hydrogel formulations controlling phenotype and fate of difficult-to-culture cell types.

1 - 15 . (canceled) 
     
     
         18 . An array having discrete volumes of cell culture microenvironments possessing different properties influencing behavior of encapsulated cells. 
     
     
         17 . The array according to claim  16 , wherein the discrete volumes of cell culture microenvironments comprise hydrogels. 
     
     
         18 . The array according to claim  16 , wherein at least two of the discrete volumes of cell culture microenvironments are differently degradable by proteinases. 
     
     
         19 . The array according to  claim 18 , wherein said proteinases are matrix metalloproteinases. 
     
     
         20 . The array according to claim  16 , wherein at least two of the discrete volumes of cell culture microenvironments;
 I) comprise different extracellular matrix-derived factors; and/or   ii) comprise different cell-cell interaction factors; and/or   iii) comprise different cell signalling factors; and/or   iv) are of different stiffness.   
     
     
         21 . The array according to claim  16 , wherein the cell culture microenvironments are built up from precursor molecules that are crosslinkable by cell-compatible reaction(s), on the basis of:
 covalent bond formation chosen from the group consisting of:
 enzymatically catalyzed reactions; and 
 not-enzymatically catalyzed and/or uncatalyzed reactions; and/or 
   non-covalent bond formation.   
     
     
         22 . The array according claim  16 , wherein the cell culture microenvironments are built up from linear or branched hydrophilic polymers. 
     
     
         23 . The array according to  claim 22 , wherein said branched hydrophilic polymers are branched poly(ethylene glycol) molecules. 
     
     
         24 . A method of making an array according to claim  16 , said method comprising the steps of:
 a) providing one or more different hydrogel precursor molecules;   b) combining and dispensing different combinations of hydrogel precursor molecules according to step a) onto discrete volumes of a substrate;   c) adding to said discrete volumes of said substrate one or more biologically active molecules and either attaching said molecules to at least one of the hydrogel precursor molecules present or the hydrogel formed in step e) or allowing them to diffuse freely;   d) adding cells onto/into said discrete volumes of the substrate; and   e) crosslinking said hydrogel precursor molecules to form a hydrogel matrix.   
     
     
         25 . The method according to  claim 24 , wherein in step c) adding one or more biologically active molecules selected from the group of extracellular matrix-derived factors, cell-cell interaction factors and cell-signalling factors. 
     
     
         26 . A combinatorial method of testing the influence of hydrogel formulations on cell growth behavior, comprising the steps of:
 a) providing one or more different hydrogel precursor molecules;   b) combining and dispensing different combinations of hydrogel precursor molecules according to step a) onto/into discrete volumes of a substrate;   c) further adding to said discrete volumes of said substrate one or more biologically active molecules and either attaching said molecules to at least one of the hydrogel precursor molecules present or the hydrogel formed in step e) or allowing them to diffuse freely;   d) adding cells onto/into said discrete volumes of the substrate;   e) crosslinking said hydrogel precursor molecules to form a hydrogel matrix;   f) allowing said cells to grow in said discrete volumes of said hydrogel matrix;   g) monitoring said cells during step f) over time;   h) determining the behavior for different cell culture microenvironments;   l) optionally, determining the synergistic and/or antagonistic effects on each other of the biologically active molecules and/or of the mechanical properties and/or susceptibilities to enzymatic degradation of the discrete cell culture microenvironments;   j) optionally, identifying a specific hydrogel formulation or range of hydrogel formulations that instructs a certain cell behaviour; and   k) optionally, isolating cells from at least one hydrogel cell culture microenvironment for further analysis or for continuous cell culture or passaging.   
     
     
         27 . A combinatorial method according to  claim 26 , wherein one or more biologically active molecules of step c) are selected from the group consisting of extracellular matrix-derived factors, cell-cell interaction factors and cell-signalling factors. 
     
     
         28 . A kit of parts for making an array having discrete volumes of hydrogel according to claim  16 , comprising as components:
 a) one or more hydrogel precursor molecules;   b) one or more biologically active molecules;   c) optionally, at least one crosslinking agent for the precursor molecules a); and   d) instructions for use of said components.   
     
     
         29 . The kit of parts according to  claim 28 , wherein the instructions for use of said component are in accordance with a method according to  claim 24 . 
     
     
         30 . The kit of parts according to  claim 28 , wherein
 the molecule(s) of component b) is/are selected from the group of extracellular matrix-derived factors, cell-cell interaction factors and/or cell-signalling factors; and/or   factor XIIIa comprises component c).   
     
     
         31 . The kit of parts according to  claim 28 , wherein the hydrogel precursor molecules of component a) comprise or consist of multiarm poly(ethylene glycol) molecules. 
     
     
         32 . The kit of parts according to  claim 28 , wherein at least two hydrogel precursor molecules are provided as component a) which are crosslinkable by an enzyme. 
     
     
         33 . The kit of parts according to  claim 32 , wherein said enzyme is included as component c). 
     
     
         34 . The kit of parts according to  claim 32 , wherein one of the at least two hydrogel precursor molecules is functionalized by a glutamine-bearing peptidic substrate and the other one by a lysine-bearing peptidic substrate for a crosslinking enzyme, and transglutaminase factor XIIIa is included as component c). 
     
     
         35 . The kit of parts according to  claim 28 , wherein at least hydrogel precursor molecules of component a) are provided pre-supplied in wells of a multi-well plate in substantially unreacted form, preferably in dried form.