Nanoliter-scale synthesis of arrayed biomaterials and screening thereof

• US 20050019747A1
• Filed: 05/12/2004
• Published: 01/27/2005
• Est. Priority Date: 08/07/2002
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Anderson, Daniel G., Langer, Robert S., Levenberg, Shulamit
• Application Number:
  US10/843,707
• Publication Number:
  US 20050019747A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00736 Non-biologic macromolecules, e.g. polymeric compounds
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

A method of screening cell-polymer interactions. The method includes depositing monomers as a plurality of discrete elements on a substrate, causing the deposited monomers to polymerize, thereby creating an array of discrete polymer elements on the substrate, incubating the substrate in a cell-containing culture medium, and characterizing a predetermined cell behavior on each polymer element.

Claims

1. A method of screening cell-polymer interactions, comprising:

depositing monomers as a plurality of discrete elements on a substrate;

causing the deposited monomers to polymerize, thereby creating an array of discrete polymer elements on the substrate;

incubating the substrate in a cell-containing cell culture medium; and

characterizing a predetermined cell behavior on each polymer element.

2. The method of claim 1, wherein at least a portion of the polymer elements include a homopolymer.

3. The method of claim 1, wherein at least a portion of the monomers are deposited as a mixture of monomers.

4. The method of claim 1, wherein at least a portion of the monomers are bifunctional or multifunctional.

5. The method of claim 1, wherein at least a portion of the monomers are mono functional.

6. The method of claim 1, further comprising coating the substrate with a cytophobic material before depositing.

7. The method of claim 2, wherein the cytophobic material comprises one or more of poly(hydroxyethyl methacrylate), a poly(alkylene glycol), co-polymers including an alkylene glycol monomer, polymers derivatized with a poly(alkylene glycol), and hydrogels.

8. The method of claim 1, wherein the cell culture medium includes a growth factor.

9. The method of claim 8, wherein the growth factor is selected from activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, platelet derived growth factor, hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α

- or β

-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

10. The method of claim 1, wherein the cell culture medium includes serum.

11. The method of claim 1, wherein at least a portion of the polymer elements are co-polymers of at least two monomer species.

12. The method of claim 11, wherein the monomers are co-polymers of diethylene glycol methacrylate and 18

13. The method of claim 11, wherein the monomer species are monomers of one or more of polymers selected from polyamides, polyphosphazenes, polypropylfumarates, synthetic poly(amino acids), polyethers, polyacetals, polycyanoacrylates, polyurethanes, polycarbonates, polyanhydrides, poly(ortho esters), polyhydroxyacids, polyesters, polyacrylates, ethylene-vinyl acetate polymers, cellulose acetates, polystyrenes, chlorosulphonated polyolefins, polyaniline, polyesters, polyamides, polymerized vinyl compounds, and polymerized vinylidine compounds.

14. The method of claim 13, wherein the monomer species are selected from 1,4 butanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, neopentyl glycol diacrylate, phenylene diacrylate 1,3, propoxylated neopentyl glycol diacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, caprolactone 2-(methacryloyloxy)ethyl ester, 5-ethyl-5-(hydroxymethyl)-β

,β

-dimethyl-1,3-dioxane-2-ethanol diacrylate, 1,6-hexanediol propoxylate diacrylate, 3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate, glycerol 1,3-diglycerolate diacrylate, glycerol dimethacrylate, mixture of isomers, tech., 85%, neopentyl glycol dimethacrylate, neopentyl glycol ethoxylate (1 EO/OH) diacrylate, trimethylolpropane benzoate diacrylate, 1,1 4-tetradecanediol dimethacrylate, tricyclo[5.2.1.0^2,6]decanedimethanol diacrylate, trimethylolpropane ethoxylate (1 EO/OH) methyl ether diacrylate, trimethylolpropane triacrylate, tech,

15. The method of claim 14, wherein the monomers are selected from 1,4 butanediol dimethacrylate, diethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, phenylene diacrylate 1,3, triethylene glycol diacrylate, triethylene glycol dimethacrylate,

16. The method of claim 1, wherein the polymer is an acrylate polymer produced by polymerizing one or more monomers having a structure selected from

17. The method of claim 1, wherein the cell behavior is one or more of adhesion, proliferation, metabolic behavior, differentiation, production of a predetermined protein, expression of a predetermined gene, and an amount of any of the above.

18. The method of claim 17, wherein the predetermined protein is selected from cytokeratin, vimentin, desmin, alpha feto protein, nestin, GFAP, and actin.

19. The method of claim 1, wherein the cells are selected from chondrocytes, fibroblasts, connective tissue cells, epithelial cells, endothelial cells, cancer cells, hepatocytes, islet cells, smooth muscle cells, skeletal muscle cells, heart muscle cells, kidney cells, intestinal cells, organ cells, lymphocytes, blood vessel cells, human embryonic stem cells, and mesenchymal stem cells.

20. A method of controlling cell behavior, comprising:

selecting a first polymer in combination with which a predetermined cell exhibits a particular cell behavior;

selecting a second polymer differing from the first polymer in cross-link density or electron density; and

seeding the predetermined cell on the second polymer.

21. The method of claim 20, wherein the second polymer differs from the first in a density of acrylate groups, a density of methacrylate groups, a density of ester groups, a density of ether groups, the presence of an electron donating group, the identity of a heteroatom, the substitution on a heteroatom, the presence of a predetermined substituent, the presence of a predetermined heteroatom, or any combination of these.

22. A method of controlling a behavior of human embryonic stem cells, comprising:

exposing human embryonic stem cells to a synthetic polymer, wherein the polymer is selected to promote a predetermined behavior of the cells.

24. The method of claims 22 or 23, wherein the polymer is photopolymerizable.

25. The method of claims 22 or 23, wherein the polymer is an acrylate polymer produced by polymerizing one or more of 1,4 butanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, neopentyl glycol diacrylate, phenylene diacrylate 1,3, propoxylated neopentyl glycol diacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, caprolactone 2-(methacryloyloxy)ethyl ester, 5-ethyl-5-(hydroxymethyl)-β

,β

-dimethyl-1,3-dioxane-2-ethanol diacrylate, 1,6-hexanediol propoxylate diacrylate, 3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate, glycerol 1,3-diglycerolate diacrylate, glycerol dimethacrylate, mixture of isomers, tech., 85%, neopentyl glycol dimethacrylate, neopentyl glycol ethoxylate (1 EO/OH) diacrylate, trimethylolpropane benzoate diacrylate, 1,14-tetradecanediol dimethacrylate, tricyclo[5.2.1.0^2,6]decanedimethanol diacrylate, trimethylolpropane ethoxylate (1 EO/OH) methyl ether diacrylate, and trimethylolpropane triacrylate, tech, 7

26. The method of claims 22 or 23, wherein the polymer is an acrylate polymer produced by polymerizing one or more monomers having a structure selected from

27. The method of claims 22 or 23, wherein the behavior of the embryonic stem cell is selected from adhesion, proliferation, metabolic behavior, differentiation, production of a predetermined protein, expression of a predetermined gene, and an amount of any of the above.

23. A method of controlling a behavior of human embryonic stem cells, comprising:

exposing human embryonic stem cells to a synthetic polymer, wherein the polymer is not a polycation, polystyrene, a poly(lactide), or a co-polymer including lactide monomers.

28. A method of controlling cell behavior, comprising:

selecting a first monomer in combination with the polymer of which cells exhibit a particular cell behavior;

selecting a second monomer, that, when co-polymerized with the first monomer, modifies the cell behavior;

co-polymerizing the first and the second monomer to produce a co-polymer; and

seeding cells on the co-polymer.

29. The method of claim 28, wherein seeding the cells on the co-polymer comprises incubating the co-polymer in a cell-containing cell culture medium containing a growth factor, wherein the growth factor modifies the cell behavior of the cells in comparison to the behavior of cells seeded on the co-polymer in the absence of the growth factor.

30. The method of claim 29, wherein the growth factor is selected from activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, platelet derived growth factor, hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α

- or β

-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

31. The method of claim 28, wherein the cell behavior is one or more of adhesion, proliferation, metabolic behavior, differentiation, production of a predetermined protein, expression of a predetermined gene, and an amount of any of the above.

32. The method of claim 28, wherein the first and second monomers are co-polymerized on a cytophobic surface.

33. The method of claim 28, wherein the first monomer is selected from monofunctional monomers, bifunctional monomers, and multifunctional monomers.

34. The method of claim 28, wherein the second monomer is selected from monofunctional monomers, bifunctional monomers, and multifunctional monomers.

35. The method of claim 28, wherein the cells are selected from chondrocytes, fibroblasts, connective tissue cells, epithelial cells, endothelial cells, cancer cells, hepatocytes, islet cells, smooth muscle cells, skeletal muscle cells, heart muscle cells, kidney cells, intestinal cells, organ cells, lymphocytes, blood vessel cells, human embryonic stem cells, and mesenchymal stem cells.

36. The method of claim 28, wherein seeding cells comprises:

culturing embryonic stem cells under conditions where embryoid bodies are formed;

dissociating the embryoid bodies;

adding the dissociated cells to a culture medium; and

incubating the co-polymer in the cell-containing culture medium.

37. The method of claim 36, wherein the cell-containing culture medium includes serum.

38. The method of claim 28, wherein the monomers are selected from monomers of polymers selected from polyamides, polyphosphazenes, polypropylfumarates, synthetic poly(amino acids), polyethers, polyacetals, polycyanoacrylates, polyurethanes, polycarbonates, polyanhydrides, poly(ortho esters), polyhydroxyacids, polyesters, polyacrylates, ethylene-vinyl acetate polymers, cellulose acetates, polystyrenes, chlorosulphonated polyolefins, polyaniline, polyesters, polyamides, polymerized vinyl compounds, and polymerized vinylidine compounds.

39. The method of claim 38, wherein the monomer species are selected from 1,4 butanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, neopentyl glycol diacrylate, phenylene diacrylate 1,3,propoxylated neopentyl glycol diacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, caprolactone 2-(methacryloyloxy)ethyl ester, 5-ethyl-5-(hydroxymethyl)-β

,β

-dimethyl-1,3-dioxane-2-ethanol diacrylate, 1,6-hexanediol propoxylate diacrylate, 3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate, glycerol 1,3-diglycerolate diacrylate, glycerol dimethacrylate, mixture of isomers, tech., 85%, neopentyl glycol dimethacrylate, neopentyl glycol ethoxylate (1 EO/OH) diacrylate, trimethylolpropane benzoate diacrylate, 1,14-tetradecanediol dimethacrylate, tricyclo[5.2.1.0^2,6]decanedimethanol diacrylate, trimethylolpropane ethoxylate (1 EO/OH) methyl ether diacrylate, and trimethylolpropane triacrylate, tech,

40. The method of claim 39, wherein the monomers are selected from 1,4 butanediol dimethacrylate, diethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, phenylene diacrylate 1,3, triethylene glycol diacrylate, triethylene glycol dimethacrylate,

41. The method of claim 39, wherein,

when the first monomer is diethylene glycol methacrylate, the second monomer is 18

when the first monomer is 1,4 butanediol dimethacrylate, the second monomer is25

when the first monomer is 7

the second monomer

is 1,6, hexanediol diacrylate or 25

when the first monomer is triethylene glycol diacrylate, the second monomer is 1,4 butanediol dimethacrylate or 21

when the first monomer is triethylene glycol dimethacrylate the second monomer is 21

when the first monomer is

the second monomer is 25

when the first monomer is

the second monomer is

and the first monomer and the second monomer are present in the polymer in a ratio of 70/30 by volume.

42. The method of claim 41, wherein seeding cells comprises incubating the co-polymer in a cell-containing cell culture medium including retinoic acid.

43. The method of claim 28, wherein the polymer is an acrylate polymer produced by polymerizing one or more monomers having a structure selected from

44. A method of controlling cell behavior, comprising:

selecting a first monomer in combination with the polymer of which cells exhibit a particular cell behavior;

selecting a growth factor that modifies the cell behavior when the cells are seeded on the polymer of the first monomer;

polymerizing the first monomer to produce a polymer; and

incubating the polymer in a cell-containing culture medium containing the growth factor.

45. The method of claim 44, wherein the cell-containing culture medium includes serum.

46. The method of claim 44, wherein the cell behavior is one or more of adhesion, proliferation, metabolic behavior, differentiation, production of a predetermined protein, expression of a predetermined gene, and an amount of any of the above.

47. The method of claim 44, wherein the first monomer is a monomer of one or more polymers selected from polyamides, polyphosphazenes, polypropylfumarates, synthetic poly(amino acids), polyethers, polyacetals, polycyanoacrylates, polyurethanes, polycarbonates, polyanhydrides, poly(ortho esters), polyhydroxyacids, polyesters, polyacrylates, ethylene-vinyl acetate polymers, cellulose acetates, polystyrenes, chlorosulphonated polyolefins, polyaniline, polyesters, polyamides, polymerized vinyl compounds, and polymerized vinylidine compounds.

48. The method of claim 47, wherein the first monomer is selected from 1,4 butanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, 1,6 hexanediol diacrylate, neopentyl glycol diacrylate, phenylene diacrylate 1,3, propoxylated neopentyl glycol diacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, caprolactone 2-(methacryloyloxy)ethyl ester, 5-ethyl-5-(hydroxymethyl)-β

,β

-dimethyl-1,3-dioxane-2-ethanol diacrylate, 1,6-hexanediol propoxylate diacrylate, 3-hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate, glycerol 1,3-diglycerolate diacrylate, glycerol dimethacrylate, mixture of isomers, tech., 85%, neopentyl glycol dimethacrylate, neopentyl glycol ethoxylate (1 EO/OH) diacrylate, trimethylolpropane benzoate diacrylate, 1,14-tetradecanediol dimethacrylate, tricyclo[5.2.1.0^2,6]decanedimethanol diacrylate, trimethylolpropane ethoxylate (1 EO/OH) methyl ether diacrylate, and trimethylolpropane triacrylate, tech,

49. The method of claim 48, wherein the first monomer is 1,4 butanediol dimethacrylate, diethylene glycol dimethacrylate, phenylene diacrylate 1,3,

50. The method of claim 44, wherein the monomer is monofunctional, bifunctional, or multifunctional.

51. The method of claim 44, wherein polymerized first monomer has a structure selected from

52. The method of claim 44, wherein the cells are selected from chondrocytes, fibroblasts, connective tissue cells, epithelial cells, endothelial cells, cancer cells, hepatocytes, islet cells, smooth muscle cells, skeletal muscle cells, heart muscle cells, kidney cells, intestinal cells, organ cells, lymphocytes, blood vessel cells, human embryonic stem cells, and mesenchymal stem cells.

53. The method of claim 44, wherein the growth factor is selected from activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, platelet derived growth factor, hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α

- or β

-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

54. The method of claim 53, wherein the growth factor is retinoic acid.

55. The method of claim 44, wherein polymerizing comprises co-polymerizing the first monomer with a second monomer.

56. A method of controlling cell behavior, comprising:

selecting cells characterized by a predetermined level of expression of a first gene;

selecting a monomer in combination with the polymer of which the cells exhibit a level of expression of the first gene different from the predetermined level;

polymerizing the monomer to produce a polymer; and

seeding the cells on the polymer.

57. The method of claim 56, wherein the cells are human embryonic stem cells.

58. A method of controlling cell behavior, comprising:

selecting cells characterized by a predetermined level of expression of a first protein;

selecting a monomer in combination with the polymer of which the cells exhibit a level of expression of the first protein different from the predetermined level;

polymerizing the monomer to produce a polymer; and

seeding the cells on the polymer.

59. The method of claim 58, wherein the cells are human embryonic stem cells.

60. The method of claim 58, wherein the first protein is cytokeratin, vimentin, or actin.

61. A method of supporting growth of C2C 12 cells in vitro, comprising culturing the C2C12 cells on a polymer produced from one or more of 1,4 butanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, phenylene diacrylate 1,3, propoxylated neopentyl glycol diacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, caprolactone 2-(methacryloyloxy)ethyl ester, 5-ethyl-5-(hydroxymethyl)-β

,β

-dimethyl-1,3-dioxane-2-ethanol diacrylate, 1,6-hexanediol propoxylate diacrylate, neopentyl glycol ethoxylate (1 EO/OH) diacrylate, trimethylolpropane benzoate diacrylate, tricyclo[5.2.1.0^2,6]decanedimethanol diacrylate,