MANUFACTURE AND USES OF REACTIVE MICROCONTACT PRINTING OF BIOMOLECULES ON SOFT HYDROGELS

• US 20100055733A1
• Filed: 09/02/2009
• Published: 03/04/2010
• Est. Priority Date: 09/04/2008
• Status: Abandoned Application

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Lutolf, Matthias P., Blau, Helen M., Doyonnas, Regis
• Application Number:
  US12/552,400
• Publication Number:
  US 20100055733A1
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2501/59 Lectins
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

Embodiments of the present disclosure encompass microfabrication methods (“reactive microcontact printing of soft matter”) for hydrated soft polymer materials and surfaces for culture platforms suitable for the culturing of isolated single primary mammalian cells in an environment approximating the natural niches of the cells. Such culture platforms may comprise arrays of microwells, or other microscopically textured features, in which individual features can comprise desired proteins or mixtures of proteins. The microfabrication methods of the disclosure allow spatial control of surface biochemistry and topography at the micrometer scale on these hydrated soft gels. The hydrogels and methods of manufacture and use of the disclosure allow the isolation of a single stem cell and the characterizing of its interaction with cytokines and morphogens, especially with regard to modulation of the proliferative capacity of the stem cell when implanted in a recipient host. The systems for isolating or culturing a eukaryotic cell comprise a hydrogel film comprising a cross-linked polymeric composition having the characteristic of hydrating to form a hydrogel and having a topographical feature or a plurality of topographical features that may have a surface capable of receiving and immobilizing at least one biomolecule species thereon.

Claims

1. A system for isolating or culturing a cell, the system comprising a hydrogel film comprising a cross-linked polymeric composition has the characteristic of hydrating to form a hydrogel and having a topographical feature or a plurality of topographical features, wherein each topographical feature has a surface capable of receiving and immobilizing at least one biomolecule species thereon.

2. The system of claim 1, wherein the hydrogel film is hydrated as a hydrogel.

3. The system of claim 1, wherein a biomolecule species is immobilized to the cross-linked polymeric composition.

4. The system of claim 3, wherein the at least one biomolecule species is selected from the group consisting of:

a polypeptide, a peptide, an oligonucleotide, and a small molecule.

5. The system of claim 3, wherein the biomolecule species is selected from the group consisting of:

Wnt3a, N-cadherin, thrombopoietin, erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growth factor, insulin, and recombinant insulin.

6. The system of claim 1, wherein the cross-linked polymeric composition is selected from the group consisting of:

a poly(ethylene glycol), a polyaliphatic polyurethane, a polyether polyurethane, a polyester polyurethane, a polyethylene copolymer, a polyamide, a polyvinyl alcohol, a polypropylene glycol, a polytetramethylene oxide, a polyvinyl pyrrolidone, a polyacrylamide, a poly(hydroxyethyl acrylate), and a poly(hydroxyethyl methacrylate).

7. The system of claim 1, wherein the cross-linked polymeric composition is formed from at least two precursor compounds in a ratio whereby, when the precursors are cross-linked to form the polymeric composition, a surface of a topographical feature adapted to immobilize a polypeptide or a tether thereto.

8. The system of claim 1, wherein the cross-linked polymeric composition is synthesized from at least two precursor compounds wherein one precursor compound comprises n nucleophilic groups, and a second precursor compound comprises m electrophilic groups, wherein n and m are each at least 2 and the sum (n+m) is at least five.

9. The system of claim 8, wherein the cross-linked polymeric composition is synthesized from at least two precursor components using a Michael-type addition reaction.

10. The system of claim 8, wherein the n nucleophilic groups are thiol groups.

11. The system of claim 8, wherein the m electrophilic groups are conjugated unsaturated groups.

12. The system of claim 1, wherein the plurality of topographical features is an array of microwells.

13. The system of claim 1, wherein a surface of a topographical feature comprises a reactive functional group of the cross-linked polymeric composition, wherein the reactive functional group is capable of binding to a biomolecule species desired to be immobilized on the surface of the topographical feature.

14. The system of claim 13, wherein the reactive functional group is selected from the group consisting of:

a succinimidyl active ester, an aldehyde, a thiol, and a thiol-selective group.

15. The system of claim 1, wherein a topographical feature has a tether immobilized thereon, wherein the tether is adapted to selectively bind to the biomolecule species desired to be immobilized on the surface of the topographical feature.

16. The system of claim 15, wherein the tether is selected from the group consisting of:

a peptide, a polypeptide, and a non-peptide linker.

17. The system of claim 15, wherein the tether is selected from the group consisting of:

a heterofunctional PEG, Protein A, Protein G, an immunoglobulin, streptavidin, neutravidin, biotin, a linker capable of forming a complex with a metal ion, and a transglutaminase substrate.

18. The system of claim 15, wherein the tether is Protein A or Protein G, and the biomolecule species bound thereto is a polypeptide comprising an immunoglobulin Fc region and a region capable of interacting with a cell disposed in the topographical feature.

19. The system of claim 1, wherein the hydrogel film is disposed in a well of a multi-well tissue culture plate.

20. A microcontact printing method of preparing a hydrogel, comprising:

(a) providing a template comprising a negative topographical feature or a plurality of negative topographical features, wherein each negative topographical feature defines a topographical feature desired to be formed in a hydrogel, and wherein the negative topographical feature or features has on a surface thereof a biomolecule species desired to be transferred to a hydrogel, a tether capable of selectively binding to said biomolecule species, or a combination thereof;

(b) delivering to the template a hydrogel polymer precursor composition;

(c) polymerizing the hydrogel polymer precursor composition to form a hydrogel film; and

(d) removing the hydrogel film from the template, thereby transferring the biomolecule species, the tether, or a combination thereof, to a surface of the topographical feature molded in the hydrogel film.

21. The method of claim 20, wherein each topographical feature molded in the hydrogel is a microwell.

22. The method of claim 20, further comprising:

hydrating the hydrogel film, thereby forming a hydrogel.

23. The method of claim 20, wherein the biomolecule species is selected from the group consisting of:

a polypeptide, a peptide, an oligonucleotide, and a small molecule.

24. The method of claim 23, wherein the biomolecule species is selected from the group consisting of:

Wnt3a, N-cadherin, thrombopoietin, erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growth factor, insulin, and recombinant insulin.

25. The method of claim 20, wherein, if a tether is transferred from the template to a surface of a topographical feature, the method further comprises delivering to the topographical feature of the hydrogel a composition, said composition comprising a biomolecule species desired to be immobilized on the surface of the microwells, thereby selectively binding the biomolecule species to the tether and immobilizing the biomolecule species to the surface of the topographical feature.

26. The method of claim 20, wherein a surface of a topographical feature or of a plurality of topographical features comprises a reactive functional group of the cross-linked polymeric composition, wherein the reactive functional group is capable of binding to the biomolecule species desired to be immobilized on the topographical feature or plurality of topographical features.

27. The method of claim 26, wherein the reactive functional group is selected from the group consisting of:

a succinimidyl active ester, an aldehyde, a thiol and a thiol-selective group.

28. The method of claim 20, wherein a surface of a topographical features has a tether immobilized thereon, wherein the tether is capable of selectively binding to the biomolecule species desired to be immobilized on the surface of the topographical feature.

29. The method of claim 28, wherein the tether is selected from the group consisting of:

a peptide, a polypeptide, and a non-peptide linker.

30. The method of claim 28 wherein the tether is selected from the group consisting of:

a heterofunctional PEG, Protein A, Protein G, an immunoglobulin, streptavidin, neutravidin, biotin, a linker capable of forming a complex with a metal ion, and a transglutaminase substrate.

31. The method of claim 28, wherein the tether is Protein A or Protein G, and the biomolecule species bound thereto comprises an immunoglobulin Fc region and a region capable of interacting with a cell disposed in the microwell of the topographical feature.

32. The method of claim 20, wherein the hydrogel polymer precursor composition comprises at least two precursor compounds in a ratio whereby when the precursors are cross-linked to form the polymer the surface of the microwells of the microwell array is capable of immobilizing a polypeptide or a tether thereto.

33. The method of claim 20, wherein the hydrogel film is disposed in a well of a multi-well tissue culture plate.

34. A method of isolating individual cells from a population of cells, comprising:

(a) providing a hydrogel system disposed in a well of a multi-well tissue culture plate, wherein the hydrogel comprises a hydrated cross-linked polymer having an array of topographical features indented therein, and wherein a surface of each of the topographical features has at least one biomolecule species immobilized thereon;

(b) delivering a cell suspension of isolated cells to the well of the multi-well tissue culture plate, whereby the cells of the suspension descend under gravity into the multiplicity of microwells, and wherein the cell density of the cell suspension is adjusted whereby at least one well of the multiplicity of wells receives a single cell; and

(c) incubating the hydrogel under conditions favorable for proliferation of the cells.

35. The method of claim 34, wherein the topographical feature is a microwell.

36. The method of claim 34, wherein the cell suspension comprises a population of stem cells.

37. The method of claim 36, wherein the stem cells of the population of stem cells is selected from the group consisting of:

a hematopoietic stem cell, a hematopoietic progenitor cell, an adult stem cell, an embryonic stem cell, and a cancer stem cell.

38. The method of claim 34, wherein a biomolecule species is immobilized by a tether to a surface of a topographical feature.

39. The method of claim 34, wherein the at least one biomolecule species is selected from the group consisting of:

a polypeptide, a peptide, an oligonucleotide, and a small molecule.

40. The method of claim 39, wherein the biomolecule species is selected from the group consisting of:

Wnt3a, N-cadherin, thrombopoietin, erythropoietin, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2, interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growth factor, insulin, and recombinant insulin.

41. The method of claim 40, wherein a surface of a topographical feature comprises a reactive functional group of the cross-linked polymeric composition, wherein the reactive functional group is capable of binding to the biomolecule species desired to be immobilized on the surface of the topographical feature.

42. The method of claim 40, wherein the reactive functional group is selected from the group consisting of:

a succinimidyl active ester, an aldehyde, a thiol and a thiol-selective group.

43. The method of claim 38, wherein the tether is selected from the group consisting of:

a peptide, a polypeptide, and a non-peptide linker.

44. The method of claim 38, wherein the tether is selected from the group consisting of:

a heterofunctional PEG, Protein A, Protein G, an immunoglobulin, streptavidin, neutravidin, biotin, a linker capable of forming a complex with a metal ion, and a transglutaminase substrate.

45. The method of claim 38, wherein the tether is Protein A or Protein G, and the biomolecule species bound thereto comprises an immunoglobulin Fc region and a region capable of interacting with a cell disposed in the microwell of the topographical feature.

46. A method for determining the proliferative outcome of transplanting a stem cell into a recipient host, comprising;

(a) delivering a population of cells to a plurality of microwells indented in a hydrogel, wherein an interior surface of each microwell of the plurality of microwells has a biomolecule species immobilized thereon, and wherein at least some of the microwells of the multiplicity of microwells receive a single cell from the population of cells;

(b) monitoring the proliferation of the isolated single cells by time-lapse photography;

(c) correlating the proliferation of the cells to the proliferative outcome of a stem cell transplanted into a recipient host; and

(d) identifying those cells in a microwell having the characteristic of regenerating a hematopoietic system when transplanted into a recipient host.

47. The method of claim 46, wherein the stem cell is a hematopoietic stem cell.