Compositions, methods, and systems for bead formation using improved polymers

• US 10,590,244 B2
• Filed: 11/01/2018
• Issued: 03/17/2020
• Est. Priority Date: 10/04/2017
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Delaney, Joshua, Gohil, Shalini, Hindson, Christopher, Lowe, Adam, Price, Andrew D.
• Primary Examiner(s): Pepitone, Michael F
• Application Number:
  US16/178,430
• Publication Number:
  US 20190100632A1
• Time in Patent Office: 502 Days
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C08J 2333/26 Homopolymers or copolymers of acrylamide or methacrylamide
• CPC Class Codes: C08J 3/075 Macromolecular gels
• CPC Class Codes: C08J 3/24 Crosslinking, e.g. vulcanising, of macromolecules mechanical aspects B29C35/00; crosslinking agents C08K

Abstract

The present disclosure provides systems and methods for making a hydrogel comprising a cell, cell nucleus, or one or more components derived from a cell or cell nucleus. A method for making a hydrogel may comprise providing a cell or cell nucleus, a first polymer, wherein the first polymer comprises a plurality of first crosslink precursors, each of the plurality of first crosslink precursors comprising an azide group; providing a second polymer, wherein the second polymer comprises a plurality of second crosslink precursors, each of the plurality of second crosslink precursors comprising an alkyne group; and crosslinking the first polymer and the second polymer via a reaction between a first section of the first crosslink precursors and a second section of the second crosslink precursors, thereby providing the hydrogel comprising the cell or cell nucleus.

Claims

1. A gel, comprising:

(a) a cell, a nucleus of said cell, or one or more constituents derived from said cell;

(b) two or more polymers, wherein a polymer of said two or more polymers comprises at least one member selected from the group consisting of a polyolefin, an olefin copolymer, an acrylic, a vinyl polymer, a polyamide, a polyimide, a formaldehyde resin, a polyurethane, a cellulosic, a thermoplastic elastomer, and a thermoplastic polyurethane; and

(c) a plurality of linkers, each of said plurality of linkers comprising a 1,2,3-triazole moiety, wherein said plurality of linkers crosslink said two or more polymers, and wherein said plurality of linkers is distributed throughout said gel.

2. The gel of claim 1, wherein a polymer of said two or more polymers is a polyacrylamide.

3. The gel of claim 2, wherein a linker of said plurality of linkers is connected to an amide of said polyacrylamide.

4. The gel of claim 1, wherein a linker of said plurality of linkers comprise a labile bond.

5. The gel of claim 4, wherein said labile bond is a chemically labile bond, a thermally labile bond, or a photo-labile bond.

6. The gel of claim 4, wherein said labile bond comprises a disulfide bond.

7. The gel of claim 1, further comprising at least one reagent for conducting a chemical or biological reaction enclosed within said gel.

8. The gel of claim 1, wherein said gel is a hydrogel.

9. The gel of claim 1, wherein said gel further comprises a charged species.

10. The gel of claim 9, wherein said charged species is attached to at least one of said two or more polymers.

11. The gel of claim 1, wherein at least one of said two or more polymers is an electrically charged polymer.

28. The gel of claim 1, wherein said polymer is a polyolefin.

29. The gel of claim 1, wherein said polymer is an olefin copolymer.

30. The gel of claim 1, wherein said polymer is an acrylic.

31. The gel of claim 1, wherein said polymer is a vinyl polymer.

32. The gel of claim 1, wherein said polymer is a polyamide.

33. The gel of claim 1, wherein said polymer is a polyimide.

34. The gel of claim 1, wherein said polymer is a formaldehyde resin.

35. The gel of claim 1, wherein said polymer is a polyurethane.

36. The gel of claim 1, wherein said polymer is a cellulosic.

37. The gel of claim 1, wherein said polymer is a thermoplastic elastomer.

38. The gel of claim 1, wherein said polymer is a thermoplastic polyurethane.

12. A method of forming a gel, comprising:

(a) providing a cell, a nucleus of said cell, or one or more constituents derived from said cell; and

(b) generating said gel comprising (i) said cell, said nucleus of said cell, or said one or more constituents derived from said cell, (ii) two or more polymers, wherein a polymer of said two or more polymers comprises at least one member selected from the group consisting of a polyolefin, an olefin copolymer, an acrylic, a vinyl polymer, a polyamide, a polyimide, a formaldehyde resin, a polyurethane, a cellulosic, a thermoplastic elastomer, and a thermoplastic polyurethane, and (iii) a plurality of linkers, each of said plurality of linkers comprising a 1,2,3-triazole moiety, wherein said plurality of linkers crosslink said two or more polymers, and wherein said plurality of linkers is distributed throughout said gel.

13. The method of claim 12, wherein (a) comprises providing (i) a first polymer, wherein said first polymer comprises a plurality of first crosslink precursors, each of said plurality of first crosslink precursors comprising an azide group and (ii) a second polymer, wherein said second polymer comprises a plurality of second crosslink precursors, each of said plurality of second crosslink precursors comprising an alkyne group, and wherein (b) comprises crosslinking said first polymer and said second polymer by reacting said plurality of first crosslink precursors with said plurality of second crosslink precursors, thereby forming said gel comprising said plurality of linkers crosslinking said two or more polymers.

14. The method of claim 13, wherein said reacting comprises forming a 1,2,3-triazole between said azide group and said alkyne group.

15. The method of claim 13, wherein said first polymer or said second polymer further comprises a labile bond.

16. The method of claim 15, wherein said labile bond is a disulfide bond.

17. The method of claim 15, wherein at least about 80% of said labile bond remains intact while reacting said plurality of first crosslink precursors with said plurality of second crosslink precursors.

18. The method of claim 13, wherein reacting said plurality of first crosslink precursors with said plurality of second crosslink precursors is catalyzed by a catalyst.

19. The method of claim 18, further comprising removing said catalyst or a derivative thereof from said gel.

20. The method of claim 12, wherein said two or more polymers comprises a first polymer and a second polymer, wherein said first polymer is different than said second polymer.

21. The method of claim 12, further comprising, subsequent to (b), lysing said cell or said cell nucleus to release one or more cell or cell nucleus constituents into said gel.

22. The method of claim 12, wherein (a) comprises providing said cell, nucleus of said cell, or one or more constituents derived from said cell is in a partition, and wherein (b) comprises generating said gel in or from said partition.

23. The method of claim 22, wherein said partition is a well.

24. The method of claim 22, wherein said partition is an aqueous droplet in an emulsion.

25. The method of claim 24, wherein said partition comprises a reagent that catalyzes a reaction for forming said gel.

26. The method of claim 25, wherein said emulsion comprises an oil phase comprising a reagent comprising a copper (II) moiety, and wherein said aqueous droplet comprises a reducing agent capable of reducing said copper (II) moiety to a copper (I) moiety, wherein said copper (I) moiety catalyzes said reaction.

27. The method of claim 26, wherein said emulsion comprises a reagent that facilitates transport of said copper (II) moiety from said oil phase into said aqueous droplet.

39. The method of claim 12, wherein said polymer is a polyolefin.

40. The method of claim 12, wherein said polymer is an olefin copolymer.

41. The method of claim 12, wherein said polymer is an acrylic.

42. The method of claim 12, wherein said polymer is a vinyl polymer.

43. The method of claim 12, wherein said polymer is a polyamide.

44. The method of claim 12, wherein said polymer is a polyimide.

45. The method of claim 12, wherein said polymer is a formaldehyde resin.

46. The method of claim 12, wherein said polymer is a polyurethane.

47. The method of claim 12, wherein said polymer is a cellulosic.

48. The method of claim 12, wherein said polymer is a thermoplastic elastomer.

49. The method of claim 12, wherein said polymer is a thermoplastic polyurethane.