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

Patent metadata

Field	Value
Publication number	US-2024271183-A1
Application number	US-202318542404-A
Country	US
Kind code	A1
Filing date	Dec 15, 2023
Priority date	Oct 4, 2017
Publication date	Aug 15, 2024
Grant date	—

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.

First claim

What is claimed is:

1 . A composition, comprising:
a partition comprising a matrix encapsulating a cell or cell nucleus, wherein the matrix is diffusively permeable to chemical reagents, wherein: i) the matrix comprises two or more crosslinked polymers, wherein at least one polymer of the two or more crosslinked polymers is 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 ii) the two or more crosslinked polymers comprise a triazole group, a dihydropyridazine group, an iminophophorane group, a Diels-Alder adduct, an iminophophorane group, or an isoxazoline group.

2 . The composition of claim 1 , wherein the partition is a droplet is among a plurality of droplets, wherein the droplet is fluidically isolated from other droplets in the plurality of droplets.

3 . The composition of claim 1 , wherein the partition is a well.

4 . The composition of claim 1 , wherein the two or more crosslinked polymers are attached to an oligonucleotide.

5 . The composition of claim 4 , wherein the oligonucleotide comprises a poly-T sequence or a poly-A sequence.

6 . The composition of claim 1 , wherein the composition is copper-free.

7 . The composition of claim 1 , wherein the matrix comprises a labile bond selected from a disulfide bond, a carbamate bond, and a peptide bond.

8 . The composition of claim 7 , wherein the labile bond is a peptide bond.

9 . The composition of claim 8 , wherein the labile bond is a carbamate linkage.

10 . The composition of claim 1 , wherein the partition further comprises a barcode molecule.

11 . The composition of claim 10 , wherein the barcode molecule is a nucleic acid barcode molecule.

12 . The composition of claim 11 , wherein the nucleic acid barcode molecule is attached to a bead in the partition.

13 . The composition of claim 1 , wherein the chemical reagents are selected from nucleic acids, enzymes, and potential cell binding ligands.

14 . A method of forming a matrix comprising:
(a) combining under copper-free click chemistry reaction conditions in a partition among a plurality of partitions: (i) a cell or cell nucleus, and (ii) two or more polymers, wherein at least one polymer of said two or more polymers is 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 (b) forming crosslinks between the two or more polymers, thereby generating a matrix encapsulating the cell or cell nucleus.

15 . The method of claim 14 , wherein (b) comprises using (i) strain-promoted azide/dibenzocyclooctyne-amine (DBCO) click chemistry; (ii) inverse electron demand Diels-Alder (IED-DA) tetrazine/trans-cyclooctene (TCO) click chemistry; (iii) inverse electron demand Diels-Alder (IED-DA) tetrazine/norbonene click chemistry; (iv) Diels-Alder maleimide/furan click-chemistry; (v) Staudinger ligation; or (vi) nitrile-oxide/norbonene cycloaddition click chemistry to form crosslinks between the two or more polymers.

16 . The method of claim 14 , further comprising degrading the matrix.

17 . The method of claim 16 , wherein the matrix comprises a carbamate bond and the method further comprises degrading the matrix using a polyamine and heat to degrade the carbamate bond.

18 . The method of claim 16 , wherein the matrix comprises a peptide bond and the method further comprises degrading the matrix using a protease to degrade the peptide bond.

19 . The method of claim 14 , wherein the partition among the plurality of partitions is selected from a well and a droplet.

20 . The method of claim 14 , further comprising using a barcode molecule in the partition and a nucleic acid from the cell or cell nucleus to generate a barcoded nucleic acid molecule.

Assignees

• 10X Genomics Inc

Inventors

• Delaney Joshua
• Gohil Shalini
• Hindson Christopher
• Lowe Adam
• Price Andrew D
• Shuga Joseph Francis

Classifications

• C08J2333/00

  Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers · CPC title

• C08J3/075 Primary

  Macromolecular gels · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• B01J13/0065

  containing an organic phase · CPC title

• B01J13/18

  In situ polymerisation with all reactants being present in the same phase · CPC title