# US 11,441,172 B2 · Compositions, methods, and systems for bead formation using improved polymers

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- Number: US 11,441,172 B2
- Document ID: US11441172B2
- Status: Granted
- Kind: B2
- Application number: 17/009,352
- Filed: 2020-09-01
- Published: 2020-12-17
- Granted: 2022-09-13
- Earliest claimed: 2017-10-04
- Assignees: 10x Genomics, Inc.
- Applicants: 10X Genomics, Inc.
- Inventors: Joshua Delaney; Shalini Gohil; Christopher Hindson; Adam Lowe; Andrew D. Price; Joseph Francis Shuga
- Web page: https://patentgrep.com/patent/US11441172B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01J 13/0065: {containing an organic phase}
- B01J 13/18: In situ polymerisation with all reactants being present in the same phase
- C08J 3/075: Macromolecular gels
- C08J 2333/00: 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
- C12Q 1/6869: Methods for sequencing
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C08J: WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H (working, e.g. shaping, of plastics B29)

## 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

### Claim 1

A method comprising:
(a) providing a live cell encapsulated in a gel matrix that is diffusively permeable to chemical or biochemical reagents, wherein the gel matrix comprises two or more crosslinked polymers, wherein at least one polymer of said 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 wherein the crosslinks are formed by click chemistry;
(b) culturing the live cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method further comprises contacting the gel matrix with a chemical or biochemical reagent.

### Claim 3 (depends on 1)

The method of claim 1, wherein the culturing of the live cell is in the presence of a chemical or a biochemical reagent.

### Claim 4 (depends on 1)

The method of claim 1, wherein the chemical or biochemical reagents are selected from nucleic acids, enzymes, potential cell binding ligands.

### Claim 5 (depends on 1)

The method of claim 1, wherein the gel matrix further comprises an activatable functional group.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method further comprises analyzing one or more components of the live cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the gel matrix is contained in a partition.

### Claim 8 (depends on 1)

The method of claim 1, wherein the live cell is selected from a prokaryotic cells, eukaryotic cells, a bacterial cell, a fungal cell, a plant cell, a mammalian cell, a mycoplasma cell, a normal tissue cell, a tumor cell, a T-cell, and a rare cell.

### Claim 9 (depends on 1)

The method of claim 1, wherein the crosslinks are distributed throughout the gel.

### Claim 10 (depends on 1)

The method of claim 1, wherein the gel matrix is contained in a partition, wherein the partition is a droplet.

### Claim 11 (depends on 1)

The method of claim 1, wherein the crosslinks comprise a picolyl moiety.

### Claim 12 (depends on 1)

The method of claim 1, wherein the click chemistry is copper-catalyzed or copper-free.

### Claim 13 (depends on 12)

The method of claim 12, wherein the click chemistry is copper-free click chemistry selected from: (a) strain-promoted azide/dibenzocyclooctyne-amine (DBCO) click chemistry; (b) inverse electron demand Diels-Alder (IED-DA) tetrazine/trans-cyclooctene (TCO) click chemistry; (c) inverse electron demand Diels-Alder (IED-DA) tetrazine/norbonene click chemistry; (d) Diels-Alder maleimide/furan click-chemistry; (e) Staudinger ligation; and (f) nitrile-oxide/norbonene cycloaddition click chemistry.

### Claim 14 (depends on 1)

The method of claim 1, wherein the crosslinks comprise a 1,2,3-triazole moiety or a dihydropyridazine moiety.

### Claim 15 (depends on 1)

The method of claim 1, wherein the crosslinks comprise a labile bond.

### Claim 16 (depends on 15)

The method of claim 15, wherein the labile bond is selected from a chemically labile bond, a thermally labile bond, an enzymatically labile bond, a photo-labile bond, a disulfide bond, a carbamate bond, a peptide bond, or a combination thereof.

### Claim 17

A composition comprising
a partition comprising a live cell encapsulated in a gel matrix that is diffusively permeable to chemical or biochemical reagents, wherein the gel matrix comprises two or more crosslinked polymers, wherein the crosslinks are formed by click chemistry and at least one polymer of said 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.

### Claim 18 (depends on 17)

The composition of claim 17, wherein the crosslinks are distributed throughout the gel matrix.

### Claim 19

A method of forming a live cell encapsulated in a gel matrix comprising:
combining in a partition under click chemistry reaction conditions (i) a live cell, and (ii) two or more polymers configured to form crosslinks by click chemistry, wherein at least one polymer of said 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.