US-12624384-B2 - Compositions, methods, and systems for bead formation using improved polymers

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.

Inventors

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

Assignees

• 10X GENOMICS, INC.

Dates

Publication Date

20260512

Application Date

20231215

Claims (20)

1. 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, a Diels-Alder adduct, or an isoxazoline group.
2. 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. 3 . The composition of claim 1 , wherein the partition is a well.
4. 4 . The composition of claim 1 , wherein the two or more crosslinked polymers are attached to an oligonucleotide.
5. 5 . The composition of claim 4 , wherein the oligonucleotide comprises a poly-T sequence or a poly-A sequence.
6. 6 . The composition of claim 1 , wherein the composition is copper-free.
7. 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. 8 . The composition of claim 7 , wherein the labile bond is a peptide bond.
9. 9 . The composition of claim 8 , wherein the labile bond is a carbamate linkage.
10. 10 . The composition of claim 1 , wherein the partition further comprises a barcode molecule.
11. 11 . The composition of claim 10 , wherein the barcode molecule is a nucleic acid barcode molecule.
12. 12 . The composition of claim 11 , wherein the nucleic acid barcode molecule is attached to a bead in the partition.
13. 13 . The composition of claim 1 , wherein the chemical reagents are selected from nucleic acids, enzymes, and potential cell binding ligands.
14. 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. 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/norbornene 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. 16 . The method of claim 14 , further comprising degrading the matrix.
17. 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. 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. 19 . The method of claim 14 , wherein the partition among the plurality of partitions is selected from a well and a droplet.
20. 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.

Description

CROSS-REFERENCE This application is a continuation of U.S. patent application Ser. No. 17/861,503, filed Jul. 11, 2022, now issued as U.S. Pat. No. 11,884,964, which is a continuation of U.S. patent application Ser. No. 17/009,352, filed Sep. 1, 2020, now issued as U.S. Pat. No. 11,441,172, which is a continuation of U.S. patent application Ser. No. 16/374, 112, filed Apr. 3, 2019, now issued as U.S. Pat. No. 10,837,047, which is a continuation-in-part of U.S. patent application Ser. No. 16/178,430, filed Nov. 1, 2018, now issued as U.S. Pat. No. 10,590,244, which is a continuation of International Pat. Appl. No. PCT/US2018/054458, filed Oct. 4, 2018, which claims priority to U.S. Provisional Patent Application No. 62/687,161, filed Jun. 19, 2018, and U.S. Provisional Pat. Appl. No. 62/568,021, filed Oct. 4, 2017, each of which is incorporated by reference herein in its entirety for all purposes. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Apr. 3, 2024, is named 43487-1004_303_SL.xml and is 23,198 bytes in size. BACKGROUND Samples may be processed for various purposes, such as identification of a type of sample of moiety within the sample. The sample may be a biological sample. The biological samples may be processed for various purposes, such as detection of a disease (e.g., cancer) or identification of a particular species. There are various approaches for processing samples, such as polymerase chain reaction (PCR) and sequencing. Biological samples may be processed within various reaction environments, such as partitions. Partitions may be wells or droplets. Droplets or wells may be employed to process biological samples in a manner that enables the biological samples to be partitioned and processed separately. For example, such droplets may be fluidically isolated from other droplets, enabling accurate control of respective environments in the droplets. Biological samples in partitions may be subjected to various processes, such as chemical processes or physical processes. Samples in partitions may be subjected to heating or cooling, or chemical reactions, such as to yield species that may be qualitatively or quantitatively processed. SUMMARY A hydrogel matrix (including a bead) can create a semi-open system capable of enclosing a large molecule within the boundary of the matrix, while allowing a small molecule to permeate the matrix. The large molecule can be a biological sample, including, for example, a cell, a large protein or a long nucleic acid. The small molecule can be, such as, for example, a reagent, a smaller protein, or a shorter nucleic acid. An enzyme, for example, may be small enough to permeate the matrix. The hydrogel matrix can also comprise a labile bond such that after the hydrogel matrix is degraded, the enclosed large molecule can be released from the confine of the matrix into the surrounding environment. Provided herein are methods, systems and compositions for the production of a hydrogel matrix capable of enclosing a large molecule and allowing a small molecule to permeate the matrix. In some aspects, the present disclosure provides a gel, comprising: (a) a cell, a cell nucleus, or one or more constituents derived from a cell; (b) two or more polymers; and (c) a plurality of linkers, each of said plurality of linkers comprising a 1,2,3-triazole moiety, wherein said linkers crosslink said two or more polymers. In some embodiments, each of said two or more polymers independently comprises at least one selected from the group consisting of a polyolefin, an olefin copolymer, an acrylic, a vinyl polymer, a polyester, a polycarbonate, a polyamide, a polyimide, a formaldehyde resin, a polyurethane, an ether polymer, a cellulosic, a thermoplastic elastomer, and a thermoplastic polyurethane. In some embodiments, each of said two or more polymers is, independently, a polyacry lamide. In some embodiments, each of said plurality of linkers is independently connected to an amide of said two or more polymers. In some embodiments, each of said plurality of linkers comprise a labile bond. In some embodiments, said labile bond is a chemically labile bond, a thermally labile bond, or a photo-labile bond. In some embodiments, said labile bond comprises a disulfide bond. In some embodiments, said 1,2,3-triazole moiety is formed by a process of treating an azide group with an alkyne group in conditions sufficient for forming said 1,2,3-triazole moiety. In some embodiments, the gel further comprises at least one reagent enclosed within said gel. In some embodiments, said gel is a hydrogel. In some embodiments, said gel further comprises a charged species. In some embodiments, said charged species is positively charged. In some embodiments, said charged species comprises trimethylammonium. In some embodiments, said charged species is negativ