# US 2021/0239683 A1 · Cell encapsulation compositions and methods for immunocytochemistry

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- Number: US 2021/0239683 A1
- Document ID: US20210239683A1
- Status: Published application
- Kind: A1
- Application number: 17/050,989
- Filed: 2019-05-03
- Published: 2021-08-05
- Earliest claimed: 2018-05-03
- Assignees: The University of British Columbia
- Applicants: The University of British Columbia
- Inventors: Hongshen Ma; Jeong-Hyun Lee
- Web page: https://patentgrep.com/patent/US20210239683A1

## CPC

- A61L 27/18: obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- G01N 33/531: Production of immunochemical test materials
- A61L 27/52: Hydrogels or hydrocolloids
- A61L 27/56: Porous materials, {e.g. foams or sponges}
- C08F 18/08: Vinyl acetate
- C08F 20/18: with acrylic or methacrylic acids
- C08F 299/00: Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers
- C08G 65/3322: {acyclic}
- C08J 3/075: Macromolecular gels
- C08J 3/28: Treatment by wave energy or particle radiation
- C08J 9/26: by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
- C08L 1/02: Cellulose; Modified cellulose
- C08L 1/08: Cellulose derivatives
- C08L 5/02: Dextran; Derivatives thereof
- C08L 5/08: Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
- C08L 5/12: {Agar or} agar-agar {, i.e. mixture of agarose and agaropectin}; Derivatives thereof
- C08L 33/14: of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen
- C08L 51/08: grafted on to macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds
- C08L 71/02: Polyalkylene oxides
- C08L 89/06: derived from leather or skin {, e.g. gelatin}
- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/554: the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- A61L 2300/62: Encapsulated active agents, e.g. emulsified droplets
- A61L 2300/64: Animal cells
- C08J 2201/026: Crosslinking before of after foaming
- C08J 2205/022: Hydrogel, i.e. a gel containing an aqueous composition
- C08J 2207/10: Medical applications, e.g. biocompatible scaffolds
- C08J 2333/06: of esters containing only carbon, hydrogen, and oxygen, the oxygen atom being present only as part of the carboxyl radical
- C08J 2333/14: of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen
- C08J 2351/08: grafted on to macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C08J 2371/02: Polyalkylene oxides
- C08J 2433/06: of esters containing only carbon, hydrogen, and oxygen, the oxygen atom being present only as part of the carboxyl radical
- C08J 2451/08: grafted on to macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C08J 2471/02: Polyalkylene oxides
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- 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)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)

## Abstract

Provided herein are compositions comprising: a scaffold polymer having one or more acryloyl groups or one or more methacryloyl groups; optionally a porogen and a crosslinking agent, compositions that upon crosslinking form a hydrogel for use in cell encapsulation and methods for immunocytochemistry of encapsulated cells. Scaffold polymers used are selected from: Poly(ethylene glycol) diacrylate (PEGDA); Poly(ethylene glycol) dimethylacrylate (PEGDMA); Poly(ethylene glycol) methyl ether acrylate (PEGMEA); Poly(ethylene glycol) methacrylate (PEGMA); and Poly(ethylene glycol) methyl ether methacrylate (PEGMEMA), and porogens selected from: Poly(ethylene glycol) (PEG); Chitosan; Agarose; Dextran; Hyaluronic acid; Poly(methyl methacrylate) (PMMA); Cellulose and derivatives thereof; Gelatin and derivatives thereof; and Acrylamide and derivatives thereof. The invention also provides, at least in part, compositions for forming a porous hydrogel around a cell suitable for immunostaining of cells within the hydrogel.

## Claims

### Claim 1

A composition, the composition comprising:
(a) a scaffold polymer, wherein the scaffold polymer:
(i) has one or more acryloyl group or one or more methacryloyl groups;
(ii) has an average molecular weight (Mn) between about 300 and about 6,000;
(iii) is water soluble and biocompatible; and
(iv) is operable to form a hydrogel following cross-linking;
(b) a porogen; and
(c) a crosslinking agent;
wherein, the composition has a density of between about 1.0 g/ml and about 1.12 g/ml at 25° C.

### Claim 2 (depends on 1)

The composition of claim 1, wherein composition has a density of between about 1.0 g/ml and about 1.10 g/ml at 25° C.

### Claim 3 (depends on 1, 2)

The composition of claim 1 or 2, wherein composition has a density of between about 1.0 g/ml and about 1.08 g/ml at 25° C.

### Claim 4 (depends on 1, 2, 3)

The composition of claim 1, 2 or 3, wherein scaffold polymer has an average molecular weight (Mn) between about 300 and about 3,000.

### Claim 5 (depends on 1, 2, 3, 4)

The composition of any one of claims 1-4, wherein the scaffold polymer is selected from the following: Poly(ethylene glycol) diacrylate (PEGDA); Poly(ethylene glycol) dimethylacrylate (PEGDMA); Poly(ethylene glycol) methyl ether acrylate (PEGMEA); Poly(ethylene glycol) methacrylate (PEGMA); and Poly(ethylene glycol) methyl ether methacrylate (PEGMEMA).

### Claim 6 (depends on 1, 2, 3, 4, 5)

The composition of any one of claims 1-5, wherein the scaffold polymer is selected from the following: PEGDA; PEGDMA; PEGMA; and PEGMEMA.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The composition of any one of claims 1-6, wherein the scaffold polymer is selected from the following: PEGDA and PEGDMA.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The composition of any one of claims 1-7, wherein the scaffold polymer is PEGDA.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The composition of any one of claims 1-8, wherein the scaffold polymer has an average Mn between about 300 and about 6,000.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The composition of any one of claims 1-9, wherein the scaffold polymer has an average Mn between about 300 and about 2,000.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The composition of any one of claims 1-10, wherein the scaffold polymer has an average Mn of about 700.

### Claim 12 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The composition of any one of claims 2-11, wherein the porogen is selected from one or more of the following: Poly(ethylene glycol) (PEG); Chitosan; Agarose; Dextran; Hyaluronic acid; Poly(methyl methacrylate) (PMMA); Cellulose and derivatives thereof; Gelatin and derivatives thereof; and Acrylamide and derivatives thereof.

### Claim 13 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The composition of any one of claims 2-12, wherein the porogen is PEG.

### Claim 14 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The composition of any one of claims 2-13, wherein the porogen is PEG and has an average Mn between 1,000 and 40,000.

### Claim 15 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The composition of any one of claims 2-14, wherein the porogen is PEG and has an average Mn of 20,000.

### Claim 16 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The composition of any one of claims 2-15, wherein:
(i) the weight ratio of the scaffold polymer to porogen is about 1:1;
(ii) the scaffold polymer is PEGDA having an average Mn of 700 and 15% w/v; and
(iii) the porogen is PEG having an average Mn of 20,000 and 15% w/v.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The composition of any one of claims 1-16, wherein the crosslinking agent is a free-radical generating compound.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The composition of any one of claims 1-16, wherein the crosslinking agent is a photo-initiator [UV] selected from TABLE 1B.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The composition of any one of claims 1-18, wherein the crosslinking agent is Irgacure 819 or Irgacure 2959.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

The composition of any one of claims 1-19, wherein the crosslinking agent is Irgacure 2959 at 0.1% w/v or Irgacure 819 at 0.1% w/v.

### Claim 21

A composition, the composition comprising:
(a) a scaffold polymer, wherein the scaffold polymer:
(i) is selected from: PEGDA; PEGMA; and PEGDMA;
(ii) has an average molecular weight (Mn) between about 500 and about 3,000;
(iii) is water soluble and biocompatible; and
(iv) is operable to form a hydrogel following cross-linking; and
(b) 2-Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone is less than or equal to 1.0% w/v of the composition;
wherein, the composition has a density of between about 1.0 g/ml and about 1.10 g/ml at 25° C.

### Claim 22 (depends on 21)

The composition of claim 21, wherein the composition further comprises a porogen.

### Claim 23 (depends on 21, 22)

The composition of claim 21 or 22, wherein the 2-Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone is less than or equal to 0.3% w/v of the composition

### Claim 24 (depends on 21, 22, 23)

The composition of claim 21, 22 or 23, wherein the 2-Hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone is less than or equal to 0.1% w/v of the composition

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

A cell encapsulation method, the method comprising:
(a) mixing a composition of claim 1-20 with a cells or a cell suspension to form a cell polymer mixture;
(b) adding the cell polymer mixture to a cell imaging container;
(c) settling the cell within the cell imaging container; and
(d) cross-linking the cell polymer mixture to form a hydrogel.

### Claim 26 (depends on 25)

The method of claim 25, wherein the method further comprises assaying of the cells encapsulated by the hydrogel using immunocytochemistry.

### Claim 27 (depends on 25, 26)

The method of claim 25 or 26, wherein settling of the cell within the cell imaging container is by centrifugation.

### Claim 28 (depends on 26, 27)

The method of claim 26 or 27, the method further comprising bleaching the fluorescence from a previous immunocytochemistry assay and assaying of the cells encapsulated by the hydrogel using a second immunocytochemistry assay.

### Claim 29 (depends on 28)

The method of claim 28, the method further comprising repeated bleaching of fluorescence and assaying of the cells encapsulated by the hydrogel using immunocytochemistry.

### Claim 30

A cell encapsulation method, the method comprising:
(a) adding a crosslinking agent to the surface of a cell imaging container;
(b) adding a composition to the cell imaging container, the composition comprising:
(i) a scaffold polymer, wherein the scaffold polymer:
has one or more acryloyl group or one or more methacryloyl groups;
has an average molecular weight (Mn) between about 300 and about 6,000;
is water soluble and biocompatible; and
is operable to form a hydrogel following cross-linking; and
(ii) a porogen;
(c) adding cells or a cell suspension to the composition to form a cell polymer mixture in the imaging container;
(d) settling the cell within the cell imaging container; and
(e) cross-linking the cell polymer mixture to form a hydrogel.

### Claim 31 (depends on 30)

The method of claim 30, wherein the method further comprises assaying of the cells encapsulated by the hydrogel using immunocytochemistry.

### Claim 32 (depends on 30, 31)

The method of claim 30 or 31, wherein the wherein settling of the cell within the cell imaging container is by centrifugation.

### Claim 33 (depends on 30, 32)

The method of claim 30 or 32, the method further comprising bleaching the fluorescence and assaying of the cells encapsulated by the hydrogel using immunocytochemistry.

### Claim 34 (depends on 33)

The method of claim 33, the method further comprising bleaching the fluorescence from a previous immunocytochemistry assay and assaying of the cells encapsulated by the hydrogel using a second immunocytochemistry assay.

### Claim 35 (depends on 30, 31, 32, 33, 34)

The method of any one of claims 30-34, wherein the hydrogel has a thickness of between about 10 μm and about 1,000 μm.

### Claim 36 (depends on 30, 31, 32, 33, 34, 35)

The method of any one of claims 30-35, wherein the hydrogel has pores between about 10 nm and about 10 μm.

### Claim 37 (depends on 30, 31, 32, 33, 34, 35, 36)

The method of any one of claims 30-36, wherein the cross-linking is by UV light.

### Claim 38 (depends on 30, 31, 32, 33, 34, 35, 36, 37)

The method of any one of claims 30-37, wherein the cross-linking is by UV light at a wavelength between about 300 nm and about 375 nm.

### Claim 39 (depends on 30, 31, 32, 33, 34, 35, 36, 37, 38)

The method of any one of claims 30-38, wherein the cross-linking is by UV light at a wavelength between about 300 nm and about 375 nm for an exposure of 5 seconds or less.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)

A cell encapsulation kit, the kit comprising:
(a) composition of any one of claims 1-24; and
(b) instructions for the compositions use in the encapsulation of cells.

### Claim 41 (depends on 40)

The kit of claim 40, further comprising immunocytochemistry reagents.

### Claim 42 (depends on 40, 41)

The kit of claim 40 or 41, further comprising an imaging container.