# US 2018/0024119 A1 · Compositions and methods of cell attachment

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- Number: US 2018/0024119 A1
- Document ID: US20180024119A1
- Status: Published application
- Kind: A1
- Application number: 15/648,319
- Filed: 2017-07-12
- Published: 2018-01-25
- Earliest claimed: 2016-07-12
- Assignees: Emulate, Inc.
- Applicants: Emulate Inc.
- Inventors: Daniel Levner; Kyung Jin Jang; Jacob Fraser; Jordan Kerns; Antonio Varone; Dongeun Huh
- Web page: https://patentgrep.com/patent/US20180024119A1

## CPC

- G01N 33/5032: {on intercellular interactions}
- C07K 2/00: Peptides of undefined number of amino acids; Derivatives thereof
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)}
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C12N 5/0658: {Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts}
- C12N 5/067: {Hepatocytes}
- G01N 33/5014: {for testing toxicity}
- C12N 2500/32: Amino acids
- C12N 2521/00: Culture process characterised by the use of hydrostatic pressure, flow or shear forces
- C12N 2533/30: Synthetic polymers
- C12N 2533/50: Proteins
- C12N 2533/52: Fibronectin; Laminin
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N 2535/10: Patterned coating
- C12N 2537/10: Cross-linking
- G01N 33/5044: {involving specific cell types}
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Compositions, devices and methods are described for improving adhesion, attachment, and/or differentiation of cells in a microfluidic device or chip. In one embodiment, one or more ECM proteins are covalently coupled to the surface of a microchannel of a microfluidic device. The microfluidic devices can be stored or used immediately for culture and/or support of living cells such as mammalian cells, and/or for simulating a function of a tissue, e.g., a liver tissue, muscle tissue, etc. Extended adhesion and viability with sustained function over time is observed.

## Claims

### Claim 1

A method of treating a microfluidic device, comprising: a) providing a microfluidic device comprising a surface; b) covalently attaching one or more proteins or peptides to said surface using a crosslinker so as to create a treated surface; and c) storing said microfluidic device.

### Claim 2 (depends on 1)

The method of claim 1, wherein said microfluidic device comprises a microchannel, said surface disposed within said microchannel.

### Claim 3 (depends on 1)

The method of claim 1, wherein said storing in step c) comprises storing said surface dry.

### Claim 4 (depends on 1)

The method of claim 1, wherein said storing in step c) comprises storing said surface wet.

### Claim 5 (depends on 1)

The method of claim 1, wherein said crosslinker comprises at least one light-reactive portion, at least one chemically reactive portion.

### Claim 6 (depends on 1)

The method of claim 1, wherein said crosslinker further comprises at least one spacer portion.

### Claim 7 (depends on 5)

The method of claim 5, wherein said at least one light-reactive portion is selected from the group consisting of a nitrophenyl, a diazirine and an azides.

### Claim 8 (depends on 5)

The method of claim 5, wherein said at least one chemically reactive portion is selected from the group consisting of NHS-ester, sulfo-NHS-ester, isocyanate, isothiocyanate, imidoester, maleimide, pyridyldithiol, and hydrazide

### Claim 9 (depends on 1)

The method of claim 1, wherein said crosslinker is selected from the list comprising: sulfo-SANPAH, SANPAH, SDA, sulfo-SDA, LC-SDA, sulfo-LC-SDA, ANB-NOS, SDAD, sulfo-SDAD.

### Claim 10 (depends on 1)

The method of claim 1, wherein said storing is done at a controlled temperature below room temperature.

### Claim 11 (depends on 1)

The method of claim 1, wherein said storing is done at between 2 and 10° C.

### Claim 12 (depends on 1)

The method of claim 1, wherein said one or more covalently attached proteins is collagen I.

### Claim 13 (depends on 12)

The method of claim 12, wherein said covalently attached collagen I is stored dry.

### Claim 14 (depends on 1)

The method of claim 1, wherein said one or more covalently attached proteins is laminin.

### Claim 15 (depends on 14)

The method of claim 14, wherein said covalently attached laminin is stored wet.

### Claim 16 (depends on 1)

The method of claim 1, wherein said method further comprises: d) seeding viable cells on said treated surface so as to create attached cells; and f) culturing said attached cells such that said cells remain attached and viable for at least 7 days.

### Claim 17 (depends on 16)

The method of claim 16, wherein said microfluidic device further comprises a microchannel, said surface disposed within said microchannel, and wherein said microchannel is in fluidic communication with a fluidic source comprising fluid, the method further comprising flowing fluid from said fluid source through said microchannel so as to create flow conditions, and wherein culturing in f) further comprises culturing said attached cells under said flow conditions.

### Claim 18 (depends on 16)

The method of claim 16, wherein said attached cells further remain viable for at least 14 days.

### Claim 19 (depends on 16)

The method of claim 16, wherein said attached cells further remain functional for at least 7 days.

### Claim 20 (depends on 16)

The method of claim 16, wherein said attached cells further remain functional for at least 14 days.

### Claim 21 (depends on 16)

The method of claim 16, wherein said attached cells are hepatocytes.

### Claim 22 (depends on 16)

The method of claim 16, wherein said surface comprises PDMS.

### Claim 23 (depends on 16)

The method of claim 16, where said surface is plasma treated prior to step b).

### Claim 24 (depends on 1)

The method of claim 1, wherein said covalently attaching one or more proteins or peptides in b) further comprises:
i) introducing said crosslinker or a solution containing said crosslinker to contact said surface and permitting said crosslinker or said solution containing said crosslinker to react with said surface; and
ii) introducing at least one protein or peptide, or a solution containing at least one protein or peptide to contact said surface.

### Claim 25 (depends on 1)

The method of claim 1, wherein said covalently attaching one or more proteins or peptides in b) further comprises exposing at least a portion of said surface to light.

### Claim 26 (depends on 25)

The method of claim 25, wherein said light comprises UV light.

### Claim 27 (depends on 25)

The method of claim 25, wherein said exposing comprises exposing a selected area or pattern for the covalent attachment of at least a portion of said one or more proteins or peptides.

### Claim 28 (depends on 25)

The method of claim 25, wherein said exposing comprises masking said light so as to select said selected area or pattern.

### Claim 29 (depends on 25)

The method of claim 25, wherein said exposing comprises projecting a light pattern so as to select said selected area or pattern.

### Claim 30 (depends on 25)

The method of claim 25, wherein said exposing comprises rastering light so as to select said selected area or pattern.

### Claim 31 (depends on 29)

The method of claim 29, wherein said selected area or pattern comprises a linear pattern.

### Claim 32 (depends on 1)

The method of claim 1, wherein said cells comprise muscle cells or muscle-liked cells that align with respect to said selected area or pattern.

### Claim 33 (depends on 1)

The method of claim 1, wherein said covalently attaching one or more proteins or peptides in b) further comprises introducing said crosslinker to contact one or more selected areas of said microfluidic device.

### Claim 34 (depends on 1)

The method of claim 1, wherein said microfluidic device further comprises a porous membrane.

### Claim 35 (depends on 34)

The method of claim 34, wherein said membrane comprises said surface.