Compositions and methods of cell attachment

Patent metadata

Field	Value
Publication number	US-2018024118-A1
Application number	US-201715648306-A
Country	US
Kind code	A1
Filing date	Jul 12, 2017
Priority date	Jul 12, 2016
Publication date	Jan 25, 2018
Grant date	—

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.

First claim

We claim:

1 . A method of culturing cells, 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; c) seeding viable cells on said treated surface so as to create attached cells; and d) culturing said attached cells such that said cells remain attached and viable for at least 7 days.

2 . The method of claim 1 , wherein said microfluidic device further comprising 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 the step of flowing fluid from said fluid source through said microchannel so as to create flow conditions, and wherein said culturing in d) further comprises culturing said attached cells under said flow conditions.

3 . The method of claim 1 , wherein said attached cells further remain viable for at least 14 days.

4 . The method of claim 1 , wherein said attached cells further remain functional for at least 7 days.

5 . The method of claim 1 , wherein said attached cells further remain functional for at least 14 days.

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

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

8 . The method of claim 6 , wherein said at least one light-reactive portion is selected from the group consisting of a nitrophenyl, a diazirine and an azide.

9 . The method of claim 6 , wherein said at least one chemically reactive portion is selected from the group consisting of an NHS-ester, a sulfo-NHS-ester, isocyanate, isothiocyanate, imidoester, maleimide, pyridyldithiol, and hydrazide.

10 . The method of claim 1 , wherein said crosslinker is selected from the group consisting of sulfo-SANPAH, SANPAH, SDA, sulfo-SDA, LC-SDA, sulfo-LC-SDA, ANB-NOS, SDAD, sulfo-SDAD.

11 . The method of claim 1 , wherein said surface comprises PDMS.

12 . The method of claim 1 , wherein said surface is plasma treated prior to step b).

13 . The method of claim 1 , wherein said cells are hepatocytes.

14 . The method of claim 1 , further comprising step e) assessing viability by measuring the level of activity of one or more cellular enzymes.

15 . The method of claim 14 , wherein said cellular enzyme is a CYP enzyme.

16 . The method of claim 14 , wherein said cellular enzyme is a transaminase.

17 . The method of claim 1 , further comprising step e) assessing viability by measuring the level of expression of one or more cellular proteins.

18 . The method of claim 1 , wherein said one or more proteins comprises collagen.

19 . The method of claim 1 , wherein said one or more proteins comprises a mixture of collagen type I, fibronectin and collagen type IV.

20 . The method of claim 1 , wherein said one or more peptides comprises RGD or a peptide comprising the RGD motif.

21 . The method of claim 20 , wherein RGD is covalently attached to said surface using N-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.

22 . The method of claim 1 , wherein said covalently attaching one or more proteins or peptides in step 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.

23 . 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.

24 . The method of claim 23 , wherein said light comprises UV light.

25 . The method of claim 23 , 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.

26 . The method of claim 23 , wherein said exposing comprises masking said light so as to select said selected area or pattern.

27 . The method of claim 23 , wherein said exposing comprises projecting a light pattern so as to select said selected area or pattern.

28 . The method of claim 23 , wherein said exposing comprises rastering light so as to select said selected area or pattern.

29 . The method of claim 28 , wherein said selected area or pattern comprises a linear pattern.

30 . The method of claim 28 , wherein said cells comprise muscle cells or muscle-like cells that align with respect to said selected area of pattern.

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

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

33 . The method of claim 32 , wherein said porous membrane comprises said surface.

Assignees

• Emulate Inc

Inventors

• Levner Daniel
• Jang Kyung Jin
• Fraser Jacob
• Kerns Jordan
• Varone Antonio
• Huh Dongeun

Classifications

• G01N33/5044

  involving specific cell types · CPC title

• C12N2533/30

  Synthetic polymers · CPC title

• C12N5/0068

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title

• C12N5/067 Primary

  Hepatocytes · CPC title

• C12N2521/00

  Culture process characterised by the use of hydrostatic pressure, flow or shear forces · CPC title