Compositions and methods of cell attachment

Patent metadata

Field	Value
Publication number	US-2018024119-A1
Application number	US-201715648319-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 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.

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

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

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

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

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

7 . 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.

8 . 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

9 . 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.

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

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

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

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

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

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

16 . 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.

17 . 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.

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

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

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

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

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

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

24 . 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.

25 . 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.

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

27 . 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.

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

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

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

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

32 . 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.

33 . 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.

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

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

Assignees

• Emulate Inc

Inventors

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

Classifications

• C12N2500/32

  Amino acids · CPC title

• C12N2533/30

  Synthetic polymers · CPC title

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title

• C12N2537/10

  Cross-linking · CPC title

• C12N5/067 Primary

  Hepatocytes · CPC title