Compositions and methods of cell attachment

Patent metadata

Field	Value
Publication number	US-11519903-B2
Application number	US-201715648319-A
Country	US
Kind code	B2
Filing date	Jul 12, 2017
Priority date	Jul 12, 2016
Publication date	Dec 6, 2022
Grant date	Dec 6, 2022

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 polydimethylsiloxane microchannel surface, said polydimethylsiloxane microchannel surface covalently attached to an ultraviolet light reactive moiety of a heterobifunctional N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate crosslinker after exposure to a source of ultraviolet light;
b) covalently attaching one or more extracellular matrix proteins to a chemically reactive moiety of said heterobifunctional crosslinker;
c) storing said microfluidic device in the absence of living cells;
d) removing said microfluidic device from said storage, wherein said microfluidic device is in fluidic connection with a fluid source comprising a fluid; and
e) attaching viable epithelial cells to said one or more extracellular matrix proteins, and said viable epithelial cells are maintained as an attached monolayer without detachment from said one or more extracellular matrix proteins under a flow of said fluid.

2. The method of claim 1 , wherein said microchannel surface is disposed within a microchannel.

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

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

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

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

7. The method of claim 1 , wherein said attached monolayer is maintained attached and viable for at least 7 days.

8. The method of claim 1 , wherein said attached monolayer is maintained attached and viable for at least 14 days.

9. The method of claim 1 , wherein said attached epithelial cells are hepatocytes.

10. The method of claim 1 , where said polydimethylsiloxane microchannel surface is plasma treated prior to step b).

11. The method of claim 1 , wherein:
said covalently attaching one or more extracellular matrix proteins in step b) comprises introducing said at least one extracellular protein, or a solution containing said at least one extracellular protein, so as to contact and react with said chemically-reactive moiety of said heterobifunctional crosslinker.

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

13. The method of claim 12 , wherein said membrane comprises said polydimethylsiloxane microchannel surface.

14. A method of treating a microfluidic device, comprising:
a) providing a microfluidic device comprising a polydimethylsiloxane microchannel surface, said polydimethylsiloxane microchannel surface attached to an ultraviolet light reactive moiety of N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate after exposure to a source of ultraviolet light;
b) covalently attaching one or more collagen or fibronectin proteins to a chemically reactive moiety of N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate;
c) storing said microfluidic device in the absence of living cells;
d) removing said microfluidic device from said storage, wherein said microfluidic device is in fluidic connection with a fluid source comprising a fluid; and
e) attaching viable epithelial cells to said one or more collagen or fibronectin proteins, and said attached viable epithelial cells are maintained as an attached monolayer without detachment from said one or more collagen or fibronectin proteins under a flow of said fluid for at least 7 days.

15. The method of claim 14 , wherein said storing is done at a controlled temperature below room temperature.

16. The method of claim 14 , wherein said storing is done at between 2 and 10° C.

Assignees

• Emulate Inc

Inventors

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

Classifications

• C12N5/0658

  Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C12M23/16 Primary

  Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093) · CPC title

• C12N5/0018

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

• C12N5/0068

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