Compositions and methods of cell attachment
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.
1. A method of culturing cells, comprising:
a) providing;
i) a microfluidic device comprising a microchannel and a polydimethylsiloxane surface disposed within said microchannel,
ii) a fluidic source comprising a fluid, wherein said fluidic source is in fluidic communication with said microchannel; and
iii) a source of ultraviolet light;
b) exposing said polydimethylsiloxane surface to ultraviolet light from said source of ultraviolent light thereby covalently attaching a UV-light linked portion of an N-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate conjugate to said polydimethylsiloxane surface;
c) covalently attaching one or more extracellular matrix proteins to a chemically-reactive portion of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate conjugate;
d) seeding viable epithelial cells on said one or more extracellular matrix proteins so as to create attached epithelial cells;
e) flowing said fluid from said fluid source through said microchannel so as to create flow conditions; and
f) culturing said attached epithelial cells under said flow conditions such that said cells remain attached and viable for at least 7 days.
2. The method of claim 1 , wherein said attached epithelial cells further remain attached and viable for at least 14 days.
3. The method of claim 1 , wherein said attached epithelial cells are functional for at least 7 days.
4. The method of claim 1 , wherein said attached epithelial cells are functional for at least 14 days.
5. The method of claim 1 , wherein said epithelial cells are hepatocytes.
6. The method of claim 5 , further comprising step g) assessing viability of said hepatocytes by measuring the level of activity of one or more cellular enzymes.
7. The method of claim 6 , wherein said cellular enzyme is a cytochrome P450 (CYP) enzyme.
8. The method of claim 6 , wherein said cellular enzyme is a transaminase.
9. The method of claim 1 , further comprising step g) assessing viability of said epithelial cells by measuring the level of expression of one or more cellular proteins.
10. The method of claim 1 , wherein said one or more extracellular matrix proteins comprises collagen.
11. The method of claim 1 , wherein said one or more extracellular matrix proteins comprises a mixture of collagen type I, fibronectin and collagen type IV.
12. The method of claim 1 , wherein said polydimethylsiloxane surface comprises a porous membrane.
13. A method of culturing cells, comprising:
a) providing;
i) a microfluidic device comprising a microchannel and a polydimethylsiloxane surface disposed within said microchannel;
ii) a fluidic source comprising a fluid, wherein said fluidic source is in fluidic communication with said microchannel;
iii) viable hepatocyte cells and a collagen; and
iv) a source of ultraviolet light and an N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate crosslinker;
b) exposing said plasma treated surface to ultraviolent light from said source of ultraviolet light thereby covalently attaching said treated surface to a UV-light linked portion of said N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate crosslinker;
c) covalently attaching said collagen to a chemically-reactive portion of said crosslinker;
d) seeding said viable hepatocyte cells on said collagen so as to create attached hepatocyte cells;
e) flowing said fluid from said fluid source through said microchannel so as to create flow conditions; and
f) culturing said attached hepatocyte cells under said flow conditions such that said hepatocyte cells remain attached and viable for at least 7 days.
14. The method of claim 13 , wherein said attached hepatocyte cells further remain attached and viable for at least 14 days.
15. The method of claim 13 , wherein said attached hepatocyte cells are functional for at least 7 days.
16. The method of claim 13 , wherein said attached hepatocyte cells are functional for at least 14 days.
17. The method of claim 13 , further comprising step g) assessing viability of said hepatocytes by measuring the level of activity of one or more cellular enzymes.
18. The method of claim 17 , wherein said cellular enzyme is a cytochrome P450 (CYP) enzyme.
19. The method of claim 17 , wherein said cellular enzyme is a transaminase.
20. The method of claim 13 , further comprising step g) assessing viability of said hepatocytes by measuring the level of expression of one or more cellular proteins.
21. The method of claim 13 , wherein said collagen is selected from the group consisting of collagen type I and collagen type IV.