Compositions and methods of cell attachment

Patent metadata

Field	Value
Publication number	US-11536714-B2
Application number	US-201715648213-A
Country	US
Kind code	B2
Filing date	Jul 12, 2017
Priority date	Jul 12, 2016
Publication date	Dec 27, 2022
Grant date	Dec 27, 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 culturing cells, comprising:
a) providing a microfluidic device comprising a microchannel comprising a polydimethylsiloxane surface, said polydimethylsiloxane surface reacted with an ultraviolet light reactive portion of a N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate heterobifunctional crosslinker using an ultraviolet light source, said microchannel in fluidic communication with a fluid source comprising a fluid;
b) covalently attaching one or more extracellular matrix proteins to a chemically-reactive portion of said heterobifunctional crosslinker so as to create a treated surface;
c) seeding viable epithelial cells on said treated surface so as to create attached epithelial cells;
d) flowing said fluid from said fluid source through said microchannel so as to create flowing conditions; and
e) culturing said attached epithelial cells under said flow conditions such that said attached epithelial cells remain attached and viable for at least 7 days.

2. The method of claim 1 , wherein said one or more extracellular matrix proteins comprises collagen.

3. The method of claim 1 , wherein said one or ore extracellular proteins comprises RGD or a RGD motif.

4. A method of culturing cells, comprising:
a) providing a microfluidic device comprising a microchannel comprising a polydimethylsiloxane surface, said surface reacted with an ultraviolet light reactive portion of a N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate crosslinker using a source of ultraviolet light, said microchannel in fluidic communication with a fluid source comprising a fluid;
b) covalently attaching one or more extracellular matrix proteins to a chemically-reactive portion of said crosslinker so as to create a treated surface;
c) seeding viable hepatocytes on said treated surface so as to create attached cells;
d) flowing said fluid from said fluid source through said microchannel so as to create flowing conditions; and
e) culturing said attached cells under said flow conditions such that said cells remain attached and viable for at least 7 days.

5. The method of claim 4 , wherein said hepatocytes are dog hepatocytes.

6. The method of claim 4 , further comprising f) 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 4 , wherein said one or more extracellular matrix proteins comprise collagen.

9. The method of claim 4 , wherein said one or more extracellular proteins comprises RGD or a RGD motif.

10. The method of claim 4 , wherein said polydimethylsiloxane surface is plasma treated prior to step b).

Assignees

• Emulate Inc

Inventors

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

Classifications

• C12N2537/10

  Cross-linking · CPC title

• C12N2535/10

  Patterned coating · CPC title

• C12N5/067 Primary

  Hepatocytes · CPC title

• C07K14/78

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C12N2500/32

  Amino acids · CPC title