Compositions and methods of cell attachment

Patent metadata

Field	Value
Publication number	US-2018024116-A1
Application number	US-201715648213-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 microchannel comprising a surface, said microchannel in fluidic communication with a fluid source comprising fluid; b) covalently attaching one or more proteins or peptides to said microchannel surface so as to create a treated surface; c) seeding viable cells on said treated surface so as to create attached cells; c) flowing fluid from said fluid source through said microchannel so as to create flowing conditions; and d) culturing said attached cells under said flow conditions such that said cells remain attached and viable for at least 14 days.

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

3 . The method of claim 2 , further comprising e) assessing viability by measuring the level of activity of one or more cellular enzymes.

4 . The method of claim 3 , wherein said cellular enzyme is a CYP enzyme.

5 . The method of claim 3 , wherein said cellular enzyme is a transaminase.

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

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

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

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

10 . The method of claim 9 , wherein RGD is covalently attached to said microchannel surface using N-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.

11 . A method of culturing cells, comprising: a) providing a microfluidic device comprising a microchannel comprising a surface, said microchannel in fluidic communication with a fluid source comprising fluid; b) covalently attaching one or more proteins or peptides to said microchannel surface so as to create a treated surface; c) seeding viable hepatocytes on said treated surface so as to create attached cells; c) flowing fluid from said fluid source through said microchannel so as to create flowing conditions; and d) culturing said attached cells under said flow conditions such that said cells remain attached and viable for at least 14 days.

12 . The method of claim 11 , wherein said hepatocytes are dog hepatocytes.

13 . The method of claim 11 , further comprising e) assessing viability by measuring the level of activity of one or more cellular enzymes.

14 . The method of claim 13 , wherein said cellular enzyme is a CYP enzyme.

15 . The method of claim 13 , wherein said cellular enzyme is a transaminase.

16 . The method of claim 11 , further comprising e) assessing viability by measuring the level of expression of one or more cellular proteins.

17 . The method of claim 11 , wherein said one or more proteins comprise collagen.

18 . The method of claim 11 , wherein said one or more proteins comprises a mixture of collagen type I, fibronectin and collagen type IV.

19 . The method of claim 18 , wherein said mixture is covalently attached to said microchannel surface using N-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.

20 . The method of claim 11 , 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 microchannel surface using N-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.

21 . The method of claim 20 , wherein said microchannel surface is PDMS and is plasma treated prior to step b).

Assignees

• Emulate Inc

Inventors

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

Classifications

• C12M23/20

  Material Coatings (immunocoatings C12M25/00) · CPC title

• G01N33/5014

  for testing toxicity · CPC title

• C12N2537/10

  Cross-linking · CPC title

• C12M25/02

  Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00) · CPC title

• C12N5/067 Primary

  Hepatocytes · CPC title