Micro- and nanopatterned substrates for cell migration and uses thereof

Patent metadata

Field	Value
Publication number	US-10435734-B2
Application number	US-201615556942-A
Country	US
Kind code	B2
Filing date	Mar 9, 2016
Priority date	Mar 9, 2015
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

Abstract

The present invention generally relates to the field of cell growth and tissue engineering, in particular, an engineered biomimetic culture platform (BCP) that has a nanotextured and micropatterned surface that provides both chemical and mechanical cues designed to mimic the structure of the in vivo extracellular micro-environment. The BCP can be used in assays to assess the migratory behavior and/or potential of a population of cells, such as tumor cells, as well as in screening assays for diagnostic and/or prognostic purposes, or to identify agents that modify the migratory behavior or the epithelial-to-mesenchymal transition (EMT) of cells. BCPs as described herein further provide a platform for the identification of protein or genetic targets for the modification of cell migratory or invasion behavior.

First claim

The invention claimed is:

1. An array for assessing cell migration comprising:
a. a nanopatterned substrate comprising parallel grooves and ridges, wherein the groove width is between 200 nm to 3000 nm, the ridge width is between 200 nm to 3000 nm, and depth of the groove is between 200 nm to 3000 nm; and
b. an array of at least one cell migration unit on the nanopatterned substrate, each cell migration unit comprising:
i. at least one migration pathway having a proximal and distal end,
ii. at least one cell non-adherent region having a proximal and distal end, and
iii. at least one cell loading region;
wherein the at least one migration pathway comprises a cell adherent surface having a width between 10 μm-500 μm, aligned parallel to the grooves and ridges,
wherein the at least one cell non-adherent region is aligned parallel to the grooves and ridges and is adjacent to the at least one migration pathway; and
wherein the at least one cell loading region is located at the proximal end of the at least one migration pathway and optionally, at the proximal end of at least one cell non-adherent region.

2. The array of claim 1 , wherein the at least one migration pathway is coated with an extracellular matrix (ECM) component coating.

3. The array of claim 2 , wherein the ECM component coating is not laminin.

4. The array of claim 2 , wherein the ECM component coating further comprises at least one growth factor or chemotaxis agent.

5. The array of claim 1 , wherein the cell migration unit comprises n migration pathways, n cell non-adherent regions, and at least one cell loading region located at the proximal end of the n migration pathways and n cell non-adherent regions.

6. The array of claim 1 , wherein the cell migration unit comprises n migration pathways and:
n+1 cell non-adherent regions, and at least one cell loading region located at the proximal end of the n migration pathways and n+1 cell non-adherent regions, or
n+2 cell non-adherent regions, and at least one cell loading region located at the proximal end of the n migration pathways and n+2 cell non-adherent regions,
wherein each of the n migration pathways has a cell non-adherent region located on either side.

7. The array of claim 6 , wherein n is selected from, 2, 3, 4, 5, 6, 7, 8, 9, 10, between 11-15, between 16-20 or more than 20 but less than 50.

8. The array of claim 6 , wherein the migration pathways are of the same width, having a width selected from 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, or greater than 150 μm but less than 500 μm.

9. The array of claim 6 , wherein the migration pathways are of different widths, wherein the different widths of the migration pathways are selected from any or a combination of: 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, or greater than 150 μm but less than 500 μm.

10. The array of claim 1 , wherein the cell non-adherent region has a width is-selected from: between 50 μm-500 μm, or 150 μm-500 μm.

11. The array of claim 1 , wherein
a. the groove width is selected from a width of: between 200 nm-800 nm, between 800 nm-1200 nm, 1000 nm-2000 nm or 2000 nm-3000 nm;
b. the ridge width is selected from a width of: between 200 nm to 800 nm, between 800 nm-1200 nm, 1000 nm-2000 nm or 2000 nm-3000 nm; and
c. groove depth is selected from a groove depth of: between 200 nm to 800 nm, between 800 nm-1200 nm, 1000 nm-2000 nm or 2000 nm-3000 nm.

12. The array of claim 1 , wherein the at least one migration pathway and the at least one cell non-adherent region is between 0.5 mm-10 mm or 10 mm-20 mm in length.

13. The array of claim 1 , wherein the array comprises at least 2 cell migration units or at least 3, 6, 8, 12, 24, 48, 96, 384, or 1536 cell migration units.

14. The array of claim 1 , wherein the array is configured as a multi-well plate, each well of the multi-well plate comprising at least one cell migration unit, wherein the multi-well plate comprises any of: 2, 3, 4, 6, 8, 12, 24, 48, 96, 384 or 1536 wells.

15. The array of claim 1 , further comprising a removable barrier located at the proximal end of the at least one migration pathway and optionally at the proximal end of the at least one cell non-adherent region, wherein the barrier prevents cells present in the cell loading region from entering the migration pathway.

16. The array of claim 15 , wherein the removable barrier is a micropatterned stamp.

17. The array of claim 1 , further comprising a population of mammalian cells, wherein the population of mammalian cells comprises cells attached to the nanopatterned substrate at least in the cell loading region.

18. A micro-well plate comprising the array of claim 1 .

19. A method for measuring cell migration, the method comprising:
a. seeding a population of cells onto a nanopatterned substrate comprising parallel grooves and ridges, wherein the groove width is between 200 nm to 3000 nm, the ridge width is between 200 nm to 3000 nm, and groove height is between 200 nm to 3000 nm, wherein the cells are seeded at a cell loading region of at least one cell migration unit; wherein the cell migration unit comprises,
i. at least one migration pathway having a proximal and distal end,
ii. at least one cell non-adherent region having a proximal and distal end, and
iii. at least one cell loading region;
wherein the at least one migration pathway is aligned parallel to the grooves and ridges and comprises a cell-adherent surface, and is between 10 μm-500 μm in width;
wherein the at least one cell non-adherent region is aligned parallel to the grooves and ridges and is adjacent to the at least one migration pathway; and
wherein the at least one cell loading region is located at the proximal end of the at least one migration pathway and optionally, at the proximal end of at least one cell non-adherent region;

b. culturing the population of cells in the cell loading region to form a monolayer;
c. optionally removing a barrier located between the proximal end of the at least one cell migration pathway and the cell loading region;
d. culturing the population of cells for a selected period of time to allow migration of the cells along the migration pathway towards the distal end;
e. measuring the distance of cell migration of the population of cells towards the distal end of the migration pathway in the selected period of time.

20. The method of claim 19 , further comprising capturing time-lapse images at desired intervals for a series of images during the selected period of time.

21. The method of claim 19 , further comprising
a. measuring the distance of cell migration of a population of cells towards the distal end of the migration pathway in the selected period of time in the presence of a test agent, relative to the distance of cell migration of a population of the same cells in the absence of the test agent; or
b. measuring the distance of cell migration of a population of tumor cells towards the distal end of the migration pathway in the selected period of time in the presence of a test agent, relative to the distance of cell migration of a population of the same tumor cells in the presence of different concentrations of the test agent, and/or the presence of an agent known to inhibit cell migration, and/or the presence of an agent known to promote cell migration; or
c. measuring the distance of cell migration of a population of tumor cells

Assignees

• Univ Washington

Inventors

• Kim Deok-Ho
• Kim Peter
• Levchenko Andre

Classifications

• C12M23/02

  Form or structure of the vessel (large containers B65D88/00) · CPC title

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• G01N33/5002 Primary

  Partitioning blood components · CPC title

• C12M23/20

  Material Coatings (immunocoatings C12M25/00) · CPC title

• C12M23/12

  Well or multiwell plates (C12M25/04 takes precedence) · CPC title