Neuronal axon mimetics for in vitro analysis of neurological diseases, myelination, and drug screening

Patent metadata

Field	Value
Publication number	US-10845360-B2
Application number	US-201916387323-A
Country	US
Kind code	B2
Filing date	Apr 17, 2019
Priority date	Feb 25, 2016
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

Aspects of the present invention provide improved methods and apparatus for use in in vitro modeling of the interaction of cells with cellular constructs/parts/axons, including axon mimetics and use of three-dimensional fibers.

First claim

What is claimed is:

1. A cell-mimetic device comprising:
a three-dimensional structure comprising a plurality of fibers composed of polyhydroxyethylmethacrylate (pHEMA), said plurality of fibers configured to mimic neuronal axons and having an average stiffness post-curing of between about 0.1 and about 300 kPa and an average diameter of between about 0.1 and about 50 micrometers; and
a support structure connected to the three-dimensional structure; wherein the stiffness is calculated as any of Young's modulus, bulk modulus, shear modulus, and dynamic modulus; and post-curing stiffness is measured after equilibration in an aqueous solution buffered at pH 7.0-7.4.

2. The device of claim 1 , wherein the post-curing stiffness is between about 0.1 and 100 kPa.

3. The device of claim 1 , wherein the post-curing stiffness is between about 0.1 and 10 kPa.

4. The device of claim 1 , wherein the post-curing stiffness is between about 0.1 and 1 kPa.

5. The device of claim 1 , wherein the average diameter is between about 0.1 and about 10 micrometers.

6. The device of claim 1 , wherein the average diameter is between about 0.1 and about 1 micrometers.

7. The device of claim 1 , wherein the fibers are modified by a surface ligand.

8. The device of claim 1 , wherein the average stiffness is constant over the three-dimensional structure.

9. The device of claim 1 , wherein the average diameter is constant over the three-dimensional structure.

10. The device of claim 7 , wherein a surface ligand density or a surface ligand type is constant over the three-dimensional structure.

11. The device of claim 7 , wherein the plurality of fibers are arranged as one or more piles in the three dimensional structure, and at least one of fiber diameter, fiber stiffness, surface ligand density, and surface ligand type varies along at least one dimension of the three dimensional structure.

12. The device of claim 1 , wherein the pHEMA fibers in the three-dimensional structure are stretchable.

13. The device of claim 12 , wherein the three-dimensional structure is elastically deformable in at least one dimension by at least 5%.

14. The device of claim 13 , further comprising a substrate material attached to, and at least as elastically deformable in the at least one dimension as, the three-dimensional structure.

15. A method of studying cells in vitro, comprising:
providing a three-dimensional structure comprising:
a plurality of fibers composed of polyhydroxyethylmethacrylate (pHEMA), said plurality of fibers configured to mimic neuronal axons and having an average stiffness post-curing of between about 0.1 and about 300 kPa and an average diameter of between about 0.1 and about 50 micrometers, and
a support structure connected to the three-dimensional structure;

providing a cell-mimetic device comprising a three-dimensional structure comprising a plurality of fibers;
contacting the cell-mimetic device with a population of cells;
studying at least one feature of an interaction of the population of cells with the cell mimetic device; and
studying at least one feature of an interaction between cells of the same cell type or of different cell types within the cell-mimetic device.

16. The method of claim 15 , wherein the cells are neural cells or oligodendrocytes.

17. An assay device comprising:
a substrate;
a fiber support attached to the substrate; and
a plurality of fibers composed of polyhydroxyethylmethacrylate (pHEMA) and configured to mimic neuronal axons, each of the plurality of fibers having a length and spanning from the substrate to the fiber support such that each fiber is suspended in air or fluid along at least part of the fiber length, and the plurality of fibers having:
an average stiffness of between about 0.1 and about 300 kPa; and
an average diameter of between about 0.1 and about 50 micrometers; wherein the stiffness is calculated as any of Young's modulus, bulk modulus, shear modulus, and dynamic modulus;
and
wherein the post-curing stiffness is measured after equilibration in an aqueous solution buffered at pH 7.0-7.4.

18. The device of claim 17 , wherein the fibers are modified by a surface ligand.

19. The device of claim 17 , wherein the average stiffness is constant over the three-dimensional structure.

20. The device of claim 17 , wherein the average diameter is constant over the three-dimensional structure.

21. The device of claim 18 , wherein a surface ligand density or a surface ligand type is constant over the three-dimensional structure.

22. The device of claim 18 , wherein the plurality of fibers are arranged as one or more piles in the three-dimensional structure and at least one of fiber diameter, fiber stiffness, and surface ligand density and type varies along at least one dimension of the three dimensional structure.

23. The device of claim 17 , wherein the pHEMA fibers in the three-dimensional structure are stretchable.

24. An assay method comprising:
given the device of claim 17 :
contacting the assay device with a population of cells; and
studying at least one feature of an interaction of the population of cells with the device.

25. The assay method of claim 24 , wherein the cells are oligodendrocytes and the at least one feature of the interaction is myelination of the plurality of fibers.

26. The assay method of claim 25 , wherein the studying comprises determining, for at least one of the plurality of fibers, both an extent of myelination along a longitudinal axis of the fiber and a thickness of myelin.

27. The assay method of claim 26 , wherein the longitudinal extent and thickness of myelin are determined from microscopy images.

Assignees

• Massachusetts Inst Technology
• Harvard College
• Massachusetts Institue Of Tech

Inventors

• Van Vliet Krystyn J
• Jagielska Anna
• Homan Kimberly
• Lewis Jennifer A
• Busbee Travis Alexander

Classifications

• C12M25/04 Primary

  in combination with well or multiwell plates, i.e. culture inserts · CPC title

• C12M25/14

  Scaffolds; Matrices (in general C12N5/0068) · CPC title

• G01N33/5058

  Neurological cells · CPC title

• C12M41/46

  of cellular or enzymatic activity or functionality, e.g. cell viability · CPC title

• C12M41/38

  of metabolites or enzymes in the cells · CPC title