U.S. patent application number 15/442530 was filed with the patent office on 2017-11-16 for neuronal axon mimetics for in vitro analysis of neurological diseases, myelination, and drug screening.
The applicant listed for this patent is Travis Alexander Busbee, Kimberly Homan, Jennifer A. Lewis, Massachusetts Institute of Technology. Invention is credited to Travis Alexander Busbee, Kimberly Homan, Anna Jagielska, Jennifer A. Lewis, Krystyn J. Van Vliet.
Application Number | 20170328888 15/442530 |
Document ID | / |
Family ID | 58387875 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170328888 |
Kind Code |
A1 |
Van Vliet; Krystyn J. ; et
al. |
November 16, 2017 |
Neuronal Axon Mimetics For In Vitro Analysis Of Neurological
Diseases, Myelination, And Drug Screening
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.
Inventors: |
Van Vliet; Krystyn J.;
(Lexington, MA) ; Jagielska; Anna; (Salem, MA)
; Homan; Kimberly; (Somerville, MA) ; Lewis;
Jennifer A.; (Cambridge, MA) ; Busbee; Travis
Alexander; (Somerville, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Homan; Kimberly
Lewis; Jennifer A.
Busbee; Travis Alexander
Massachusetts Institute of Technology |
Cambridge |
MA |
US
US
US
US |
|
|
Family ID: |
58387875 |
Appl. No.: |
15/442530 |
Filed: |
February 24, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62299964 |
Feb 25, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 41/38 20130101;
G01N 33/5032 20130101; C12M 25/14 20130101; G01N 33/5058 20130101;
C12M 41/46 20130101; C12M 25/04 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/50 20060101 G01N033/50 |
Claims
1. A cell-mimetic device comprising: a three dimensional structure
comprising a plurality of fibers, said plurality of fibers 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. (canceled)
3. The device of claim 1, wherein the post-curing stiffness is
between about 0.1 and 100 kPa.
4. The device of claim 1, wherein the post-curing stiffness is
between about 0.1 and 10 kPa.
5. The device of claim 1, wherein the post-curing stiffness is
between about 0.1 and 1 kPa.
6. The device of claim 4, wherein the average diameter is between
about 0.1 and about 10 micrometers.
7. The device of claim 4, wherein the average diameter is between
about 0.1 and about 1 micrometers.
8. (canceled)
9. (canceled)
10. The device of claim 6, wherein the fibers are formed of
compliant polymers or hydrogels.
11. (canceled)
12. The device of f claim 10, wherein the fibers are PEG, pHEMA,
PDMS, polyacrylamide, hyaluronic methacrylate, or any viscoelastic
polymer, or a derivative of the foregoing.
13. The device of claim 12, wherein the fibers are modified by a
surface ligand.
14. The device of claim 6, wherein the average stiffness is
constant over the three dimensional structure.
15. The device of claim 6, wherein the average diameter is constant
over the three dimensional structure.
16. The device of claim 6, wherein the surface ligand density or
type is constant over the three dimensional structure.
17. The device of claim 6, 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.
18. The device of claim 6, wherein the three dimensional structure
represents at least one of a model of a tissue, and a model of
neuronal axons.
19. The device of claim 6, wherein the fibers in the three
dimensional structure are formed of a stretchable fiber
material.
20. The device of claim 19, wherein the stretchable fiber material
is PDMS, pHEMA, hyaluronic methacrylate, or any viscoelastic
polymer.
21. The device of claim 20, wherein the three dimensional structure
can be elastically deformed in at least one dimension by at least
5%.
22. The device of claim 21, further comprising a substrate material
attached to, and at least as elastically deformable in the at least
one dimension as, the three dimensional structure.
23. A method of studying cells in vitro, comprising: providing a
three dimensional structure comprising a plurality of fibers, said
plurality of fibers 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.
24. The method of claim 23, wherein the cells are neural cells or
oligodendrocytes.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. An assay device comprising: a substrate; a fiber support
attached to the substrate; and a plurality of fibers, 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; wherein
the post-curing stiffness is between about 0.1 and 10 kPa; wherein
the average diameter is between about 0.1 and about 10 micrometers;
and wherein the post-curing stiffness is measured after
equilibration in an aqueous solution buffered at pH 7.0-7.4.
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. The device of claim 30, wherein the fibers are formed of
compliant polymers or hydrogels.
40. (canceled)
41. The device of claim 39, wherein the fibers are PEG, pHEMA,
PDMS, polyacrylamide, hyaluronic methacrylate, or any viscoelastic
polymer or derivatives thereof.
42. The device of claim 30, wherein the fibers are modified by a
surface ligand.
43. The device of claim 30, wherein the average stiffness is
constant over the three dimensional structure.
44. The device of claim 30, wherein the average diameter is
constant over the three dimensional structure.
45. The device of claim 30, wherein the surface ligand density or
type is constant over the three dimensional structure.
46. The device of claim 41, 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.
47. The device of claim 46, wherein the three dimensional structure
represents at least one of a model of a tissue, and a model of
neuronal axons.
48. The device of claim 30, wherein the fibers in the three
dimensional structure are formed of a stretchable fiber
material.
49. An assay method comprising: given the device of claim 30:
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.
50. The assay method of claim 49, wherein the cells are
oligodendrocytes and the at least one feature of the interaction is
myelination of the plurality of fibers.
51. The assay method of claim 50, 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.
52. The assay method of claim 51, wherein the longitudinal extent
and thickness of myelin are determined from a microscopy
images.
53. (canceled)
54. The device of claim 5, wherein the average diameter is between
about 0.1 and about 1 micrometers.
55. The device of claim 17, wherein the three dimensional structure
represents at least one of a model of a tissue, and a model of
neuronal axons.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/299,964, filed on Feb. 25, 2016. The entire
teachings of the above application(s) are incorporated herein by
reference.
BACKGROUND
[0002] Currently, devices available to study neural cell responses
are comprised chiefly of either two-dimensional (2D), stiff
polystyrene tissue culture dishes or organotypic culture using
tissue slices. There can be certain limitations to both approaches.
The first format can be limited, for example, in eliciting cell
responses that are recapitulated in vivo, because the smooth, flat,
stiff surface provides a very different environment from the 3D,
topographically complex, and mechanically compliant neural tissue
comprising a network of neuronal axons and other features. The
latter format can represent in vivo environments more credibly, but
there can still be inherent variability between tissue slices.
Further, organotypic cultures may not afford systematic isolation
of the individual physical and chemical cues, limiting its
usefulness in facilitating understanding of the role of each in
disease characteristics or in drug screening responses.
[0003] Although certain electrospun nanofibers have been described
as a model to study myelination (Lee et al. 2012, Li et al., 2014),
these fibers were made from stiff polymers (polystyrene or
polycaprolactone), therefore providing very different mechanical
cues than does the compliant environment characterizing the central
nervous system (CNS) in vivo. Moreover, electrospinning techniques
do not provide adequate control of fiber alignment and construct
geometry, and thus are not applicable for building complex fiber
architectures or for creating controlled chemical and mechanical
gradients via arrangement of different fiber types. Accordingly,
there is a need for new and improved methods and apparatus for use
in in vitro modeling of the interaction of cells with cellular
constructs, including nerve cells and axons.
SUMMARY
[0004] Aspects of the present invention address this need by
providing improved methods and apparatus for use in in vitro
modeling of the interaction of cells with cellular
constructs/parts/axons.
[0005] In one embodiment, the invention relates to a cell-mimetic
device comprising: a three dimensional structure comprising a
plurality of fibers, said plurality of fibers 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.
[0006] In some embodiments, the invention further relates to one,
or to combination of the following:
[0007] the stiffness is calculated as any of Young's modulus, bulk
modulus, shear modulus, and dynamic modulus;
[0008] the post-curing stiffness is between about 0.1 and 100
kPa;
[0009] the post-curing stiffness is between about 0.1 and 10
kPa;
[0010] the post-curing stiffness is between about 0.1 and 1
kPa;
[0011] the average diameter is between about 0.1 and about 10
micrometers;
[0012] the average diameter is between about 0.1 and about 1
micrometers;
[0013] the post-curing stiffness is measured after equilibration in
an aqueous solution buffered at pH 7.0-7.4;
[0014] the plurality of fibers are arranged as one or more piles in
the three dimensional structure;
[0015] the fibers are formed of compliant polymers or
hydrogels;
[0016] the support structure is formed of glass, polystyrene,
tissue culture dish, tissue culture plate, or molded
polydimethylsiloxane (PDMS);
[0017] the fibers are polyethylene glycol (PEG),
polyhydroxyethylmethacrylate (pHEMA), polydimethylsiloxane (PDMS),
polyacrylamide, hyaluronic methacrylate, or any viscoelastic
polymer, or any derivative of the foregoing;
[0018] the fibers are modified by a surface ligand;
[0019] the average stiffness is constant over the three dimensional
structure;
[0020] the average diameter is constant over the three dimensional
structure;
[0021] the surface ligand density or type is constant over the
three dimensional structure;
[0022] 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, including, for
example, fiber arrangements that create gradients of one or more of
fiber diameter, fiber stiffness, surface ligand density, and
surface ligand type;
[0023] the three dimensional structure represents at least one of a
model of a tissue, and a model of neuronal axons; and
[0024] the fibers in the three dimensional structure are formed of
a stretchable fiber material, optionally wherein any one or a
combination of the following is true: (a) the stretchable fiber
material is PDMS, pHEMA, hyaluronic methacrylate, or any
viscoelastic polymer; (b) the three dimensional structure can be
elastically deformed in at least one dimension by at least 5%; and
(c) the device further comprises a substrate material attached to,
and at least as elastically deformable in the at least one
dimension as, the three dimensional structure.
[0025] In some embodiments, the invention relates to a method of
studying cells in vitro, comprising: providing a three dimensional
structure comprising a plurality of fibers, said plurality of
fibers 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 (either of the
same cell type or of different cell types) within the cell-mimetic
device.
[0026] In some embodiments, the invention further relates to one or
any combination of the following:
[0027] the cells are neural cells;
[0028] the cells are oligodendrocytes; and
[0029] the at least one feature of the interaction is production of
myelin by the oligodendrocytes.
[0030] In some embodiments, the invention relates to a method of
manufacturing a cell-mimetic device, comprising: creating a three
dimensional structure comprising a plurality of fibers, the fibers
having an average stiffness of between about 0.1 and about 300.0
kPa; and an average diameter of between about 0.1 and about 50
micrometers; and optionally wherein one or both of the following
are true: three dimensional printing (3DP) is used to create the
three dimensional structure; and the three dimensional structure
represents any of: a model of a tissue, and a model of neuronal
axons.
[0031] In some embodiments, the invention relates to an assay
device comprising: a substrate; a fiber support attached to the
substrate; and a plurality of fibers, each of the plurality of
fibers: having a length and spanning from the substrate to the
fiber support such that each fibers 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
300.0 kPa and an average diameter of between about 0.1 and about 50
micrometers.
[0032] In some embodiments, the invention further relates to one or
any combination of the following:
[0033] the stiffness is calculated as any of Young's modulus, bulk
modulus, shear modulus, and dynamic modulus;
[0034] the post-curing stiffness is between about 0.1 and 100
kPa;
[0035] the post-curing stiffness is between about 0.1 and 10
kPa;
[0036] the post-curing stiffness is between about 0.1 and 1
kPa;
[0037] the average diameter is between about 0.1 and about 10
micrometers;
[0038] the average diameter is between about 0.1 and about 1
micrometers;
[0039] the post-curing stiffness is measured after equilibration in
an aqueous solution buffered at pH 7.0-7.4;
[0040] the plurality of fibers are arranged as one or more piles in
the three dimensional structure;
[0041] the fibers are formed of compliant polymers or
hydrogels;
[0042] the support structure is formed of: (a) glass, polystyrene,
or molded polydimehtylsiloxane (PDMS); (b) a tissue culture dish or
a tissue culture plate; (c) or combination of the foregoing;
[0043] the fibers are PEG, pHEMA, PDMS, polyacrylamide, hyaluronic
methacrylate, or any viscoelastic polymer, or a derivative of any
of the foregoing;
[0044] the fibers are modified by a surface ligand;
[0045] the average stiffness is constant over the three dimensional
structure;
[0046] the average diameter is constant over the three dimensional
structure;
[0047] the surface ligand density or type is constant over the
three dimensional structure;
[0048] 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, including, for
example, fiber arrangements that create gradients of fiber
diameter, fiber stiffness, surface ligand density, surface ligand
type, or a combination of the foregoing.
[0049] the three dimensional structure represents at least one of a
model of a tissue, and a model of neuronal axons; and
[0050] the fibers in the three dimensional structure are formed of
a stretchable fiber material.
[0051] In some embodiments, the invention relates to an assay
method comprising: given any of the assay devices as described
herein or above: contacting the assay device with a population of
cells as described herein or above; and studying at least one
feature of an interaction of the population of cells with any of
the cell-mimetic devices recited herein or above, optionally
wherein any one or a combination of the following is true: the
cells are oligodendrocytes and the at least one feature of the
interaction is myelination of the plurality of fibers; 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; the longitudinal extent and
thickness of myelin are determined from a single microscopy image;
and the interaction of cells with plurality of fibers is measured
by factors secreted by cells or/and by analysis of cell gene
expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0053] The foregoing will be apparent from the following more
particular description of example embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating embodiments of the present invention.
[0054] FIG. 1A depicts a device schematic, in accordance with
aspects of the present invention, including, for this particular
embodiment, a log-pile configuration of artificial axon fibers, and
depicting an interaction of oligodendrocytes therewith.
[0055] FIG. 1B depicts a process of 3D-printing on a glass slide,
in accordance with aspects of the present invention.
[0056] FIG. 1C depicts an example of a three-dimensionally printed
(3DP) device, in accordance with aspects of the present
invention.
[0057] FIG. 1D depicts a higher magnification of the device
pictured in FIG. 1C.
[0058] FIG. 1E depicts oligodendrocyte precursor cells (OPCs)
attaching to and migrating on a polyHEMA neuron mimetic device of
10 .mu.m fiber diameter, in accordance with aspects of the present
invention.
[0059] FIGS. 1F and 1G depict cells differentiated into
oligodendrocytes and wrapping neuron mimetic fibers with layers of
myelin, in accordance with aspects of the present invention.
[0060] FIG. 1H depicts an example of a multimaterial scaffold,
consisting of two types of polyHEMA fibers of 10 .mu.m diameter,
having different stiffnesses, in accordance with aspects of the
present invention.
[0061] FIG. 2A is schematic representation of gradients
incorporated into fiber devices, in accordance with aspects of the
present invention, namely gradients of fiber diameter.
[0062] FIG. 2B is a schematic representation of gradients
incorporated into fiber devices, in accordance with aspects of the
present invention, namely gradients of fiber stiffness, with E1-E3
representing different Young's moduli.
[0063] FIG. 2C is a schematic representation of gradients of ligand
concentration in accordance with aspects of the present invention,
with red and blue dots representing two different ligands.
[0064] FIG. 3A shows a schematic representation of a stretchable
log pile structure printed in wells of a stretchable PDMS plate, in
accordance with aspects of the present invention.
[0065] FIG. 3B shows an image of a PDMS printed scaffold within the
well of PDMS stretchable plate, in accordance with aspects of the
present invention.
[0066] FIG. 3C shows a magnified scaffold with a fiber diameter of
5 .mu.m, and a 100 .mu.m spacing between fibers, in accordance with
aspects of the present invention.
[0067] FIG. 3D shows a schematic representation of a stretched
plate and scaffolds, in accordance with aspects of the present
invention.
[0068] FIG. 3E shows a stretchable plate mounted on a stretcher
device, in accordance with aspects of the present invention.
[0069] FIG. 3F shows a strain of 10% applied to a PDMS plate, the
strain being transferred to the fiber scaffold, in accordance with
aspects of the present invention.
[0070] FIG. 3G shows oligodendrocyte producing cells (OPCs) (green)
adhered to the PDMS scaffold (red), in accordance with aspects of
the present invention.
[0071] FIG. 3H shows differentiated oligodendrocytes (green) on a
PDMS scaffold (red), in accordance with aspects of the present
invention.
[0072] FIG. 4A shows a schematic of a 3DP neuron mimetic
myelination device, which includes a multi-well plate.
[0073] FIG. 4B shows a schematic of structure (in one example)
within a single well within the multi-well plate, according to
aspects of the present invention.
[0074] FIG. 4C shows a schematic of structure (in another example)
within a single well within the multi-well plate, according to
aspects of the present invention.
[0075] FIG. 5A depicts a myelination assay device according to
aspects of the present invention.
[0076] FIG. 5B depicts a magnification of the device from FIG.
5A
[0077] FIG. 5C provides another view of the device.
[0078] FIG. 5D provides an image of suspended fibers with colors
scale corresponding to height above glass slide surface, according
to aspects of the present invention.
DETAILED DESCRIPTION
[0079] For clarity of description, example embodiments are
presented in the figures and below discussions. These examples are
for purposes of illustration and not a limitation of the principles
of the present invention. A description of example embodiments of
the invention follows.
[0080] Cell-Mimetic Devices
[0081] The invention relates, in some embodiments, to the provision
of cell-mimetic devices. As used herein a "cell-mimetic" is a
structure that mimics one or more relevant features of a cell or a
portion thereof. In some embodiments the cell mimetic is a
"neuron-mimetic," mimicking features of a neuron (e.g, a peripheral
neuron, a central nervous system (CNS) neuron, e.g., from brain or
spinal cord). In some embodiments, the neuron-mimetic is an
"axon-mimetic," mimicking a neuronal axon (e.g., a CNS neuronal
axon).
[0082] Relevant Features
[0083] Relevant features of cells that can be mimicked by
cell-mimetics include, without limitation: physical, mechanical,
and biochemical properties and gradients of any of these
properties. Physical properties include size, length, width, and
density. Mechanical properties include stiffness. Biochemical
properties include surface chemistry/ligand modification.
Additional relevant properties may include biocompatibility and
cell adherence. For example, it may be desirable for a neuron
mimetic, or artificial axon, to be formed of materials that are
biocompatible and cell adherent. These features can enable other
cells such as glial cells from the CNS to be seeded within the
mimetic, adhere, grow, and respond to cues such as administered
drugs.
[0084] Form and Construction
[0085] Cell-mimetic devices can have a wide variety of forms. In
some embodiments, they are comprised of groups of fibers (e.g.,
polymeric fibers) arranged in a desired 2D or 3D pattern. In some
embodiments, the fibers are laid down by 3D printing. In some
embodiments, axon mimetics are constructed to mimic one or more
relevant features of neurons in the central nervous system
("neuron-mimetics").
[0086] Cell mimetics can be constructed of a wide variety of
materials, including for example, polymers. As one example, neuron
mimetics can be created by 3D printing, e.g., using a printing
technique developed in the Lewis labs (Barry et al., 2009; Sun et
al., 2012; Kolesky et al., 2014) that is specialized for printing
of compliant materials at micrometer-scale dimensions. In some
embodiments, this provides 3D mimetics of neuronal axons as defined
by feature geometry, mechanical properties, and/or biochemical
functionalization. In some embodiments, these devices enable
neurological studies in both reductionist and complex environments,
where individual cues can be studied separately or be considered
together.
[0087] In some embodiments, 3D printing allows for one or a
combination of the following: (a) creation of neuronal axon
mimetics (artificial axons) having flexible design; (b) fabrication
of multiple fiber geometries and structure architectures; and (c)
the representation of particular biophysical microenvironments,
including defined magnitudes and gradients of fiber mechanical
stiffness, diameter, and/or ligand/molecule presentation at the
fiber surface. One embodiment includes a stretchable version of the
neuronal axon mimetic, to incorporate effects of mechanical strain
and/or fiber diameter changes on the responses of the cell types
adhered to those fibers.
[0088] 3D-Printed Artificial Axons Device
[0089] For example, as described below, certain elements of this
product and process include neuron mimetic devices consisting of
multilayers of 3D-printed fibers, with varying diameters and
spacing between fibers. In some embodiments, a cell-mimetic device
(e.g., a neuron mimetic/3DP neuron mimetic/artificial axon device)
consists of 3DP fibers (e.g., fibers mimicking neuronal axons)
organized into multilayered structures (e.g., FIG. 1A). For
example, as shown with respect to FIGS. 1A-1D (further discussed
below), the fibers can have a diameter of 10 and a tunable spacing
between fiber centers in the range of 20-50 There is also the
capacity to print multimaterial devices, such as, for example,
where fibers within the same array are printed with different
materials (FIG. 1H) (e.g., up to four different materials).
[0090] The ability of oligodendrocyte precursor cells (OPCs)--which
act in vivo to protect axons by wrapping them in a myelin
sheath--to adhere, survive, migrate and differentiate into
oligodendrocytes that produce myelin was confirmed in embodiments
of the invention. OPCs thrived on these scaffolds, and were able to
migrate along and between the fibers (FIG. 1E). After several days
in culture within the neuron mimetic device, OPCs differentiated
into oligodendrocytes that wrapped myelin around the fibers,
remarkably similar to wrapping of axons that is observed in vivo
(FIGS. 1F-G).
[0091] In some embodiments, neuron mimetic devices have multilayers
of 3D-printed fibers with diameter of 5-10 and tunable spacing
between axon fiber centers (20-150 .mu.m).
[0092] In some embodiments, devices according to the present
invention have one, or any combination of, the following
characteristics: [0093] Fibers represent neuronal axons. [0094]
Fibers can be printed at various mechanical stiffnesses of the
solid printed material, including a very low range of Young's
elastic modulus E.about.0.1-1 kPa, corresponding to the low
stiffness of mammalian neuronal axons, as well as higher stiffness,
above 1 kPa, corresponding to neuron pathologies. [0095]
Controllable fiber diameters, within the range of 0.5-10 are
employed, being comparable to the diameters of neuronal axons.
[0096] Fiber surfaces can be functionalized with proteins
representing extracellular matrix (ECM) components and ligands.
[0097] Multifiber, multilayer configurations are used to
systematically position fiber axis orientations within and among
multiple layers, allowing for formation of mechanical and
biochemical gradients. [0098] High optical transparency materials
are used to enable observations of structures and cell-structure
interactions via optical methods such as confocal microscopy.
[0099] A reductionist design is used to allow for
isolating/studying individual biochemical and mechanical factors.
[0100] Design allows for additional components to be added, e.g.,
components characteristic of disease environment, such as other
neural cell types, ECM proteins, growth factors, and variation of
tissue stiffness, axon diameter, etc. [0101] The system is
permissible for fluids and gases. [0102] Device allows for cell
migration along and across the fibers. [0103] Device allows for
facile fluid/media exchange. [0104] System is stable in biological
conditions (e.g., one or more of 37.degree. C., 5% CO.sub.2, high
salinity, and high humidity). [0105] System can be made by an
inexpensive manufacturing process that is amenable to rapid design
changes in printed device features. [0106] Individual devices can
be easily multiplexed by printing in standard multi-well plates
(e.g., from 6- to 384-well plates)
[0107] FIG. 1A shows a schematic of an example of a 3D-printed
artificial axon device, showing fibers representing axons in a
log-pile configuration, and oligodendrocytes interacting with
artificial axon fibers.
[0108] FIG. 1B shows a process of 3D-printing devices on a glass
slide. (Taken from "Organs on demand", The Scientist, Sep. 1, 2013,
courtesy of J. A. Lewis.)
[0109] FIG. 1C depicts an example of the 3DP device consisting of
four layers of polyHEMA (polyhydroxyethylmethacrylate) fibers of
material elastic modulus 100 kPa, diameter of 10 .mu.m, and spacing
of 20 .mu.m between fibers. A rim printed of polydimethylsiloxane
(PDMS) creates the well for fluid culture media. FIG. 1D shows a
higher magnification of the device pictured in FIG. 1C.
[0110] FIG. 1E shows how oligodendrocyte precursor cells (OPCs) can
attach to and migrate on a polyHEMA neuron mimetic device, shown
with 10 .mu.m fiber diameter. FIGS. 1F-G show OPCs cultured in the
device, differentiated into oligodendrocytes, and wrapping the
neuron mimetic fibers with layers of myelin.
[0111] FIG. 1H depicts a multimaterial scaffold, consisting of two
types of polyHEMA fibers of 10 .mu.m diameter, with fiber-material
Young's elastic modulus of E.about.2.7 kPa (green) and 4.5 kPa
(red).
[0112] In some embodiments, 3D printing is done using compliant
fibers with desired small diameters and low stiffness, using, for
example, optimized fiber material properties and selected printing
parameters. For example, in one embodiment, for HEMA-based inks,
the list of varied components include: [0113] pHEMA (1,000,000 Da)
[0114] pHEMA (300,000 Da) [0115] HEMA monomer [0116] Water [0117]
PBS without Ca and Mg [0118] Glycerol [0119] Ethanol [0120] EGDMA
(comonomer) [0121] Lysine [0122] DMPA (initiator) [0123] Irgacure
(initiator)spacing [0124] Fibrinogen (340,000 Da) [0125] Xanthum
Gum (filler) [0126] Nile Blue [0127] Rhodamine
[0128] In other embodiments, aspects of the invention relate to
printing structurally stable fibers with very low stiffness, at
very small diameters, e.g., low stiffness of fibers while
maintaining small diameter. One example of a pHEMA formulation is
as follows: 10 wt % 1 MDa pHEMA, 25 wt % 300 kDa pHEMA, 5 wt % HEMA
monomer, 33.5 wt % water, 1 wt % EGDMA, 25 wt % ethanol, and 0.5 wt
% irgacure; printed in a humid environment; and cured
post-printing. This can result in fine features (down to 1 .mu.m in
diameter) with a low final cured stiffness (1.5 kPa).
[0129] Feature Gradients
[0130] In some embodiments, properties of cell mimetics are
constant over a region of space (e.g., over the device). In other
embodiments, one or more properties vary over a region of the
device. The region over which the variation occurs can be a volume
region, area region, or linear (segment) region. For example, a
volume region can be defined by a cube having a linear dimension of
1 .mu.m, 5 .mu.m, 10 .mu.m, 20 .mu.m, 50 .mu.m, 100 .mu.m, 200
.mu.m, 500 .mu.m, 1 mm, 2 mm, 5 mm, or 10 mm per edge; an area
region can be defined by a square having, per side, any of the
foregoing linear dimensions; and a linear (segment) region, can
have any of the foregoing linear dimensions. The region could also
be defined, for example, as a fraction of a linear dimension of the
device, for example, 1%, 5%, 10%, 25%, or 50%, with corresponding
area and volume regions being defined by the fraction in two, or
three, device dimensions, respectively.
[0131] Average properties can be calculated within one or more
subregions within the regions, and changes in average properties
from one subregion to the next can be calculated. For example, the
region can be divided along each available dimension into, for
example, 3 parts--yielding 3 subregions for a linear region, 9
subregions for an area region, and 27 subregions for a volume
region. In other examples, the region can be divided into 2, 4, 5,
8, 10, 20, 50, or 100 parts per available dimension.
[0132] In some embodiments, changes in average properties are
reported as a gradient within the region (e.g., as a rate of change
in an average property of subregions across a linear dimension
within the region). In some embodiments, the gradient is such that
one or more properties change by a factor of, for example, about
1.1, 1.3, 1.5, 2, 5, 10, 50, 100, or 500 across the region.
[0133] For example, there can be a gradient of one or more
properties (e.g., diameter of fibers from which cell mimetic is
formed, stiffness of fibers, density of fibers, composition of
fibers, surface density of a chemical or biochemical surface marker
or modifier). In some embodiments, the cell-mimetic (e.g., axon
mimetic) incorporates gradients of material stiffness, axon
diameter, and/or ligand presentation. In some embodiments, e.g.,
those using 3D printing, these features are easily "tuned" during
construction of the device, allowing for the facile construction of
custom assemblies. Such assemblies can be, for example, models of
generically healthy or generic disease states, or more
specifically, models of a disease state or region in an individual
subject (e.g., human patient) or a tissue/cellular region
thereof.
[0134] In some embodiments, 3DP can enable construction of
biochemical and mechanical gradients within printed fibers or among
an array of printed fibers that will serve as the neuron mimetic.
Such gradients are often present in both healthy and diseased
environments and strongly affect responses of other CNS cell types
(Jagielska et al., 2012, Jagielska et al., 2013). In accordance
with aspects of the invention, 3DP structures incorporate tunable
gradients of material stiffness, axon diameter, and ligand
presentation.
[0135] In some embodiments, the invention relates to incorporating
pre-designed, well controlled gradients, created by fibers with
different features, such as gradients of fiber diameter (FIG. 2A),
fiber stiffness (FIG. 2B), and ligand density (FIG. 2C) presented
to a cell that is in contact with the neuron-mimetic fibers. In
FIG. 2B, E1-E3 represent different Young's moduli, and in FIG. 2C,
red and blue dots represent two different ligands.
[0136] Applications of Cell Mimetics
[0137] Cell/tissue mimetic structures (e.g., neuron mimetics)
according to aspects of the present invention can be used in a wide
variety of applications, including, without limitation for the
following:
[0138] (a) in vitro investigation of disease, e.g., neurological
diseases (e.g., demyelinating disease, multiple sclerosis);
[0139] (b) in vitro study of interactions of cells with the
cell/tissue mimetic structure, and/or with other cells and cell
types within an environment of the cell mimetic structure,
including:
[0140] (1) how the interaction is modified by the application of
one or more substances (e.g., drugs);
[0141] (2) for neuron-mimetics, an in vitro study of myelination of
axons (represented by the printed fibers) by other neural cell
types; and
[0142] (c) high-throughput drug screening for cells adhered within
the cell-mimetic architecture and environment (e.g., within
neuron-mimetic architecture).
Stretchable Embodiments
[0143] This invention also includes a subset of cell mimetics
(e.g., 3DP neuron mimetics) that are stretchable, such as, for
example by using a stretchable version of the printed fibers. This
can be used to enable a study of the effects of strain on neuronal
cells. Here, the length and diameter of the artificial axon will
change as a function of strain applied to the entire device. This
can enable a controlled study of these physical cues on the
adherent glial cells' response, e.g., by using devices to study
such mechanical strain effects in architectures and environments
similar to neural tissue. The roles of the natural and pathological
strains (e.g., those associated with axon growth, myelin wrapping,
or axon swelling due to inflammation) in CNS development and
diseases can thereby be studied.
[0144] Stretchable Artificial Axons.
[0145] Some embodiments relate to a stretchable version of axon
mimetic devices, by printing the stretchable fibers, made of
polydimethylsiloxane elastomer (PDMS), inside depressed wells of
stretchable PDMS plates.
[0146] FIG. 3A depicts a schematic representation of a stretchable
log pile structure printed in wells of the stretchable PDMS plate.
FIG. 3B shows an image of the actual printed axon fiber structure.
FIG. 3C shows a magnified structure with fiber diameter of 5 .mu.m
and 100 .mu.m spacing between fibers. FIG. 3D is a schematic
representation of a stretched plate and axon fiber structure. FIG.
3E is a stretchable plate mounted on a stretcher device. FIG. 3D
depicts a strain of 10% applied to the PDMS plate, efficiently
transferred to the axon fiber structure, resulting in an average
strain .epsilon.1=10% (elongation of the fibers in the direction of
applied strain). FIG. 1G shows OPCs (green) adhered to the PDMS
axon fiber structure (red). FIG. 3H shows differentiated
oligodendrocytes (green) on the PDMS axon fiber structure (red).
Green fluorescence in cells is generated by green fluorescent
protein (GFP) fused to myelin basic protein (MBP)--the
differentiation marker expressed in oligodendrocytes.
[0147] In some embodiments the invention relates to a PDMS
custom-made plate (e.g., 6-well), wherein in each well is printed
multiple layers of PDMS fibers, covering an area, e.g, 3 mm.times.3
mm area. PDMS fibers can be covalently functionalized with
poly-D-lysine or surface ligands that are present in a neural
environment in vivo (fibronectin, laminin). These PDMS plates can
include, for example, those developed in the Van Vliet lab at MIT
(see Zeiger 2013), and can be stretched by applying uniaxial
tensile strain with a customized strain device developed in the Van
Vliet lab at MIT (see, e.g., FIGS. 3D, 3E).
[0148] It was confirmed via optical microscopy that strain applied
to the plate is transferred completely to the 3DP neuron mimetic
printed within each well, resulting in elongation of the 3DP fibers
in the direction of applied force (FIG. 3F), and that the decrease
of diameter of fibers aligned with the strain axis. It was
demonstrated that OPCs thrive on these PDMS-based 3DP neuron
mimetic devices, and differentiate into oligodendrocytes that
wrapped the fibers with myelin. These devices can be used to
quantify the effect of strain in 3D environments on oligodendrocyte
differentiation, to gain better understanding of how physiological
and pathological strains contribute to this process, and how such
dynamic changes in axon properties can affect drug metabolism and
response. Therefore, in some embodiments, the invention relates to
a compliant axon mimicking device allowing the study of effects of
axonal strain on glial cell biology.
[0149] Assay for Quantification of Myelination.
[0150] Myelin production during human development, and stimulation
of myelin repair (remyelination) in demyelinating diseases such as
multiple sclerosis, are central unsolved problems in neuroscience
and neuromedicine. The current lack of a facile, adaptable, and
high throughput assay to compare myelin production or repair
efficacy under conditions mimicking in vivo environments is
considered a major obstacle to progress in therapy development. The
recently developed assay for myelination (Mei et al., 2014, Chan
and Lee, 2014) although providing high throughput myelin
quantification, uses glass as a cell substrate, which is orders of
magnitude stiffer than nervous tissue, therefore providing very
different mechanical cues than those present in vivo.
[0151] In some embodiments, this invention relates to a version of
an artificial axon device for use in a myelination assay. In some
embodiments, the assay enables a simultaneous imaging of fiber
cross-section to quantify myelin thickness and fiber length to
quantify length of myelin segment. In other embodiments, each is
measured separately or at different times/orientations of the
device. Fibers with a range of different mechanical or/and
biochemical properties can be provided in a single assay, e.g., to
represent healthy and pathological axons. In some embodiments,
drugs/compounds are added to enable quantification of myelination
in response to those drugs/compounds, such as, for example, in
different mechanical/biochemical conditions representative of a
disease. Moreover, arranging the fibers with different
mechanical/biochemical properties such that they recreate in vivo
mechanical/biochemical gradients can be used to investigate the
impact of gradients on myelination.
[0152] FIG. 4 shows an example of a device used in connection with
aspects of the invention, a 3DP neuron mimetic myelination device.
Shown in FIG. 4A is a schematic of a multi-well plate. FIGS. 4B and
4C show two examples of a schematic of a single well within a
multi-well plate. Oligodendrocytes grow in the plate well (green)
in the vicinity of polymer artificial axon-fibers, representing
different biophysical features of an axon such as stiffness,
diameter, or ligand presentation, relevant to different disease
environments. The extent of fiber myelination by oligodendrocytes
can vary depending on these conditions, allowing for discrimination
between environments beneficial or detrimental for myelination.
Drug screening in a multi-well plate format of this device can be
performed, allowing, for example, assessment of drug effects in a
multiplexed platform.
[0153] For example, a 3DP axon mimetic approach can be used to
create a biomimetic myelination assay that takes advantage of
uniform fiber geometry to facilitate rapid quantification of myelin
thickness and segment length, while providing compliant axon-like
polymer fibers with diversity of ligand coatings. The device can
consist of a multi-well plate (e.g., 6, 12, 24, 48, 96, 384-well),
wherein each well contains multiple polymer axon-mimicking printed
fibers (FIG. 4). The fibers can, for example, have low stiffness
mimicking that in vivo, and in some embodiments can be anchored at
the top of a stiffer supporting polymer core. In some embodiments,
they can maintain an angled, extended position despite the
mechanical compliance of the fiber material (two examples of fiber
arrangements on the supporting core are shown, FIG. 4). Use of
optically transparent polymers (e.g., PEG (polyethylene glycol),
PDMS or poly-HEMA) and this angled fiber geometry can allow for
simultaneous acquisition of fluorescent images of myelin rings on
the fiber cross-section to assess myelin thickness, and the
assessment of size of myelin segment (e.g., length) on each fiber
via optical microscopy, to conduct rapid quantification of
myelination amount and distribution.
[0154] With reference to FIG. 5, a prototype of a 3DP neuron
mimetic myelination device is shown. FIG. 5A depicts a myelination
assay device consisting of a PDMS support beam (length: 10 mm,
height 0.4 mm) printed on the glass slide and pHEMA fibers that are
attached to the glass support slide along 1 mm of the fiber
lengths, on both sides of the beam, whereas the fiber middle
section (2 mm) is suspended in air and span over the PDMS beam.
FIG. 5B depicts a magnification of the device from FIG. 5A. FIG. 5C
shows an image of the device through the bottom of the glass slide,
to visualize suspended fibers over the central beam; top and bottom
PDMS beams stamp fibers to the glass slide. FIG. 5D shows an image
of the suspended fibers with color scale corresponding to height
above glass slide surface. pHEMA was UV cured and PDMS was
thermally cured at 80.degree. C. for 2 h.
[0155] In some embodiments of the device, each fiber can be printed
according to a specific design to represent different biological
conditions of an axon, such as stiffness, diameter, or ligand
expression. This presents OPCs grown in the plate well with
various, controlled biological conditions, and the cells will
choose the biological conditions that are optimal for myelination,
by myelinating some types of fibers more than others (which is not
possible with other assays that provide one type of pillar or fiber
material). The same myelination assay can be performed after
application of a drug and multiplexed in all wells of the plate to
test effects of different drug compositions and dosages on
myelination efficiency. Therefore, this device embodiment enables
simultaneous high throughput drug screening and testing of drug
effects in different biological conditions, represented by fibers
with different characteristics.
[0156] In some embodiments, the myelination assay involves one or
any combination of the following: [0157] uses different
materials--biocompatible, biomimetic polymer fibers (PEG, pHEMA,
PDMS) that are similar to real axons in their stiffness and
diameters. This can provide an advantage over assays that use
etched glass micropillars (or other stiff plastics), which are
orders of magnitude stiffer than real axons, and may not represent
the axon mechanical environment correctly. [0158] has a geometry
designed to maintain suspended compliant fibers at the angle
sufficient to image simultaneously the fiber cross-section and
length for rapid myelin quantification, e.g. wherein the beginning
and the end of the suspended section of the fiber defines a line
segment having an angle to the substrate surface of about
5.degree., 10.degree., 10.degree., 20.degree., 30.degree.,
45.degree., 50.degree., 60.degree., 70.degree., 80.degree., or
85.degree.. This can provide an advantage over assays using stiff
micropillars vertically rising from the glass surface, and of
cross-sectional geometry that is not constant along the pillar
length. [0159] enables the creation in a single assay of
combinations of fibers with diverse mechanical and biochemical
properties that represent a complex disease environment, such as
local changes in axon stiffness or ligands expressed on an axon
surface. This allows for studying drug responses in credible
biomimetic environments that are fully controlled and
reproducible.
[0160] Other aspects of some embodiments include, alone, or in any
combination, the following: [0161] Biocompatible, neuronal axon
mimetic (fiber) with tunable diameter and stiffness within the
range of biological neural cell properties. [0162] Suitable for
research on myelin diseases. [0163] Amenable to oligodendrocyte
precursor cell culture, adhesion, and differentiation to produce
myelin. [0164] Enables study of axon myelination of fibers,
advantageous over 2D dish format. [0165] Enables high throughput
myelination assay of drug responses in complex, designable
mechano/biochemical environments that mimic real disease
microenvironments. [0166] Suitable for research on a wide spectrum
of neuronal diseases that involve axons--amenable to cell culture
of other glial cells that interact with axons (microglia,
astrocytes). [0167] 3D-printing of fibers with a wide range of
stiffness, including very low stiffness of 0.1-1 kPa characteristic
of nervous tissue. [0168] 3D-printing of gradients of fiber length,
diameter and/or mechanical stiffness, controllable within a single
printed device footprint. [0169] 3D-printing of multi-molecular
compositions/molecular gradients, controllable presentation within
a single printed device footprint. [0170] Enables separation of
mechanical and biochemical cues on myelination and other processes
involving axons, which is not possible to separate in tissue slice
cultures. [0171] Enables creation of mechanical and biochemical
gradients by design, to mimic the disease environment features
expected in tissue. [0172] Mechanical and biochemical variation can
be introduced independently, at the level of single fibers (axons)
or entire fiber network (tissue). [0173] Suitable for research on
the effect of strain in neurological diseases--amenable to
mechanical stretching and modulation of fiber diameter as
described. [0174] Novel printing materials--improved 3DP ink
formulations for hydrogels and UV-curable elastomers (PEG-, pHEMA,
PDMS-based inks). [0175] Provides potential to study effects of
dynamic change of axon diameter on myelination, which is not
possible with existing devices. [0176] Provides high throughput,
low cost in vitro drug screening assays for axon/myelin associated
diseases.
Additional Embodiments
[0177] In some embodiments, the "artificial axon device" consists
of axon mimicking fibers, which can have various stiffness within
the range corresponding to stiffness of neural tissue and neural
cells, 0.1-1 kPa, and stiffness above 1 kPa.
[0178] In some embodiments, the axon fibers can have a range of
diameters of micrometer scale, including a range of 0.1-20
micrometers.
[0179] In some embodiments, the axon fibers can have different
molecules and molecule densities on their surface.
[0180] In some embodiments, the biomimetic fibers can be produced
from various compliant polymers and hydrogels, including PEG,
pHEMA, PDMS, and poly-acrylamide, and their derivatives. The fibers
are arranged in a predefined order to create a desired
representation of the disease microenvironments.
[0181] In some embodiments, methods/devices can be used to
screen/investigate drugs/therapies/candidates or create
models/model the progression of disease, e.g., demyelinating
disease, multiple sclerosis.
[0182] Such arrangements can include gradients of properties, such
as fiber stiffness, ligand density on the fiber surface, and fiber
diameter. Such arrangements can be achieved by e.g., various 3D
printing methods (including direct printing.)
[0183] In some embodiments, the invention relates to a device in
the "stretchable artificial axon" form. In this version of the
device, the fibers described above can be printed from a
stretchable material (e.g. PDMS or pHEMA), and deposited on a
stretchable medium (e.g. PDMS plate where the fibers are printed in
each well of the plate), which facilitates stretching of the fibers
using external stretching device.
[0184] In some embodiments, fibers are printed in geometries that
include a supporting polymer core and compliant fibers spanning the
distance from the device bottom to the top of the core, such that
the angle between fibers and the device bottom is sufficient to
enable simultaneous microscope imaging of both the fiber
cross-section and fiber length. Such geometry allows for fast
quantification of myelin thickness and length to enable fast
screening for myelin drugs in various disease conditions
represented by different mechanical and biochemical properties of
the fiber arrangements.
[0185] In some embodiments, pharmaceutical companies developing
drugs for neurological diseases, especially those working on
remyelination therapies and myelin diseases (e.g., multiple
sclerosis), can use devices and methods described herein.
[0186] In some embodiments, devices are multiplexed, such as
multi-well plates (e.g., 6, 12, 24, 48, 96, 384-well plates), petri
dishes, glass or plastic slides.
[0187] In some embodiments, the invention relates to materials
optimized for 3D printing. These include PEG-, pHEMA, PDMS, and
poly-acrylamide based materials optimized for 3D printing.
[0188] The teachings of all patents, published applications and
references cited herein, incorporated by reference herein in their
entirety.
[0189] Also incorporated herein and forming a part of this
application is the material attached hereto as an appendix.
[0190] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
REFERENCES
[0191] Barry, R. A., Shepherd, R. F., Hanson, J. N., Nuzzo, R. G.,
Wiltzius, P. & Lewis, J. A. Direct-Write Assembly of 3D
Hydrogel Scaffolds for Guided Cell Growth. Adv. Mater. 21,
2407-2410, doi:10.1002/adma.200803702 (2009). [0192] Chan, J., Lee,
S. Patent WO 2014/100199 A1 "Micropillar arrays for assaying
myelination". [0193] Hanson-Shepherd J, Parker S T, Shepherd R F,
Gillette M U, Lewis J A, and Nuzzo R G, 3D Microperiodic Hydrogel
Scaffolds for Robust Neuronal Cultures, (2011), Adv. Funct. Mater.,
21, 47-54. [0194] Jagielska A, Norman A L, Whyte G, Vliet K J, Guck
J, Franklin R J, Mechanical Environment Modulates Biological
Properties of Oligodendrocyte Progenitor Cells, (2012), Stem Cells
Dev., November 1; 21(16):2905-14. [0195] Jagielska A, Wilhite K D,
Van Vliet K J, Extracellular Acidic pH Inhibits Oligodendrocyte
Precursor Viability, Migration, and Differentiation, (2013), PLoS
ONE 8(9): e76048. doi:10.1371. [0196] Kolesky, D. B., Truby, R. L.,
Gladman, A. S., Busbee, T. A., Homan, K. A. & Lewis, J. A. 3D
Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue
Constructs. Adv. Mater. 26, 3124-3130, doi:10.1002/adma.201305506
(2014). [0197] Lee, S., Leach, M. K., Redmond, S. A., Chong, S. Y.,
Mellon, S. H., Tuck, S. J., Feng, Z. Q., Corey, J. M. & Chan,
J. R. A culture system to study oligodendrocyte myelination
processes using engineered nanofibers. Nat. Methods 9, 917-922,
doi:10.1038/nmeth.2105 (2012). [0198] Li, Y., Ceylan, M., Shrestha,
B., Wang, H., Lu, Q. R., Asmatulu, R. & Yao, L. Nanofibers
support oligodendrocyte precursor cell growth and function as a
neuron-free model for myelination study. Biomacromolecules 15,
319-326, doi:10.1021/bm401558c (2014). [0199] Mei, F., Fancy, S.
P., Shen, Y. A., Niu, J., Zhao, C., Presley, B., Miao, E., Lee, S.,
Mayoral, S. R., Redmond, S. A., Etxeberria, A., Xiao, L., Franklin,
R. J., Green, A., Hauser, S. L. & Chan, J. R. Micropillar
arrays as a high-throughput screening platform for therapeutics in
multiple sclerosis. Nat. Med., doi:10.1038/nm.3618 (2014). [0200]
Sun, L, S T Parker, D Syoji, X Wang, J A Lewis, and D L Kaplan.
2012. Direct-Write Assembly of 3D Silk/Hydroxyapatite Scaffolds for
Bone Co-Cultures, Advanced Healthcare Materials, no. 1: 729-735.
[0201] Zeiger A S, Ph.D. thesis, Chemomechanics at Cell-Cell and
Cell-Matrix Interfaces Critical to Angiogenesis, (2013), Department
of Materials Science and Engineering, Massachusetts Institute of
Technology.
* * * * *