U.S. patent application number 16/093036 was filed with the patent office on 2019-05-02 for implantable living electrodes and methods for use thereof.
The applicant listed for this patent is Thomas Jefferson University, The Trustees Of The University of Pennsylvania. Invention is credited to H. Isaac Chen, Daniel Kacy Cullen, James P. Harris, Mijail Serruya, Douglas H. Smith, John A. Wolf.
Application Number | 20190126043 16/093036 |
Document ID | / |
Family ID | 60042253 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190126043 |
Kind Code |
A1 |
Cullen; Daniel Kacy ; et
al. |
May 2, 2019 |
IMPLANTABLE LIVING ELECTRODES AND METHODS FOR USE THEREOF
Abstract
In one aspect, the invention comprises an implantable living
electrode comprising a substantially cylindrical extracellular
matrix core; one or more neurons implanted along or within the
substantially cylindrical extracellular matrix core, the one or
more neurons including one or more optogenetic or magnetogenetic
neurons proximal to a first end of the implantable living
electrode.
Inventors: |
Cullen; Daniel Kacy; (Media,
PA) ; Harris; James P.; (Philadelphia, PA) ;
Wolf; John A.; (Philadelphia, PA) ; Chen; H.
Isaac; (Penn Valley, PA) ; Smith; Douglas H.;
(Boothwyn, PA) ; Serruya; Mijail; (Philadelphia,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees Of The University of Pennsylvania
Thomas Jefferson University |
Philadelphia
Philadelphia |
PA
PA |
US
US |
|
|
Family ID: |
60042253 |
Appl. No.: |
16/093036 |
Filed: |
April 14, 2017 |
PCT Filed: |
April 14, 2017 |
PCT NO: |
PCT/US2017/027705 |
371 Date: |
October 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62322434 |
Apr 14, 2016 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61N 5/0601 20130101;
A61N 5/0622 20130101; A61N 1/0531 20130101; A61B 5/01 20130101;
A61N 1/36067 20130101; A61B 5/4094 20130101; A61B 5/4082 20130101;
A61B 5/6868 20130101; A61B 5/0478 20130101; A61B 5/4088 20130101;
A61N 1/05 20130101; A61N 1/0536 20130101; A61N 1/36182 20130101;
A61B 5/04001 20130101; A61B 5/0059 20130101 |
International
Class: |
A61N 1/36 20060101
A61N001/36; A61N 1/05 20060101 A61N001/05; A61N 5/06 20060101
A61N005/06; A61B 5/0478 20060101 A61B005/0478 |
Claims
1. An implantable living electrode comprising: a substantially
cylindrical extracellular matrix core; one or more neurons
implanted along or within the substantially cylindrical
extracellular matrix core, the one or more neurons including one or
more optogenetic or magnetogenetic neurons proximal to a first end
of the implantable living electrode.
2. The implantable living electrode of claim 1, wherein the
implantable living electrode is capable of bidirectional
stimulation and bidirectional recording.
3. The implantable living electrode of claim 1, wherein the
implantable living electrode is capable of unidirectional
stimulation and unidirectional recording.
4. The implantable living electrode of claim 1, wherein the
implantable living electrode is capable of unidirectional
stimulation and bidirectional recording.
5. The implantable living electrode of claim 1, wherein the neurons
are stimulated and recorded using different wavelengths of
light.
6.-9. (canceled)
10. The implantable living electrode of claim 1, wherein the one or
more neurons include a plurality different phenotypes that target a
plurality of targets and a plurality of different optogenetic or
magnetogenetic phenotypes for responding to or emitting distinct
wavelengths of light.
11. The implantable living electrode of claim 1, wherein the one or
more neurons include one or more selected from the group consisting
of: primary cerebral cortical neurons, dorsal root ganglion
neurons, glutamatergic neurons, GABAergic neurons, cholinergic
neurons, dopaminergic neurons, serotonergic neurons, peptidergic
neurons, neurons from the thalamus, neurons from the striatum,
neurons from the hippocampus, neurons from the substantia nigra,
neurons from the peripheral nervous system, and spinal motor
neurons.
12. The implantable living electrode of claim 11, wherein the
primary cerebral cortical neurons include one or more selected from
the group consisting of: neurons from layer I of the cortex,
neurons from layer II of the cortex, neurons from layer III of the
cortex, neurons from layer IV of the cortex, neurons from layer V
of the cortex, neurons from layer VI of the cortex, neurons from
the visual cortex, neurons from the motor cortex, neurons from the
sensory cortex, and neurons from the entorhinal cortex.
13. The implantable living electrode of claim 1, further
comprising: one or more non-neuronal cells selected from the group
consisting of: endothelial cells, myocytes, myoblasts, astrocytes,
olfactory ensheathing cells, oligodendrocytes, or Schwann
cells.
14. The implantable living electrode of claim 1, wherein the
neurons are derived from stem cells.
15. The implantable living electrode of claim 1, wherein the
neurons are derived from neuronal progenitor cells.
16. The implantable living electrode of claim 1, wherein the
hydrogel sheath comprises agarose.
17. The implantable living electrode of claim 1, wherein the one or
more neurons implanted along or within the substantially
cylindrical extracellular matrix core are formed via forced cell
aggregation.
18. A method comprising: implanting one or more implantable living
electrodes of claim 1 in a subject's brain; and placing a
compatible stimulator in proximity to at least one of the one or
more implantable living electrodes.
19.-33. (canceled)
34. A method comprising: implanting one or more implantable living
electrodes of claim 1 in a subject's brain; and placing a
compatible sensor in proximity to at least one of the one or more
implantable living electrodes.
35.-37. (canceled)
38. A method comprising: implanting one or more implantable living
electrodes of claim 1 in a subject's brain; and applying stimulus
to the implantable living electrode to selectively excite or
inhibit one or more selected from the group consisting of:
glutamatergic neurons, GABAergic neurons, cholinergic neurons,
serotonergic neurons, peptidergic neurons, neurons from the
thalamus, neurons from the striatum, neurons from the hippocampus,
neurons from the substantia nigra, neurons from the peripheral
nervous system, spinal motor neurons, cerebral cortical neurons,
dopaminergic neurons, and dorsal root ganglion neurons.
39. (canceled)
40. An implantable living electrode comprising: a substantially
cylindrical extracellular matrix core; a hydrogel sheath coaxially
surrounding the substantially cylindrical extracellular matrix
core; one or more selected from the group consisting of:
electrodes, optrodes, magnetic actuators, heating probes, cooling
probes, or chemical applicators positioned between the
substantially cylindrical extracellular matrix core and the
hydrogel sheath; and one or more neurons implanted along or within
the substantially cylindrical extracellular matrix core.
41. An implantable living electrode comprising: a substantially
cylindrical extracellular matrix core; and a plurality of
aggregated neurons implanted along or within the substantially
cylindrical extracellular matrix core.
42.-46. (canceled)
47. A method of treating Parkinson's disease in a patient,
comprising implanting a living electrode according to claim 41 into
the substantia nigra of the patient.
48. A method of manufacturing an implantable living electrode
comprising: providing an extracellular matrix core; and contacting
at least one end of the extracellular matrix core with a plurality
of aggregated neurons.
49. (canceled)
50. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 62/322,434, filed Apr. 14, 2016, the content
of which is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Brain Machine Interfaces (BMIs) allow the nervous system to
directly communicate with external devices in order to mitigate
deficits associated with neurodegeneration or to drive peripheral
prosthetics. There has been substantial progress using penetrating
microelectrode arrays and optogenetics strategies; however, these
approaches are limited in that they generally rely on placing
non-organic electrodes/optrodes into the brain, inevitably leading
to an inflammatory foreign body response that ultimately diminishes
the quality of the recording and stimulation. Current BMI
strategies suffer from impermanence, non-specificity, and/or a
significant foreign body response upon implantation.
BRIEF SUMMARY OF THE INVENTION
[0003] In one aspect, the invention comprises an implantable living
electrode comprising a substantially cylindrical extracellular
matrix core; one or more neurons implanted along or within the
substantially cylindrical extracellular matrix core, the one or
more neurons including one or more optogenetic or magnetogenetic
neurons proximal to a first end of the implantable living
electrode.
[0004] In various embodiments the implantable living electrode is
capable of bidirectional stimulation and bidirectional
recording.
[0005] In various embodiments the implantable living electrode is
capable of unidirectional stimulation and unidirectional
recording.
[0006] In various embodiments the implantable living electrode is
capable of unidirectional stimulation and bidirectional
recording.
[0007] In various embodiments the neurons are stimulated and
recorded using different wavelengths of light.
[0008] In various embodiments the substantially cylindrical
extracellular matrix core has a largest cross-sectional dimension
selected from the group consisting of: between about 10 .mu.m and
about 20 .mu.m, between about 25 .mu.m and about 50 .mu.m, between
about 50 .mu.m and about 100 .mu.m, between about 100 .mu.m and
about 150 .mu.m, between about 150 .mu.m and about 200 .mu.m,
between about 200 .mu.m and about 250 .mu.m, between about 250
.mu.m and about 300 .mu.m, between about 300 .mu.m and about 400
.mu.m, between about 400 .mu.m and about 500 .mu.m, and between
about 500 .mu.m and about 700 .mu.m, and between about 700 .mu.m
and about 1000 .mu.m.
[0009] In various embodiments the implantable living electrode
further comprises a hydrogel sheath coaxially surrounding the
substantially cylindrical extracellular matrix core.
[0010] In various embodiments the hydrogel sheath has a largest
cross-sectional dimension selected from the group consisting of:
between about 20 .mu.m and about 50 .mu.m, between about 50 .mu.m
and about 100 .mu.m, between about 100 .mu.m and about 200 .mu.m,
between about 200 .mu.m and about 250 .mu.m, between about 250
.mu.m and about 300 .mu.m, between about 300 .mu.m and about 350
.mu.m, between about 350 .mu.m and about 400 .mu.m, between about
400 .mu.m and about 450 .mu.m, between about 450 .mu.m and about
500 .mu.m, between about 500 .mu.m and about 600 .mu.m, between
about 600 .mu.m and about 800 .mu.m, and between about 800 .mu.m
and about 1200 .mu.m.
[0011] In various embodiments the implantable living electrode has
a length of about 100 .mu.m to 10 cm or greater.
[0012] In various embodiments the one or more neurons include a
plurality different phenotypes that target a plurality of targets
and a plurality of different optogenetic or magnetogenetic
phenotypes for responding to or emitting distinct wavelengths of
light.
[0013] In various embodiments the one or more neurons include one
or more selected from the group consisting of: primary cerebral
cortical neurons, dorsal root ganglion neurons, glutamatergic
neurons, GABAergic neurons, cholinergic neurons, dopaminergic
neurons, serotonergic neurons, peptidergic neurons, neurons from
the thalamus, neurons from the striatum, neurons from the
hippocampus, neurons from the substantia nigra, neurons from the
peripheral nervous system, and spinal motor neurons.
[0014] In various embodiments the primary cerebral cortical neurons
include one or more selected from the group consisting of: neurons
from layer I of the cortex, neurons from layer II of the cortex,
neurons from layer III of the cortex, neurons from layer IV of the
cortex, neurons from layer V of the cortex, neurons from layer VI
of the cortex, neurons from the visual cortex, neurons from the
motor cortex, neurons from the sensory cortex, and neurons from the
entorhinal cortex.
[0015] In various embodiments the implantable living electrode
further comprises one or more non-neuronal cells selected from the
group consisting of: endothelial cells, myocytes, myoblasts,
astrocytes, olfactory ensheathing cells, oligodendrocytes, or
Schwann cells.
[0016] In various embodiments the neurons are derived from stem
cells.
[0017] In various embodiments the neurons are derived from neuronal
progenitor cells.
[0018] In various embodiments the hydrogel sheath comprises
agarose.
[0019] In various embodiments the one or more neurons implanted
along or within the substantially cylindrical extracellular matrix
core are formed via forced cell aggregation.
[0020] In various embodiments the invention comprises a method
comprising implanting one or more implantable living electrodes in
a subject's brain; and placing a compatible stimulator in proximity
to at least one of the one or more implantable living
electrodes.
[0021] In various embodiments method further comprises controlling
the compatible stimulator to actuate at least one of the
implantable living electrodes to activate or excite brain
activity.
[0022] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to activate or excite host
synaptic activity.
[0023] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to activate or excite neuronal
activity.
[0024] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to activate or excite neural
network activity.
[0025] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to inhibit brain activity.
[0026] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to inhibit host synaptic
activity.
[0027] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to inhibit neuronal activity.
[0028] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to inhibit neural network
activity.
[0029] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to modulate host synaptic
activity.
[0030] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to modulate neuronal
activity.
[0031] In various embodiments the method further comprises
controlling the compatible stimulator to actuate at least one of
the implantable living electrodes to modulate neural network
activity.
[0032] In various embodiments at least one of the one or more
implantable living electrodes are implanted in the subject's
central nervous system, peripheral nervous system, cerebral cortex,
striatum, hippocampus, spinal cord, and/or peripheral nerves.
[0033] In various embodiments he method further comprises
selectively exciting or inhibiting cerebral cortical neurons.
[0034] In various embodiments the method further comprises
selectively exciting or inhibiting dopaminergic neurons.
[0035] In various embodiments the method further comprises
selectively exciting or inhibiting dorsal root ganglion
neurons.
[0036] In various embodiments the invention comprises a method
comprising implanting one or more implantable living electrodes in
a subject's brain; and placing a compatible sensor in proximity to
at least one of the one or more implantable living electrodes.
[0037] In various embodiments the method further comprises
reporting activity of excitatory neurons.
[0038] In various embodiments the method further comprises
reporting activity of inhibitory neurons.
[0039] In various embodiments the method further comprises
simultaneously reporting activity of excitatory and inhibitory
neurons.
[0040] In various embodiments the invention comprises a method
comprising implanting one or more implantable living electrodes of
in a subject's brain; and applying stimulus to the implantable
living electrode to selectively excite or inhibit one or more
selected from the group consisting of: glutamatergic neurons,
GABAergic neurons, cholinergic neurons, serotonergic neurons,
peptidergic neurons, neurons from the thalamus, neurons from the
striatum, neurons from the hippocampus, neurons from the substantia
nigra, neurons from the peripheral nervous system, spinal motor
neurons, cerebral cortical neurons, dopaminergic neurons, and
dorsal root ganglion neurons.
[0041] In various embodiments the method further comprises
controlling the quantity of synaptic inputs.
[0042] In another aspect the invention comprises an implantable
living electrode comprising a substantially cylindrical
extracellular matrix core; a hydrogel sheath coaxially surrounding
the substantially cylindrical extracellular matrix core; one or
more selected from the group consisting of: electrodes, optrodes,
magnetic actuators, heating probes, cooling probes, or chemical
applicators positioned between the substantially cylindrical
extracellular matrix core and the hydrogel sheath; and one or more
neurons implanted along or within the substantially cylindrical
extracellular matrix core.
[0043] In another aspect the invention comprises an implantable
living electrode comprising: a substantially cylindrical
extracellular matrix core; and a plurality of aggregated neurons
implanted along or within the substantially cylindrical
extracellular matrix core.
[0044] In various embodiments the plurality of aggregated neurons
comprise dopaminergic neurons.
[0045] In various embodiments the extracellular matrix core
comprises collagen-laminin.
[0046] In various embodiments the aggregated neurons are formed by
centrifugation in pyramidal wells.
[0047] In various embodiments the implantable living electrode
comprises a distinct neuronal body section and a distinct axonal
section.
[0048] In various embodiments the implantable living electrode is a
unidirectional or bidirectional implantable living electrode.
[0049] In various embodiments the extracellular matrix core
comprises collagen-laminin.
[0050] In various embodiments the invention comprises a method of
treating Parkinson's disease in a patient, comprising implanting a
living electrode according to any one of claims into the substantia
nigra of the patient.
[0051] In another aspect, the invention comprises a method of
manufacturing an implantable living electrode comprising providing
an extracellular matrix core; and contacting at least one end of
the extracellular matrix core with a plurality of aggregated
neurons.
[0052] In various embodiments the method further comprises
maintaining the implantable living electrode under conditions that
promote axon growth within or along the extracellular matrix
core.
[0053] In various embodiments the method further comprises
preforming the plurality of aggregated neurons prior to contacting
the at least one extracellular matrix core.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] For a fuller understanding of the nature and desired objects
of the present invention, reference is made to the following
detailed description taken in conjunction with the accompanying
drawing figures wherein like reference characters denote
corresponding parts throughout the several views.
[0055] FIG. 1 is a picture illustrating an implantable living
electrode (designated by arrow number 100), comprising a
cylindrical extracellular matrix core (102) coaxially surrounded by
a hydrogel sheath (104) and implanted with neurons (106a, 106b)
according to an embodiment of the invention. In one embodiment,
certain populations of neural cells (112a) can be excited by one
wavelength of light (114), while application of another wavelength
of light (116) inhibits another population of neural cells (112b).
In another embodiment, two different populations of neural cells
can be excited by two different wavelengths of light. In yet
another embodiment, two different populations of neural cells can
be inhibited by different wavelengths of light.
[0056] FIGS. 2A-2C depict cross-sections of electrodes comprising
an extracellular matrix core (102) surrounded by a hydrogel sheath
according to an embodiment of the invention.
[0057] FIG. 3 depicts methods for use of implantable living
electrodes according to an embodiment of the invention.
[0058] FIGS. 4A-4C illustrate micro-TENN structure and functional
analysis. Micro-TENNs consist of tight clusters of neuronal somata
with dense axonal tracts extending across in the central column.
FIG. 4A illustrates a unidirectional micro-TENN: a single neuron
(MAP-2+) population spanned by tau+ axonal tracts. FIG. 4B
illustrates a bidirectional micro-TENN showing two neuron
populations spanned by beta-tubulin-III+ (Tuj-1+) axon tracts. FIG.
4C illustrates optical stimulation and recording (via genetically
encoded opsins and/or fluorescent Ca.sup.2+ reporters) and
traditional electrophysiology paradigms in vitro. Micro-TENN
activity was assessed by stimulating one population of neurons and
recording the resulting action potentials in the other population
electrophysiologically as well as optically based on
Ca.sup.2+-sensitive reporters.
[0059] FIGS. 5A-5E demonstrate micro-TENN structure, phenotype, and
maturation for multiple architectures varying based on neuronal
somatic distribution and axonal penetration. FIG. 5A shows a
confocal reconstruction of a unidirectional dopaminergic micro-TENN
at 2 weeks in vitro (green: all axons (beta-tubulin-III+); red:
dopaminergic neurons (tyrosine hydroxylase+); blue: all nuclei).
FIG. 5B depicts a phase contrast micrograph of a bidirectional
cerebral cortical neuron micro-TENN. FIGS. 5C and 5D depict dorsal
root ganglia neuron unidirectional micro-TENNs. FIG. 5E depicts a
dense unidirectional cortical neuron micro-TENN with the somatic
region externalized relative to the hydrogel micro-column. Control
of structure, phenotype, maturation/plasticity, and function of
micro-TENN living electrodes has been demonstrated in vitro.
[0060] FIG. 6 illustrates an extra-long micro-TENN. In vitro
immunohistochemistry of a bidirectional micro-TENN demonstrated
robust axonal outgrowth across the neuron populations. Neuronal
cell body and axonal staining show axon outgrowth up to 2 cm.
[0061] FIG. 7 illustrates micro-TENN survival, ingrowth and
integration in vivo. FIG. 7, Panel A illustrates the finding that
at 3 days after delivery into the rat brain, micro-TENN neurons
survived and maintained their axonal architecture within the
hydrogel tube. FIG. 7, Panels B-D illustrate the finding that at 28
days post-implant, micro-TENN neurons survived and integrated with
the brain. FIG. 7, Panel B illustrates transplanted neurons
extended neurites into host tissue. FIG. 7, Panels C and D
illustrate a magnification of a region from FIG. 7, Panel B showing
putative dendritic spines along ingrowing neurites with (FIG. 7,
Panel D) synapsin-positive puncta in immediate proximity (circles)
to neurites, suggesting synaptic integration. Scale bars are as
follows: Panel A=50 .mu.m, Panel B=40 .mu.m, Panel C and D=20
.mu.m.
[0062] FIG. 8 describes theoretical advantages of axon-based
"living electrodes" for neuromodulation: mechanisms and specificity
of neuronal stimulation for "living electrodes" (left) versus
conventional electrodes (center) and optrodes (right). Living
electrodes provide engineered axonal tracts with a controlled
cytoarchitecture and fully differentiated neurons that if
pretransfected in vitro, constrain the spatial extent of
transfected cells while the 3D attrition issues from delivery of
cell suspensions, both advantages for clinical deployment. Living
electrodes could offer high specificity, as the constructs can be
designed to synapse with specific neuronal subtypes in a given
anatomical region (as shown by living electrode axons synapsing
with neurons of a certain color) as opposed to conventional
electrodes that inherently stimulate or record from a relatively
large 3D volume around the electrode (as shown by large aura of
stimulation affecting many layers and neurons). While micro-fiber
optrodes can achieve a high level of specificity, the in vivo
delivery of opsins generally relies on injection of virus that may
diffuse and affect non-target regions (spread of optogenetic
transduction is illustrated by lightly shaded neurons straying from
layer V into layer VI). Also, optical methods may have a limited
benefit due to tissue absorption of light. Finally, living
electrodes provide a soft pathway to route signals to/from deep
brain structures compared to rigid materials used in
electrodes/optrodes, thus minimizing signal issues due to
mechanical mismatch/micromotion and glial scarring.
[0063] FIG. 9 describes the concepts of living electrode target
specificity and synaptic integration. Living electrodes offer high
specificity, as the constructs can be designed to synapse with
specific neuronal subtypes, as demonstrated by micro-TENN axons
synapsing with only circle neurons, not star neurons in the
conceptual rendition.
[0064] FIG. 10 illustrates a recording and stimulation test
paradigm for the use of living electrodes in the cerebral
cortex.
[0065] FIG. 11 illustrates the living electrode concept.
Micro-TENNs are used for corticofugal recording (right panel) or
corticopetal stimulation (left panel) interface with neural
circuits. Micro-TENNs act as a living electrode by penetrating the
brain to a prescribed location with the other end at the brain
surface. Deep micro-TENN neurons/axons within the brain are then
able to synaptically integrate with local host neurons while axonal
projections spanning the construct serve as a functional relay to
and from the cortical surface, where information is exchanged using
optical and/or electrical interfaces. The output (corticofugal)
paradigm enables projection of a facsimile of deep activity to the
cortical surface via local synaptic integration. The input
(corticopetal) paradigm permits controlled excitation or inhibition
of specific neural circuitry.
[0066] FIG. 12 depicts examples of living electrode structure and
applications in the brain (purple: tractography of general axonal
tracts in the brain). Micro-tissue engineered neural networks
(TENNs), initially developed to physically reconstruct lost
long-distance axonal connections in the brain, are miniature
preformed constructs grown in vitro that consist of discrete
neuronal population(s) spanned by long axonal tracts. Micro-TENNs
may consist of uni- or bidirectional axonal tracts.
Cortical-thalamic micro-TENNs can be applied as living electrodes
to record or modulate sensory-motor information in the cortex or
thalamus, vulnerable in brain trauma and stroke. Dopaminergic (DA)
micro-TENNs can be used to provide/restore dopaminergic inputs to
the striatum, important to mitigate motor symptom in Parkinson's
disease. Cortical-hippocampal micro-TENNs can be used to modulate
or encode information exchange between the cortex and hippocampus,
which is crucial for learning and memory formation.
[0067] FIG. 13 provides a perspective view of a macro-TENN
electrode according to an embodiment of the invention.
[0068] FIG. 14 depicts neural growth within a macro-TENN structure
according to embodiments of the invention. Cell nuclei are stained
blue with DAPI. Axons are stained green with beta-tubulin-III
(Tuj1). FIG. 14, Panels A-C provide confocal slices along planes
illustrated in FIG. 14, Panel D. FIG. 14, Panel A is a bottom-most
confocal slice showing cell nuclei resting at bottom of macro-TENN
construct. FIG. 14, Panel B is a confocal slice just above the
slice shown in FIG. 14, Panel A, showing axons (green) growing
along the inner edge of the macro-TENN construct. FIG. 14, Panel C
is a confocal slice just above the slice shown in FIG. 14, Panel B,
showing axons (green) growing only at the edges of the interface
only at the north or south side of the tube. FIG. 14, Panel D is a
schematic looking down length of macro-TENN to diagrammatically
describe confocal slices shown in FIG. 14, Panels A-C. FIG. 14,
Panel E depicts a reconstruction of confocal images in FIG. 14,
Panels A-C as shown in cross section, similar to the viewpoint of
FIG. 14, Panel D.
[0069] FIGS. 15A-15C depict deployment of electrodes in a
defascicularization device according to an embodiment of the
invention. FIG. 15A provides a view looking down a nerve. FIG. 15B
provides a view looking along a length of a nerve. FIG. 15C depicts
a tapered device where ends match nerve size, but interior of
construct is larger to increase surface area and fascicular
separation. (Electrodes are not shown in FIG. 15C for clarity).
[0070] FIG. 16 provides phase contrast micrographs of micro-TENNs
built using (Panels A and B) neuronal suspension delivery versus
(Panels C-E) forced neuronal aggregate delivery. Panels A and B
show an example of a micro-TENN with neuronal somata infiltration
throughout the micro-column interior, a consequence of imperfect
extracellular-matrix (ECM) continuity in the core. In cases where
this occurs, this results in a deviation from the ideal micro-TENN
cytoarchitecture. In Panels C-E, in contrast, when precisely formed
neuronal aggregates are used to seed the micro-columns, the
idealized distribution of somatic (Panel D) and axonal (Panel E)
zones is consistently maintained.
[0071] FIG. 17 shows phase contrast and confocal micrographs of
neuronal somatic and axonal distribution in forced neuronal
aggregate micro-TENNs. As seen in Panels A and B, neuronal
aggregates can be precisely seeded at an end of the micro-column.
In Panel C, over several days in vitro, dense axonal outgrowth can
be observed projecting from the neurons in the aggregate. Panel D
provides a confocal micrograph following immunocytochemistry to
label these aggregate micro-TENNs using antibodies recognizing all
axons (beta-tubulin III; red) and all cell nuclei (Hoechst; blue),
and synapses (synapsin; green). The hydrogel comprising the
micro-column is non-specifically labeled as purple. This
demonstrates defined, distinct somatic (Hoechst+) and axonal
(beta-tubulin III+) regions, whereas the synapsin+ puncta
demonstrates functional maturation and electrochemical activity in
the micro-TENNs. The neuronal aggregate seeding methodology
consistently resulted in the formation of uni- or bi-directional
micro-TENNs of the idealized cytoarchitecture consisting of a
defined zone with neuronal somata as aggregates at one or both ends
of the micro-column and a defined zone with axonal projections
running longitudinally to span the central portion of the
micro-column.
[0072] FIG. 18 depicts a longitudinal cross-section of a living
electrode according to an embodiment of the invention.
[0073] FIG. 19 depicts long-projecting unidirectional axonal-based
living electrodes for neuromodulation: Panel A depicts confocal
reconstruction of a cerebral cortical neuron living electrode at 28
DIV, immunolabeled for axons (.beta.-tubulin-III; red) and neuronal
somata/dendrites (MAP-2; green), with nuclear counterstain
(Hoechst; blue). Insets of the aggregate (a') and axonal (a'')
regions are outlined and shown to the right. Scale bars: 100 .mu.m.
Panel B depicts confocal reconstruction of a ventral mesencephalic
(dopaminergic) living electrode at 28 DIV, immunolabeled for axons
(.beta.-tubulin-III; green) and tyrosine hydroxylase (dopaminergic
neurons/axons; red), with nuclear counterstain (Hoechst; blue).
Insets of the aggregate (b') and axonal (b'') regions are outlined
and shown to the left. Scale bars: 250 .mu.m.
[0074] FIG. 20 depicts potential applications of axon-based living
electrodes: custom engineered living electrodes consisting of a
phenotypically-controlled population of neurons extending long
axonal tracts through a biocompatible micro-column may be
stereotactically transplanted to span various regions to treat
particular disease processes. In panel A, axons projecting from
dopaminergic living electrodes will form synapses within local
striatal architecture, and, due to in vitro functionalization with
channelorhodopsins, may release dopamine upon optical stimulation
of the perikaryal segment at the brain surface. This mimics the
substantia nigra pars compacta input to the striatum in a manner
that can be externally controlled. In panel B, axons from
glutamatergic living electrodes may preferentially synapse on to
layer IV neurons within primary sensory cortex to convey illusory
haptic feedback via surface optical stimulation to achieve
closed-loop control of neuromotor prosthetics in patients with
paralysis. In panel C, axons from GABAergic living electrodes could
be implanted to appose seizure foci such that optical stimulation
would cause net suppression of seizure activity in patients with
lesional epilepsy.
[0075] FIG. 21 panels A-C diagram possibilities for exploiting
"biological multiplexing" in living electrodes. More sophisticated
living electrodes may be developed to further exploit so-called
biological multiplexing. By fabricating the constructs in vitro
using microprinting and micropatterning techniques, specific
synaptic architectures can be achieved to yield certain
fine-grained signal manipulations linking the construct to the
brain. Panel a shows that, in the simplest form, "channel select"
bundles of axons can transmit signals to select which other bundles
transmit signals into the brain, and which are silenced. In panel
b, multiple channels that converge on to one final common output
can likewise be toggled by the "channel select" in a biological
instantiation that most resembles the kind of multiplexing used in
telecommunications. In panel c likewise, a single input channel can
be selected or diverted to one or more parallel outputs to
"demultiplex" that signal. Panel d shows the potential for
time-division "biological multiplexing" in living electrodes.
Living electrodes may exploit delay lines emanating from a single
"clock" circuit formed by a cluster of neurons linked by gap
junctions (coupled damped oscillators) and micropatterned
inhibitory and excitatory connections. Thus, multiple parallel
input channels can be multiplexed serially with each clock cycle to
a single target output neuron that in turn links to the brain. The
rate of the clock (and hence the multiplexing sampling duration)
can be altered by driving the clock circuit directly.
[0076] FIG. 22 depicts a diffusion tensor imaging representation of
the long-distance axonal tracts that connect discrete populations
of neurons in the human brain. This conceptual rendition shows how
a unidirectional micro-TENN--consisting of a population of
dopaminergic neurons extending long, aligned processes--can be used
to recreate the nigrostriatal pathway that degenerates in
Parkinson's disease. Axons in the substantia nigra are expected to
functionally integrate with the transplanted dopaminergic neurons
in the micro-TENN, while the transplanted dopaminergic axons are
expected to functionally integrate with neurons in the striatum.
After receiving appropriate inputs from the substantia nigra, the
transplanted neurons will release dopamine in the striatum, thereby
recreating the circuitry lost in Parkinson's disease.
[0077] FIG. 23 depicts improved micro-TENN cytoarchitecture using
forced aggregation method as applied in compositions and methods
that employ these embodiments. Phase contrast and confocal
reconstructions of micro-TENNs plated with primary dopaminergic
neurons at 14 DIV. Panel A depicts representative micro-TENN plated
with dissociated neurons labeled via immunocytochemistry to denote
neurons/axons (.beta.-tubulin III) and cell nuclei (Hoechst).
Dissociated micro-TENNs did not demonstrate the desired
cytoarchitecture as they showed cell infiltration throughout the
entire length of the inner core. (B,C) Phase contrast images
depicting micro-TENNs plated with engineered dopaminergic neuron
aggregates. Based upon plating technique, aggregates either (B)
attached directly outside the agarose micro-column, or (C) inside
the inner core. Higher magnification images from demonstrative
regions in (B,C) show that while the (D1,D3) cell body regions
differed between the two aggregate plating techniques, their
(D2,D4) axonal regions were similar. (E) Representative aggregate
micro-TENN labeled via immunocytochemistry to denote all
neurons/axons (.beta.-tubulin III) and dopaminergic neurons/axons
(TH), with cell nuclei counterstain (Hoechst). Aggregate
micro-TENNs demonstrated the ideal cytoarchitecture, with (E1)
discrete cell body regions and (E2,E3) axonal regions. (F) A higher
magnification reconstruction from a demonstrative region in (E)
depicts the aggregated cell bodies. (G) Micro-TENNs generated using
aggregates demonstrated a greater extent of axonal outgrowth than
micro-TENNs plated with dissociated neurons (n=13 micro-TENNs each
group; Mann-Whitney test, p<0.0001). Data are presented as
mean.+-.standard deviation. Scale bar (A)=250 .mu.m. Scale bar
(B,C)=500 .mu.m. Scale bar (D1)=200 .mu.m. Scale bar (D2-D4)=100
.mu.m. Scale bar (E)=250 .mu.m. Scale bar (F)=50 .mu.m.
[0078] FIG. 24 depicts the effect of extracellular matrix on axonal
outgrowth within micro-TENNs. Representative confocal
reconstructions of dopaminergic micro-TENNs plated with different
ECM cores. At 14 DIV, all micro-TENNs were labeled via
immunocytochemistry to denote all neurons/axons (.beta.-tubulin
III) and dopaminergic neurons/axons (TH), with nuclear counterstain
(Hoechst). The type of ECM strongly influenced axonal outgrowth,
with (A) collagen I (n=12 micro-TENNs) and a (C) collagen I and
laminin cocktail (n=12) supporting the longest axonal outgrowth.
Micro-TENNs with (B) empty cores (n=9) or (D) crosslinked collagen
cores (n=11) demonstrated significantly less outgrowth. (a-d)
Higher magnification reconstructions from demonstrative regions in
(A-D) show similar expression of TH across groups. Panel E is a
graph showing that a one way ANOVA (p<0.0001) followed by a
post-hoc Tukey's test determined that collagen I and collagen
I-laminin cocktail cores were statistically equal (p=0.8590), and
that they each supported axonal outgrowth that was statistically
longer then outgrowth in empty (p<0.0001), laminin-coated
(p<0.0001), or crosslinked collagen (p<0.0001) cores (*
denotes significance). Panel F is a graph showing that, as
determined by a Mann-Whitney test, the lengths of TH+ axons as a
percentage of total axonal length were statistically equivalent
between the collagen I (n=12) and collagen I and laminin (n=12)
inner cores (p=0.9723). Data are presented as mean.+-.standard
deviation. Scale bar (A,C,D)=500 .mu.m. Scale Bar (B)=250 .mu.m.
Scale bar (E-H)=50 .mu.m.
[0079] FIG. 25 depicts long-projecting dopaminergic micro-TENNs.
Panels A-F are confocal reconstructions of a representative
micro-TENN plated with a dopaminergic aggregate and collagen I
inner core at 28 DIV. Micro-TENN labeled via immunocytochemistry to
denote all neurons/axons (.beta.-tubulin III) and dopaminergic
neurons/axons (TH), with nuclear counterstain (Hoechst). In panels
A-D, long-term dopaminergic micro-TENNs showed robust survival and
axonal extension over 28 DIV. Panels E-F are higher magnification
reconstructions from demonstrative regions in panel C and show
healthy TH+ neurons and axons, with apparent axonal varicosities
suggesting sites of dopamine release. Panel G shows micro-TENN
length measurements taken at 28 DIV (n=7 micro-TENNs) demonstrated
TH+ axons measuring 6046.+-.670 .mu.m, and a total TH+ length of
7264.+-.672 .mu.m with the inclusion of the dopaminergic aggregate.
Importantly, these lengths are more than sufficient to span the
nigrostriatal pathway in rats. Data are presented as
mean.+-.standard deviation. Scale bar (A-D)=250 .mu.m. Scale bar
(E-F)=50 .mu.m.
[0080] FIG. 26 depicts synapse formation between micro-TENN
dopaminergic axons and striatal neurons in vitro. Panel A depicts
representative confocal reconstruction at 14 DIV of a dopaminergic
micro-TENN plated with an aggregated striatal end target. The
micro-TENN was labeled via immunocytochemistry to denote
dopaminergic neurons/axons (TH), striatal (medium spiny) neurons
(DARPP-32), and synapses (synapsin), with nuclear counterstain
(Hoechst). Panels B-E show higher magnification reconstructions
from demonstrative regions in (A) depict the (B) dopaminergic
neuron aggregate, (C) robust, aligned TH+ axons, and (D) neurite
outgrowth from the striatal neuron population. (E) A high degree of
synapsin labeling along the trajectory of TH+ axons suggests that
dopaminergic axons formed synapses with the striatal neurons. Panel
F is a graph showing that micro-TENNs containing striatal end
targets did not result in statistically longer axonal outgrowth
when compared to unidirectional dopaminergic micro-TENNs with no
end target (n=9) micro-TENNs each group; Mann-Whitney test,
p=0.9182). Data are presented as mean.+-.standard deviation. In
panels G-H synapsin+ puncta can be seen decorating putative
dendrites projecting from striatal neurons shown with dopaminergic
axonal varicosities, further suggesting synaptic integration. Scale
bar (A)=250 .mu.m. Scale bar (B-E)=50 .mu.m. Scale bar (G-H)=20
.mu.m.
[0081] FIG. 27 depicts micro-TENN neuronal survival and maintenance
of axonal cytoarchitecture in vivo. Panel A depicts micro-TENN
implant trajectory and dimensions drawn to scale (adapted from
Gardoni F, Bellone C. 2015, Modulation of the glutamatergic
transmission by Dopamine: a focus on Parkinson, Huntington and
Addiction diseases, Frontiers in cellular neuroscience, 9: 25.).
Panel B depicts micro-TENN orientation (not to scale). Panel C
depicts a representative sagittal section at 1 week post-implant,
showing a longitudinal view of a dopaminergic micro-TENN with all
neurons expressing GFP on the synapsin promoter and labeled via
immunohistochemistry to denote dopaminergic neurons/axons (TH).
This demonstrates that micro-TENN neurons survived and the
longitudinally aligned cytoarchitecture was maintained. Panel D
depicts, at 1 month post-implant, a representative oblique section
providing a cross-sectional view of a GFP+ dopaminergic micro-TENN
labeled via immunohistochemistry to denote dopaminergic
neurons/axons (TH) and all neurons/axons (.beta.-tubulin III). This
demonstrates healthy transplanted neurons/axons with robust
dopaminergic axonal projections at 1 month in vivo. Scale bar
(A)=20 .mu.m. Scale bar (B)=50 .mu.m.
[0082] FIG. 28 depicts micro-TENNs as living electrodes for a
Neuroprosthetic Interface. Panel a is a conceptual schematic of
micro-TENNs. Panel A depicts a micro-TENN three-dimensional
construct up to several millimeters in length consisting of
hydrogel cylinder encasing an extracellular matrix core of collagen
and laminin. Current micro-TENNs have a 300-400 micron outer
diameter with a micron inner diameter, but may be made at any size.
Neuronal populations are placed at one or both ends of the
cylinder, with axonal tracts penetrating the ECM and spanning the
cylinder length. Panel B depicts that neurons from unidirectional
micro-TENN neurons may synapse with host neurons, allowing for the
transmission of signal inputs to targeted cortical regions. Panel C
depicts that host neurons may synapse and integrate with
bidirectional micro-TENNs, allowing for the transmission of signal
outputs from targeted cortical regions to the dorsal neuronal
population. Panel D depicts the delivery and integration of
micro-TENNs in vivo as "living electrodes". Micro-TENNs are
preformed in vitro; upon implantation in the brain, these living
microconduits may serve as input/output channels for sensorimotor
information. For inputs, an LED array (1) optically stimulates a
unidirectional micro-TENN with channelrhodopsin-positive neurons
(2), which synapse with host Layer IV neurons (3). For outputs,
host neurons from Layer V (4) synapse with the neurons of a
bidirectional micro-TENN (5); neuronal activity is recorded by a
microelectrode array (6).
[0083] FIG. 29 depicts aggregate fabrication of micro-TENNs,
comparing Traditional vs. Aggregate Cortical Micro-TENNs. Living
electrodes are fabricated in two steps: formation of the agarose
microcolumn, and cortical neuronal aggregation. Panel (a) depicts
agarose microcolumn formation. 1: A custom-designed, reusable
acrylic mold is used to generate agarose microcolumns with a
specified inner and outer diameter. 2: Top view of the assembled
mold. Dashed lines indicate the outer (middle) and inner diameters
(top; bottom). 3: Needles of the specified inner diameter are
inserted into the mold. 4: Molten agarose is introduced into the
mold and allowed to cool. 5: The needles are removed, the mold
disassembled and the microcolumns removed. Panel (b) depicts
cortical neuronal aggregation. 1: Square pyramidal wells are cast
in PDMS from a 3D-printed positive mold. 2: Image of the PDMS
pyramidal wells. 3: Single-cell suspensions of rodent embryonic
neurons are introduced into the wells and centrifuged into neuronal
aggregates. 4: Phase image of an aggregate 24 hours after plating.
5: Confocal reconstruction of aggregate at 72 hours, stained for
live and dead neurons. Panel (c) depicts agarose microcolumns being
filled with an extracellular matrix (1 mg/ml laminin and collagen;
pH 7.2-7.4). Neuronal aggregates are then placed at one or both
ends of the microcolumn, and allowed to grow in vitro. All scale
bars: 100 .mu.m. (d, e) Micro-TENNs in prior work were fabricated
with dissociated neurons. Dissociated micro-TENNs exhibited axonal
growth and network formation over several days in vitro, but
control and reproducibility of micro-TENN architecture was
inherently limited. (f, g, h) With the aggregate method, one or two
neuronal aggregates (for unidirectional or bidirectional
micro-TENNs, respectively) are used to seed the microcolumns. Shown
is a representative bidirectional micro-TENN after 3 days in vitro.
Aggregate micro-TENNs exhibit robust axonal growth and more
controllable architecture. Specifically, aggregation results in
reliably discrete regions populated either by cell bodies (g) or
neuritic projections (h).
[0084] FIG. 30 depicts axonal growth in aggregate micro-TENNs over
time. Both unidirectional (a) and bidirectional (b) micro-TENNs
displayed robust axonal outgrowth along the ECM core over the first
few DIV. Unidirectional micro-TENNs, lacking a distal target,
exhibited axonal retraction after about 7-8 DIV. Conversely,
bidirectional micro-TENN axons crossed the length of the
microcolumn (2-2.5 mm), synapsing with the opposing aggregate by 5
DIV. Representative micro-TENNs shown at 1, 3, 5, and 8 DIV. (c)
Longer bidirectional micro-TENNs (5 mm) took longer to develop, but
still showed robust growth. Representative micro-TENN shown at 1,
3, and 5 DIV. (d) Quantified growth rates for 2 mm unidirectional,
2 mm bidirectional, 5 mm bidirectional, and 2 mm
dissociated/traditional micro-TENNs at 1, 3, 5, 8, and 10 DIV.
Growth rates were quantified by identifying the longest neurite
from an aggregate in phase microscopy images (10.times.
magnification) at the listed timepoints. Sample sizes: n=6
(Unidirectional--2 mm), 9 (Bidirectional--2 mm), 7
(Bidirectional--5 mm), and 7 (Dissociated--2 mm). Error bars denote
s.e.m. Scale bars: 100 .mu.m.
[0085] FIG. 31 depicts aggregate-specific growth with fluorescent
labeling. Confocal reconstructions of bidirectional micro-TENNs
labeled with GFP and mCherry to observe axonal growth from each
aggregate in vitro. (a, b, c) A micro-TENN at 1 (a), 3 (b), and 7
(c) DIV. By 3 DIV there is putative axon-axon contact from each
aggregate, followed by more robust outgrowth by 5 DIV. (d) Another
micro-TENN at 6 DIV, with insets showing axons from each aggregate
growing along each other (e) and axons from one aggregate making
contact with the opposite population (f). Scale bars: 500 .mu.m (a,
d); 100 .mu.m (e, f).
[0086] FIG. 32 illustrates micro-TENN viability. Viability for
unidirectional and bidirectional micro-TENNs and age-matched
two-dimensional controls was quantified via live-dead
(calcein-AM/ethidium homodimer) staining at 10 and 28 DIV. (a, b,
c) Representative confocal live-dead images showing live cells,
dead cells, and an overlay of a unidirectional micro-TENN at 10
DIV, with outlined insets below. (d, e, f) Representative confocal
live-dead image of a bidirectional micro-TENN at 28 DIV, with
outlined insets below. All scale bars: 100 .mu.m. (g) Graph denotes
the average proportion of live cell body area to total (live+dead)
cell area for each experimental group and timepoint. Two-way ANOVA
and post-hoc analysis revealed several statistically relevant
pairwise differences (*=p<0.05; **=p<0.01; ***=p<0.001).
Error bars denote s.e.m. Sample sizes: n=4 and 4 (unidirectional);
7 and 4 (bidirectional); 9 and 5 (controls) for 10 and 28 DIV,
respectively. (h) A live-dead confocal image of a micro-TENN
stained at 40 DIV. Scale bar: 100 .mu.m.
[0087] FIG. 33 depicts micro-TENN architecture and synaptogenesis.
Confocal reconstructions of representative bidirectional
micro-TENNs at 4 DIV (a), 10 DIV (b), and 28 DIV (d); immunolabeled
for cell nuclei (Hoechst), axons (Tuj-1), and synapses (synapsin).
Insets in (b) and (d) refer to callout boxes (c) and (e) showing
zoom-ins of synapses, axonal networks, and the overlay of the two.
(f) Confocal reconstruction of a representative unidirectional
micro-TENN at 28 DIV. Scale bars: 200 .mu.m.
[0088] FIG. 34 depicts corticothalamic micro-TENN implantation.
Cross-sections of brain one month following GFP-positive micro-TENN
implantation. Implantation here mimics the "living electrode"
application (FIG. 28), with a large dorsal population of neurons
extending axons ventrally into the brain. Brains were sectioned and
stained to identify micro-TENN neurons (GFP), dendrites and somata
(MAP-2), and axons (Tuj-1). (a) Dorsal view of micro-TENN, with
insets referring to callout boxes showing the aggregate (b) and
lumen of the micro-TENN, containing axons (c). Similarly,
cross-sections of another micro-TENN implantation reveal the
aggregate (d) and axons within the lumen (e) of the micro-column.
Scale bars: 200 .mu.m (a); 100 .mu.m (b); 50 .mu.m (d); 25 .mu.m
(c, e).
DEFINITIONS
[0089] The instant invention is most clearly understood with
reference to the following definitions.
[0090] As used herein, the singular form "a," "an," and "the"
include plural references unless the context clearly dictates
otherwise.
[0091] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from context, all numerical values
provided herein are modified by the term about.
[0092] As used in the specification and claims, the terms
"comprises," "comprising," "containing," "having," and the like can
have the meaning ascribed to them in U.S. patent law and can mean
"includes," "including," and the like.
[0093] As used herein, the term "cylinder" or "cylindrical"
includes a surface consisting of each of the straight lines that
are parallel to a given straight line and pass through a given
curve. In some embodiments, cylinders have an annular profile. In
other embodiments, the cylinder has a cross-section selected from
the group consisting of: a square, a rectangle, a triangle, an
oval, a polygon, a parallelogram, a rhombus, an annulus, a
crescent, a semicircle, an ellipse, a super ellipse, a deltoid, and
the like. In other embodiments, the cylinder is the starting point
of a more complex three-dimensional structure that can include, for
example, complex involutions, spirals, branching patterns, multiple
tubular conduits, and any number of geometries that can be
implemented in computer-aided design, 3-D printing, and/or in
directed evolutionary approaches of secretory organisms (e.g.,
coral), including of various fractal orders.
[0094] As used herein, the term "living scaffolds" refers to
biological scaffolds comprised of living neural cells in a
preformed, often anisotropic, three-dimensional (3-D) architecture.
Living scaffolds can physically integrate with existing host
tissue. Living scaffolds may facilitate targeted neural cell
migration and axonal pathfinding by mimicking key developmental
mechanisms. Living scaffolds can act based on the simultaneous
presentation of structural and soluble cues, and/or
electrophysiological, ionic, or neurotransmitter based
signaling.
[0095] As used herein, the term "living electrode" refers to a
living construct including neural cells, generally but not
exclusively neurons, with a defined architecture generally
comprised of discrete somatic region(s) with protruding neurite
tracts (axonal or dendritic) designed to probe or modulate the
nervous system.
[0096] Unless specifically stated or obvious from context, the term
"or," as used herein, is understood to be inclusive.
[0097] As used herein, "synapse" refers to a junction between a
neuron and another cell, across which chemical communication
flows.
[0098] As used herein, "synapsed" refers to a neuron that has
formed one or more synapses with one or more cells, such as another
neuron or a muscle cell.
[0099] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the
context clearly dictates otherwise).
DETAILED DESCRIPTION OF THE INVENTION
[0100] Aspects of the invention utilize advanced micro-tissue
engineering techniques to create the first biological living
electrodes for chronic BMI and/or neuromodulation. Novel
micro-Tissue Engineered Neural Networks (micro-TENNs) serve as the
living electrodes, which are composed of discrete population(s) of
neurons connected by long axonal tracts, generally contained within
miniature tubular hydrogels. These living micron-scale constructs
are able to penetrate the brain to a prescribed depth for
integration with local neurons/axons, with the latter portion
remaining externalized on the brain surface where functional
information is inputted/controlled and/or outputted/gathered using
a next-generation optical and electrical interface.
[0101] Micro-TENN neurons survive, integrate with local host
neurons, and maintain their axonal architecture. These features are
exploited to advance living electrodes as a functional relay to and
from deep regions of the brain. In this radical paradigm, only the
biological component of these constructs penetrates the brain, thus
attenuating a chronic foreign body response. Moreover, through
custom cell and tissue engineering techniques, the specific host
neuronal subtypes with which the micro-TENN neurons form synapses
may be influenced, thereby adding a level of specificity in local
stimulation and recoding not currently attainable with conventional
microelectrodes.
[0102] Electrophysiological, optogenetic, and advanced microscopy
techniques reveal evidence of micro-TENN synaptic integration with
brain neural networks and cross-communication with micro-TENN
neurons on the cortical surface in rats. This versatile platform
technology will read out local brain activity and provide input to
affect neural activity and function, thereby providing the first
demonstration of tissue engineered living electrodes to
functionally integrate into native neural networks and to serve as
a conduit for bidirectional stimulation and recording. This
potentially transformative technology at the interface of
neuroscience and engineering lays the foundation for preformed
implantable neural networks as a viable alternative to conventional
electrodes.
[0103] Referring now to FIG. 1, one embodiment of the invention
provides an implantable living electrode (100). The electrode can
include a substantially cylindrical extracellular matrix core (102)
and a hydrogel sheath (104) coaxially surrounding the substantially
cylindrical extracellular matrix core (102). One or more neurons
(106a, 106b) can be implanted along or within the substantially
cylindrical extracellular matrix core (102). The one or more
neurons (106a, 106b) can include one or more optogenetic or
magnetogenetic neurons proximal to a first end (108) of the
implantable living electrode.
[0104] The extracellular matrix core (102) can comprise proteins,
nucleic acids, small molecules, hormones, growth factors, and the
like that enhance axonal growth, promote survival, reduce host
inflammation, or promote integration of the composition into host
tissue. Exemplary proteins include collagen, laminin, fibrin, and
fibronectin. The extracellular matrix core (102) can additionally
or alternatively include hyaluronic acid. The extracellular matrix
core can be unilayer (a single material cured around living axons),
bilayer (as depicted herein), or tri-layer.
[0105] The hydrogel sheath (104) can provide mechanical support for
the extracellular matrix core (102) sufficient to protect the
extracellular matrix core (102) from bending, buckling, collapsing,
and the like before, during, and/or after implantation. For
example, the hydrogel sheath (104) can have sufficient mechanical
rigidity to allow for loading within a needle and advancement from
the needle within a subject's tissue. The hydrogel sheath (104)
can, in some embodiments, be impermeable or substantially
impermeable to axonal projections in order to guide and confine
axonal growth within and/or substantially parallel to a central
axis of the hydrogel sheath (104). In some embodiments, the
hydrogel sheath (104) dissolves, degrades, and/or is absorbed after
a period of time (e.g., one month).
[0106] In some embodiments, both the extracellular matrix core 102
and the hydrogel sheath (104) are fabricated from hydrogels,
although the hydrogels can have different mechanical and/or
chemical properties.
[0107] Additionally, the sheath (104) can be omitted and the core
(102) can be fabricated from a hydrogel and/or an extracellular
matrix embedded with cells and axons and having sufficient strength
to resist bending, buckling, collapsing, and the like before,
during, and/or after implantation.
[0108] The one or more neurons (106a, 106b) can be implanted in
various locations within the implantable living electrode (100). In
one embodiment, the neurons (106) are implanted at one end (e.g.,
first end 108 or second end 110) and grow to the other end of the
electrode after implantation. In another embodiment, the neurons
are implanted in the center of the implantable living electrode
(100) and grow axially in both directions. In still other
embodiments, the neurons are placed on an end surface of the
electrode (100) and grow into and through the extracellular matrix
core (102). In still other embodiments, the neurons (106) are mixed
or placed throughout the extracellular matrix core (102) prior to
implantation and form axonal projections that connect with adjacent
neurons to facilitate communication across the electrode (100). In
one embodiment, the neurons lie at an interface between the
extracellular matrix core (102) and the hydrogel sheath (104). In
other embodiments, glial cells, or other neural or non-neural
phenotypes, are implanted to facilitate growth and phenotypic
differentiation of neurons.
[0109] The neurons useful for the compositions and methods provided
herein include all neuronal subtypes, including but not limited to
PNS motor or sensory, CNS, and stem cells (e.g., induced
pluripotent stem cells, embryonic stem cells, and the like)
differentiated into a neuronal phenotype. In one embodiment of the
present invention, neurons are derived from any cell that is a
neuronal cell (e.g., cortical neurons, dorsal root ganglion neurons
or sympathetic ganglion neurons) or is capable of differentiating
into a neuronal cell (e.g., stem cell). The neurons may be
autologous, allogenic, or xenogenic with reference to the
subject.
[0110] In certain embodiments, the neurons are peripheral or spinal
cord neurons including dorsal root ganglion neurons or motor
neurons. In certain embodiments, the neurons are from brain,
including but not limited to, neurons from the cerebral cortex,
thalamus, hippocampus, striatum, substantia nigra and cerebellum.
In certain embodiments, primary cerebral cortical neurons include
but are not limited to neurons from layers I, II, III, IV, V,
and/or VI of the cortex (separately or in any combination thereof),
neurons from the visual cortex, neurons from the motor cortex,
neurons from the sensory cortex, and neurons from the entorhinal
cortex. The neurons may be excitatory or inhibitory neurons. The
neurons may be glutamatergic, dopaminergic, GABAergic,
serotonergic, cholinergic, or any other type of neuron as
classified based upon its primary neurotransmitter.
[0111] Neurons useful in the invention may be derived from cell
lines or other mammalian sources, such as donors or volunteers. In
one embodiment, the neurons are human neurons. In one embodiment,
the neurons are non-human mammalian neurons, including neurons
obtained from a mouse, rat, dog, cat, pig, sheep, horse, or
non-human primate. In one embodiment, the neurons are cortical
neurons, hippocampal, neurons, dorsal root ganglion neurons or
sympathetic ganglion neurons. In another embodiment, neurons are
derived from immortalized cell lines that are induced to become
neuron-like (e.g., NT2, PC12). In one embodiment, the neurons are
neurons derived from a cadaver. In another embodiment, the neurons
are neurons derived from patients who have undergone
ganglionectomies, olfactory epithelium biopsy, temporal lobectomy,
tumor margin resection, peripheral nerve biopsy, brain biopsy,
ventricular shunt implantation with biopsy, or other clinical
procedure. Furthermore, the neurons may be singular, integrated
neurons or a plurality of integrated neurons (i.e., an integrated
nerve bundle).
[0112] In certain embodiments, the glial cells (e.g., astrocytes
that may extend processes to modulate host synaptic, axonal,
dendritic, somatic, and/or host network activity) are incorporated
in addition or as an alternative to neurons 106. These can be brain
or spinal cord derived astrocytes. In certain embodiments, the
cells are olfactory ensheathing cells, oligodendrocytes, Schwann
cells, endothelial cells, or myocytes/myoblasts.
[0113] The number or density of the cells positioned at either end
of the construct is dependent upon the type of neuron being used
and the eventual use of the construct. For example, in certain
embodiments, 1, 100, 1,000, 10,000, 1,000,000, 100,000,000, or more
cells are positioned at an end of the construct.
[0114] In certain embodiments, the neurons are cultured in vitro or
ex vivo. Culture of the neurons can be performed under suitable
conditions to promote the growth of axons through the core of the
construct. Those conditions include, without limitation, the
appropriate temperature and/or pressure, electrical and/or
mechanical activity, force, the appropriate amounts of O.sub.2
and/or CO.sub.2, an appropriate amount of humidity, and sterile or
near-sterile conditions. For example, the cells may require a
nutritional supplement (e.g., nutrients and/or a carbon source such
as glucose), exogenous hormones or growth factors, differentiation
factors, and/or a particular pH. Exemplary cell culture media that
can support the growth and survival of the neuron includes, but is
not limited to, NEUROBASAL.RTM. media, NEUROBASAL.RTM. A media,
Dulbecco's Modified Eagle Medium (DMEM), and Minimum Essential
Medium (MEM). In certain embodiments, the culture medium is
supplemented with B-27.RTM. supplements. In certain embodiments,
the culture medium may contain fetal bovine serum or serum from
another species at a concentration of at least 1% to about 30%, or
about 5% to about 15%, or about 10%. In one embodiment, the culture
medium comprises NEUROBASAL.RTM. supplemented with about 2% B-27
and about 500 .mu.M L-glutamine.
[0115] As depicted in FIG. 1, a single electrode 100 can support
multiplexing of a plurality of different types of neurons (106a,
106b). For example, the neurons (106a, 106b) can have different
phenotypes that are designed to target distinct structures (112a,
112b) and/or facilitate communication along different channels. In
one embodiment, the neurons respond to or emit different
wavelengths of energy for optogenetic control and/or monitoring of
nerves. For example, the electrode (100) can support bidirectional
stimulation and recording, e.g., by applying a first wavelength of
light from a light source (114) and detecting a second, different
wavelength using a detector (116).
Dimensions
[0116] Referring now to FIGS. 2A-2C, electrodes (100) can include a
plurality of layers (102, 104, 206) that can have varying diameters
and/or thicknesses.
[0117] The extracellular matrix core (102) can have a
largest-cross-sectional dimension between about 10 .mu.m and about
1,000 .mu.m. For example, the largest cross-sectional dimension can
be selected from the group consisting of: between about 10 .mu.m
and about 20 .mu.m, between about 25 .mu.m and about 50 .mu.m,
between about 50 .mu.m and about 100 .mu.m, between about 100 .mu.m
and about 150 .mu.m, between about 150 .mu.m and about 200 .mu.m,
between about 200 .mu.m and about 250 .mu.m, between about 250
.mu.m and about 300 .mu.m, between about 300 .mu.m and about 350
.mu.m, between about 350 .mu.m and about 400 .mu.m, between about
400 .mu.m and about 500 .mu.m, between about 500 .mu.m and about
700 .mu.m, and between about 700 .mu.m and about 1,000 .mu.m.
[0118] The hydrogel sheath (104) can have a largest-cross-sectional
dimension between about 20 .mu.m and about 1,200 .mu.m. For
example, the largest cross-sectional dimension can be selected from
the group consisting of: between about 20 .mu.m and about 50 .mu.m,
between about 50 .mu.m and about 100 .mu.m, between about 100 .mu.m
and about 200 .mu.m, between about 200 .mu.m and about 250 .mu.m,
between about 250 .mu.m and about 300 .mu.m, between about 300
.mu.m and about 350 .mu.m, between about 350 .mu.m and about 400
.mu.m, between about 400 .mu.m and about 450 .mu.m, between about
450 .mu.m and about 500 .mu.m, between about 500 .mu.m and about
600 .mu.m, between about 600 .mu.m and about 800 .mu.m, and between
about 800 .mu.m and about 1,200 .mu.m. Stated another way, the
thickness of the hydrogel sheath (104) can be about between about 5
.mu.m and about 400 .mu.m.
[0119] The hydrogel sheath (104) can be further surrounded by a
layer of carboxymethyl cellulose (CMC). CMC is a cellulose
derivative with carboxymethyl groups bound to hydroxyl groups. The
functional properties depend on degree of substitution of cellulose
structure and degree of polymerization. CMC possesses unique
properties in that it is stiff in a dehydrated state and gel-like
hydrated state, with a short transition period between states at
micro-dimensions. Also, CMC is nontoxic to humans and animals,
inexpensive, and widely available. A CMC layer 206 having a
thickness of about 15 .mu.m provides sufficient initial rigidity to
enable needleless insertion into a subject's brain.
[0120] In some embodiments, the hydrogel sheath (104) is replaced
entirely by a CMC sheath as depicted and described in International
Publication No. WO 2015/066627.
[0121] Living electrodes (100) can have varying depths to reflect
clinical and anatomical needs. For example, the living electrodes
(100) can be sized for insertion to sufficient depth such that
second end (110) lies adjacent to a neuronal
population/nuclei/layer while first end (108) lies adjacent to
(e.g., flush with, slightly below, or slightly proud of) an outer
surface of the subject's brain (e.g., for manipulation by light
and/or magnetic fields). For example, the electrodes (100) can have
a length of about 100 .mu.m to about 2 cm or greater. In some
embodiments, the living electrodes can be implanted to a desired
depth (e.g., based on imaging and/or feedback) before being trimmed
in situ to a desired length relative to the outer surface of the
subject's brain.
Neurons
[0122] Embodiments of the invention can include optogenetic neurons
that enable the use of light to control cells in living tissue that
have been genetically modified to express light-sensitive ion
channels. For example, the neurons can express one or more
optogenetic actuators such as channelrhodopsin, halorhodopsin, and
archaerhodopsin and/or one or more optogenetic sensors for calcium
(e.g., Aequorin, Cameleon, GCaMP), chloride (e.g., Clomeleon) or
membrane voltage (e.g., Mermaid).
[0123] Embodiments of the invention can include magnetogenetic
neurons which can be controlled in living tissue through the
application of an alternating magnetic field. Magnetogenetic
techniques are described in Xiaoyyang Long et al.,
"Magnetogenetics: remote non-invasive magnetic activation of
neuronal activity with a magnetoreceptor," 60(24) Sci. Bull.
2107-19 (2015).
[0124] In certain embodiments, the neurons are genetically modified
to secrete factors to modulate disease pathophysiology, to allow
transplant cells to be resistant to underlying disease
pathophysiology, or to express novel ion channels and receptors to
allow for nuanced biological control. For example, genes can be
added/modified that allow the neurons to better process/degrade
protein accumulations such as pathological alpha-synuclein, tau, or
amyloid-beta.
[0125] Embodiments of the invention can additionally or
alternatively include primary cerebral cortical neurons and/or
dorsal root ganglion neurons.
Biocompatibility
[0126] The structures described herein can be biocompatible. For
example, the structures, when implanted, should not generate an
adverse chronic immunogenic or inflammatory response in the
subject. In certain embodiments, the one or more elements of the
structures degrade over time, thereby leaving the encapsulated axon
tracts within the subject. In one embodiment, the living electrodes
are generated using allogeneic neurons. Allogeneic neurons should
not elicit an overt immunogenic or inflammatory response. In one
embodiment, the living electrodes are generated using autologous
neurons derived from a patient's own stem cells (e.g., induced
pluripotent stem cells) or endogenous stem cell populations such as
those found in olfactory epithelium, lingual, ventricular
ependymal, or dentate gyrus).
Hydrogels
[0127] Hydrogels can generally absorb a great deal of fluid and, at
equilibrium, typically are composed more than about 60% fluid and
less than about 40% polymer. In a preferred embodiment, the water
content of hydrogel is about 80-99.9%. Hydrogels are particularly
useful due to the inherent biocompatibility of the cross-linked
polymeric network (Hill-West, et al., 1994, Proc. Natl. Acad. Sci.
USA 91:5967-5971). Hydrogel biocompatibility can be attributed to
hydrophilicity and ability to imbibe large amounts of biological
fluids (Preparation and Characterization of Cross-linked
Hydrophilic Networks in Absorbent Polymer Technology,
Brannon-Peppas and Harland, Eds. 1990, Elsevier: Amsterdam, pp
45-66; Preparation Methods and Structure of Hydrogels in Hydrogels
in Medicine and Pharmacy, Peppas, Ed. 1986, CRC Press: Boca Raton,
Fla., pp 1-27).
[0128] Hydrogels can be prepared by crosslinking hydrophilic
biopolymers or synthetic polymers. Examples of the hydrogels formed
from physical or chemical crosslinking of hydrophilic biopolymers,
include, but are not limited to, hyaluronans, chitosans, alginates,
collagen, dextran, pectin, carrageenan, polylysine, gelatin,
hyaluronic acid, or agarose. (Hennink and van Nostrum, 2002, Adv.
Drug Del. Rev. 54, 13-36 and Hoffman, 2002, Adv. Drug Del. Rev. 43,
3-12). These materials consist of high-molecular-weight-backbone
chains made of linear or branched polysaccharides or polypeptides.
Examples of hydrogels based on chemical or physical crosslinking
synthetic polymers include, but are not limited to,
(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,
poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO),
PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene),
poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A
copolymers, poly(ethylene imine), etc. (Hoffman, 2002, Adv. Drug
Del. Rev, 43, 3-12). In some embodiments, the hydrogel comprises
poly(ethylene glycol) diacrylate (PEGDA).
[0129] In one embodiment, the hydrogel comprises at least one
biopolymer. In other embodiments, the hydrogel scaffold further
comprises at least two biopolymers. In yet other embodiments, the
hydrogel scaffold further comprises at least one biopolymer and at
least one synthetic polymer.
[0130] In one embodiment, the hydrogel comprises agarose. The
concentration of agarose may, in certain instances, be dependent
upon the type of neuron ultimately being cultured, the mechanical
properties, desired, or the like. For example, increasing
concentrations of agarose enhances neuronal survival and neurite
outgrowth. In one embodiment, the concentration of agarose is about
0.1% to about 10%. In one embodiment, the concentration of agarose
is about 0.5% to about 5%. In one embodiment, the concentration of
agarose is about 4%.
[0131] Hydrogels closely resemble the natural living extracellular
matrix (Ratner and Hoffman. Synthetic Hydrogels for Biomedical
Applications in Hydrogels for Medical and Related Applications,
Andrade, Ed. 1976, American Chemical Society: Washington, D.C., pp
1-36). Hydrogels can also be made degradable in vivo by
incorporating PLA, PLGA or PGA polymers. Moreover, hydrogels can be
modified with fibronectin, laminin, vitronectin, or, for example,
RGD for surface modification, which can promote cell adhesion and
proliferation (Heungsoo Shin, 2003, Biomaterials 24:4353-4364;
Hwang et al., 2006 Tissue Eng. 12:2695-706). Indeed, altering
molecular weights, block structures, degradable linkages, and
cross-linking modes can influence strength, elasticity, and
degradation properties of the instant hydrogels (Nguyen and West,
2002, Biomaterials 23(22):4307-14; Ifkovits and Burkick, 2007,
Tissue Eng. 13(10):2369-85).
[0132] Molecules that can be incorporated into the hydrogel matrix,
for example via covalent linkage, encapsulation, or the like,
include, but are not limited to, vitamins and other nutritional
supplements; glycoproteins (e.g., collagen); fibronectin; peptides
and proteins; neurotransmitters; growth or neurotrophic factors;
differentiation factors; carbohydrates (both simple and/or
complex); proteoglycans; antigens; oligonucleotides (sense and/or
antisense DNA and/or RNA); antibodies (for example, to infectious
agents, tumors, drugs or hormones); and gene therapy reagents.
Hydrogels may be modified with functional groups for covalently
attaching a variety of proteins (e.g., collagen) or compounds such
as therapeutic agents. Therapeutic agents which can be incorporated
to the matrix include, but are not limited to, analgesics,
anesthetics, antifungals, antibiotics, anti-inflammatories,
anthelmintics, antidotes, antihistamines, antihypertensives,
antimalarials, antimicrobials, antipsychotics, antipyretics,
antiseptics, antiarthritics, antituberculotics, antivirals,
chemotherapeutic agents, a colored or fluorescent imaging agent,
corticoids (such as steroids), antidepressants, depressants,
diagnostic aids, enzymes, hormones, hypnotics, minerals,
nutritional supplements, parasympathomimetics, potassium
supplements, radiation sensitizers, a radioisotope, an imaging
contrast agent, sedatives, sulfonamides, stimulants,
sympathomimetics, tranquilizers, vasoconstrictors, vasodilators,
vitamins, xanthine derivatives, and the like. The therapeutic agent
may also be other small organic molecules, naturally isolated
entities or their analogs, organometallic agents, chelated metals
or metal salts, peptide-based drugs, or peptidic or non-peptidic
receptor targeting or binding agents. It is contemplated that in
certain embodiments, linkage of the therapeutic agent to the matrix
may be via a protease sensitive linker or other biodegradable
linkage.
[0133] Other suitable hydrogel components are described in
International Publication No.
[0134] WO 2015/066627.
Methods of Implantation
[0135] Referring now to FIG. 3, another aspect of the invention
provides a method (300).
[0136] In step S302, one or more implantable living electrodes are
implanted within a subject. Suitable implantation regions include
the subject's brain, spinal cord, peripheral nervous system (e.g.,
peripheral neurons, peripheral axons, axonal pathways, ganglia,
dorsal root ganglia, autonomic ganglia, and the like), and muscles.
The electrodes can be implanted with or without the aid of imaging
and with or without the aid of stereotactic manual or stereotactic
automated delivery systems. In some embodiments, multiple
electrodes are implanted in a region of interest.
[0137] In step S304, the implantable living electrode is allowed to
grow and couple with one or more deep adjacent host structures
(e.g., host neurons or neural networks), over hours, days, weeks,
months, years, or decades. Although initial integration will take
place on the order of hours to days, living electrodes can form a
stable interface for weeks, months, years, decades, and/or over the
lifetime of the implanted individual in contrast to conventional
electrodes that that lose effectiveness over time due to chronic
foreign body response, mechanical separation issues, reduced
neuronal density in electrode vicinity, and glial scarring.
Additionally, because the living electrode includes living neurons
and axons, synapses formed between construct neurons and host
neurons can be permanent and/or experience a natural
turnover/plasticity consistent with similar synapses in the
subject's body. Other cellular communication points can be formed
between the living electrode and host, including but not limited to
gap junctions, electrical synapses, and ephaptic links.
[0138] In step S306, a compatible stimulator is placed in proximity
to at least one of the one or more implantable living electrodes.
For example, a light source producing a compatible wavelength of
light can be placed at the surface of the subject's brain adjacent
to the implanted electrode or outside of the subject's skull, or
through the skull across a custom-fit transparent skull-replacement
piece. Likewise, compatible magnetic sources can be placed at the
surface of the subject's brain or outside of the subject's skull,
or through the skull across a custom-fit skull-replacement piece.
Likewise, an acoustic source such as ultrasound can be placed at
the surface of the subject's brain or outside of the subject's
skull.
[0139] In steps S308 and S310, the compatible stimulator can be
controlled to actuate at least one of the implanted electrodes to
excite (S308), inhibit (S310), and/or otherwise modulate (S312)
brain activity.
[0140] In step S314, a compatible sensor is placed in proximity to
at least one of the one or more implantable living electrodes. For
example, a light sensor can be placed at the surface of the
subject's brain adjacent to the implanted electrode. Likewise, a
compatible magnetic sensor can be placed at the surface of the
subject's brain or outside of the subject's skull.
[0141] In step S316, one or more signals emitted by the implanted
electrode(s) are detected by the sensor(s).
[0142] In certain embodiments, implantable living electrodes are
used to interface with the brain to record or stimulate neurons.
Stimulation of neurons can be used for treatment, or as a
diagnostic of a particular function or dysfunction of the nervous
system. Examples of nervous system dysfunctions can include, but
are not limited to Parkinson's disease, Alzheimer's disease,
dementia with Lewy bodies, frontotemporal dementia, Huntington's
disease, prion disease, motor neurone diseases, spinocerebellar
ataxia, spinal muscular atrophy, amyotrophic lateral sclerosis
(ALS), encephalitis, epilepsy, head and brain malformations,
hydrocephalus, seizures, chronic pain, traumatic brain injury,
spinal cord injury, stroke, anoxic brain injury, cerebral palsy,
obesity, depression, multiple sclerosis, inflammation, migraines,
diabetic neuropathy, locked-in syndrome, glioblastoma,
oligodendroglioma, metastases, and the like.
Macro-TENNs
[0143] Another embodiment of the invention provides peripheral
nerve interface (PM) electrodes and techniques that provide greater
access to different fascicles/axons. A macro-tissue engineered
neural network (macro-TENN) is larger and modified specifically for
PNS applications. Embodiments of the invention guide axonal
regeneration to allow the fascicles of a nerve to spread out
circumferentially around the inner surface of the macro-TENN. The
separation has the functional utility of being able to separate
fascicles and/or axons to gain a level of neural selectivity
unavailable with previous technologies.
[0144] In addition to use in the peripheral nervous system,
macro-TENNs can be used to interface with cranial nerves.
[0145] The macro-TENN employs similar techniques to the micro-TENN
technology described herein, but a larger hydrogel column results
in a tubular construct where the axons only grow along the
periphery of the tube. In this manner, the embodiment facilitates
the defasciculization of the nerve and/or the spreading out of the
axons so that the axons could interface more intimately with a
multitude of electrodes in the interior or exterior of the tubular
column (arranged circumferentially). Leveraging this method of
growth into the tubular construct to spread out the host axons to
bring them closer to contacting circumferential surface electrodes
would allow for highly selective interfacing (recording or
stimulation) with peripheral host axons.
[0146] Similar to the micro-TENNs described herein, the macro-TENN
can have a bipartite construction of a tubular hydrogel shell (as
described above) with an extracellular matrix (ECM) core as
depicted in FIGS. 15A-15C. The ECM core can consist of several
different components, including, but not limited to laminin and/or
collagen (as described above). The axons can grow into the core
while the hydrogel allows for transmission of soluble factors into
and out of the core channel.
[0147] Exemplary dimensions include an extracellular matrix core
diameter A of between about 1 mm and about 5 mm (e.g., between
about 1 mm and about 2 mm, between about 2 mm and about 3 mm,
between about 3 mm and about 4 mm, between about 4 mm and about 5
mm, and the like). Other exemplary dimensions include hydrogel
sheath diameter C of between about 2 mm and about 7 mm (e.g.,
between about 2 mm and about 3 mm, between about 3 mm and about 4
mm, between about 4 mm and about 5 mm, between about 5 mm and about
6 mm, between about 6 mm and about 7 mm, and the like). In some
embodiments, the thickness of hydrogel sheath B is between about 1
mm and about 2 mm.
Aggregate TENNs
[0148] Another aspect of the invention provides aggregate-TENNs
including a substantially cylindrical extracellular matrix core and
a plurality of aggregated neurons implanted along or within the
substantially cylindrical extracellular matrix core.
[0149] Another aspect, of the invention includes a method of
manufacturing an implantable living electrode including providing
an extracellular matrix core; and contacting at least one end of
the extracellular matrix core with a plurality of aggregated
neurons.
[0150] In various embodiments, aggregate TENNs exhibit higher
axonal growth rates than their dissociated counterparts. The term
aggregate TENN may encompass TENNS wherein the neurons have been
concentrated to form an aggregate prior the construction of the
TENN. The term may encompass any method of forming the aggregate.
Centrifugation of neurons in a pyramidal well is one method of
forming such aggregates. TENNS manufactured using this technique
may be referred to as centrifuged TENNs and the terms are used
interchangeably herein although a person of skill in the art will
appreciate that they are not necessarily coextensive. The observed
growth rates for dissociated neurons were similar to those reported
in literature; however, axonal growth rates from aggregates greatly
exceeded previous reports. Generally, cortical axons reach lengths
of 100 to 1000 .mu.m over 3 days in planar culture. The exact
causes for this increased growth from aggregates are still under
investigation; however, without wishing to be bound by theory, the
absence of additional growth factors in the medium and relatively
slower growth of dissociated micro-TENNs implicate features of the
aggregation method, such as more controlled, in vivo-like neuronal
microenvironment and self-reinforcing, more directed axonal
extension occurring via growing axon "bundles".
[0151] In various embodiments, the extracellular matrix core is a
collagen-laminin extracellular matrix core. Laminin is an adhesion
molecule, and as such may support rather than accelerate axonal
growth. Multiple studies have investigated the relationship between
growth cone behavior and axonal growth rate in the mammalian CNS.
Notably, axonal branching tends to occur in areas where the growth
cone temporarily ceases forward movement, transiently slowing the
outgrowth rate. Again, without wishing to be limited by theory,
upon emerging from an aggregate, a growth cone within a
bidirectional micro-TENN is presented with two classes of
targets--neurons within either its own or the opposing aggregate.
The combined support/growth promotion of the ECM and limited
selection of targets may reduce growth cone pausing/axonal
branching, accelerating growth within the zone between the
aggregates until the appropriate target is reached. In various
embodiments, the distance between axons and their targets may also
influence the speed at which they grow, as micro-TENNs within the
LE.sub.BI,5 mm group exhibited faster growth compared to the
LE.sub.BI,2 mm group at 5 DIV. The continued growth up to 10 DIV
(albeit at lower rates) of the dissociated micro-TENNs may be
attributed to the random distribution of neurons throughout the
construct--rather than grow en masse towards an aggregate (a large
target for axonal growth and synaptogenesis), neurons continued to
grow individually towards their closest neighbors.
[0152] In addition to rapid axonal growth, structural evidence of
synapses was visualized as early as 4 DIV. Measuring synaptogenesis
is often used to determine the functional maturity of neuronal
cultures, since synapses are the primary points of contact and
communication between neurons. Synapsinpositive puncta were
observed between 4 DIV and 28 DIV, suggesting that neurons within
micro-TENNs begin to form functional connections soon after plating
which greatly increased over time (FIG. 33). Indeed, micro-TENNs at
later timepoints qualitatively expressed higher levels of synapsin,
consistent with literature for planar cortical cultures where
synapse formation tends to increase as a function of time. The
presence of synapsin as early as 4 DIV may indicate that micro-TENN
neurons are forming functional connections earlier--as may be
expected from the high growth rates. Alternatively, many of these
synapses may be forming within the aggregates themselves--as the
experiments that provided the data for FIG. 33 were performed, no
distinctions were made between intra-aggregate and inter-aggregate
synapses. Intra-aggregate synapses being made before
inter-aggregate synapses may explain the high synapsin expression
at earlier DIV. Overall, micro-TENN neurons are capable of quickly
and consistently forming the desired living electrode architecture
in vitro. This allows for the rapid, reproducible production and
characterization of aggregate-based micro-TENNs. In various
embodiments comprising the aggregate TENN forming method,
controlling the aggregate sizes (and, concomitantly, the number of
neurons per construct) permits more repeatable studies and
standardized production methods than when working with single-cell
suspensions. Experimental details regarding aggregate TENNs and
methods for their manufacture are presented in FIGS. 16, 17, 19,
23, 25, 26, 29-31 and 34 as well as in the Examples.
Integral Sensors
[0153] Embodiments of the invention can integrate sensory receptors
within living electrodes, described herein, to provide integral
sensors that can both detect stimulus and transmit a corresponding
signal to the subject's nervous system or an external system. For
example, a living electrode can include sensory dorsal root
ganglion cells and/or cells with specialized sensors such as
baroreceptors, chemoreceptors (e.g., type I/glomus cells and type
II glial-like cells), electromagnetic receptors (e.g., infrared
receptors, photoreceptors, ultraviolet receptors),
electroreceptors, hydroreceptors, magnetoreceptors,
mechanoreceptors, nociceptors, odor receptors, osmoreceptors,
proprioceptors, taste receptors, thermoceptors, and the like.
Living electrodes can also include cochlear and/or vestibular hair
cells.
Integration with External Sensors
[0154] Embodiments of the invention can also be utilized to
transmit signals from external sensors to a subject's nervous
system or to an external system. For example, a man-made sensor can
generate electrical, optical, and/or magnetic signals indicative of
conditions such as pressure, temperature, position, force, sound,
smell, light, and the like. Embodiments of living electrode
structures described herein can receive these signals and
transmit/transduce the signals to the subject's nerves.
Integration of Electromechanical Devices within Living
Electrodes
[0155] Although some embodiments of the invention are described in
the context of external electromechanical devices such as surface
optrodes that may be adjacent to, but distinct from the living
electrodes described herein, other embodiments of the invention can
incorporate such devices within the living electrode. For example,
electrical contacts can be grown or inserted within the living
electrode. Likewise, optrodes, electromagnetic devices,
thermoelectric (Joule-Thomson resistive) heaters, thermoelectric
(Peltier) coolers, and/or chemical applicators can be grown or
inserted within the living electrode. For example, chemical
applicators can be adapted and configured to release neural
transmitters, ligands to which neurons respond, and/or ions in
response to an electrical signal.
Biological Multiplexing
[0156] Biological multiplexing can be defined broadly to encompass
biological versions of the types of channel selection, multiplexing
and demultiplexing used in telecommunications. In some embodiments,
biological multiplexing comprises both convergent and divergent
signaling: signal processing within many neurons of the construct
can converge on to single host parenchyma targets, and one
construct neuron can have axons divergently branching to target
many host parenchyma neurons (FIG. 21, Panels A-C). Likewise,
because neurons in the host brain are themselves embedded in
endogenous neural networks, the ability of the living construct to
send axonal outputs to one of these host neurons allows a specific,
stable, activation of that endogenous neural network. Because one
axon can in principle synapse onto thousands of target neurons, a
relatively small population of neurons within the construct could
achieve a widespread effect. By deploying micropatterning
techniques, living electrodes can be forged in vitro to enable
fine-grained time-division multiplexing when implanted in vivo
(FIG. 21, Panel D).
Living Electrodes Formed From Stem Cells
[0157] In its various aspects and embodiments, the compositions of
the invention may be generated from stem cells. In various
embodiments, deleterious immune response may be mitigated through
the use of autologous cells from patients. Neurons,
oligodendrocytes, astrocytes, and Schwann cells can be
differentiated from human embryonic stem cells, induced pluripotent
stem cells, and adipose-derived stem cells. Although direct in vivo
delivery of stem cells may replace lost cells and encourage neural
regeneration through the release of trophic factors, the mechanism
by which they stimulate the nervous system remains unclear, and
they have the potential to differentiate into undesirable
phenotypes and/or result in tumorigenesis. In comparison, there are
notable advantages to the use of differentiated neurons within
living electrodes. Existing protocols to differentiate stem cells
into specific neuronal sub-types--such as cortical projection
neurons, interneurons, dopaminergic A9 neurons, spinal motor
neurons--can be used to engineer living electrodes with specific
neuronal compositions. Because neurons are both terminally
differentiated and physically constrained by the 3D architecture of
the engineered construct, this approach likely carries less risk
for tumorigenesis. In various embodiments, differentiated neurons
can be genetically modified to enhance regenerative responses.
Prior studies suggest that the low survival of transplanted cells
can be due to delivery into a degenerating or "hostile" injured
environment. In various embodiments, by using transfection
techniques or viral transduction, the durability and regenerative
potential of differentiated neurons may be augmented through the
overexpression of trophic factors. This approach may make
engineered tissue resistant to the underlying pathophysiology of
neurodegenerative disease.
Controlled Programmed Cell Death Living Electrodes
[0158] In various embodiments, the living electrodes herein
described may be engineered to contain a controlled "kill switch"
driving programmed cell death of living electrode neurons and hence
axons. Indeed, the ability to employ different strategies to induce
programmed cell death in transplanted constructs is a potentially
important method to enhance the safety of living electrodes. There
are multiple suicide-gene technologies in development that can be
embedded into living electrode constructs. These suicide genes are
biologically inert until activated by the introduction of a
prodrug, and two clinically validated constructs, iCasp9 and
HSV-TK, are well-suited for different situations based on rapid
versus gradual apoptosis, respectively as may be appropriate in
various embodiments.
Potential Applications for Axon-Based Living Electrodes
[0159] In various embodiments, living electrodes may be deployed to
augment or replace traditional forms of neuromodulation, or may be
applied for more far-reaching drug delivery applications. For
instance, living electrodes may be precisely delivered to key
locations to influence the strength of specific connections. In
various embodiments, inhibitory (e.g., GABAergic) living electrodes
may be designed to form synapses to modulate pathways that are
exerting too much influence and causing detrimental functional
effects, for example to dampen hypersynchronous activity in a
circuitry exhibiting epileptiform activity (described in more
detail below). Conversely, in some embodiments, excitatory (e.g.,
glutamatergic) living electrodes may form synapses to augment weak
pathways, for example with axons from the construct releasing
glutamate at the target of a degenerating pathway. In various
embodiments, living electrodes may also act by bulk release of
neurotransmitters at the axonal terminal, either via tonic
(self-pacing/continuous) activity, by responding to inputs from the
host to the living electrode neuronal somata/dendrites, or
controlled from externalized hardware or computer.
[0160] This type of biological neuromodulation can provide direct
(i.e. synaptically-mediated) excitatory or inhibitory inputs--or
both simultaneously--or can provide controlled release of diffuse
modulatory neurotransmitters (e.g., dopamine) to augment circuit
function. Axon-based living electrodes can uniquely fulfill this
role--over more common neuronal transplants for instance--by acting
based on network feedback relayed and processed reciprocally
between the construct and the surrounding brain with the potential
for computer-controlled regulation/feedback. A sample of
applications for the compositions and methods of the invention is
detailed below:
Friedrich's Ataxia
[0161] In most cases of Friedrich's ataxia, the expansion of the
trinucleotide (GAA) repeat in intron 1 of both alleles of the
frataxin gene on chromosome 9q13 leads to reduced transcription of
the gene (ie, silencing), decreased expression of the gene product
frataxin, and ultimate destruction of the dorsal column pathways.
Patients consequently develop severe motor impairments in the
absence of proprioceptive and epicritic signals from the periphery.
In various embodiments, living electrodes could provide an
artificial sensory arc: by tapping into signals from periphery
(such as strain gauges, accelerometers and gyroscopes worn at
joints in all four limbs, or from implanted cuff recordings of
peripheral nerves), living electrodes implanted into primary
sensory cortices could provide sensory feedback and allow improved
voluntary movement and functional independence. Grown with
glutamatergic neurons, these living electrodes could be implanted
to terminate in layer IV of the post-central gyrus; because living
electrodes are themselves quite small, in various embodiments
multiple constructs could be implanted corresponding to different
joints (e.g., gyros from the left knee driving a living electrode
implanted in the right medial sensory cortex, left elbow and
shoulder to right lateral sensory cortex, and vice versa for the
right extremities and left hemisphere).
Severe Motor Impairment and Sensory Feedback.
[0162] In brainstem stroke, spinal cord injury, muscular dystrophy
and amyotrophic lateral sclerosis, people are rendered paralyzed
because the substrate of voluntary motor control (primary motor
cortex) is functionally disconnected from the skeletal muscles (and
in certain cases bulbar-pharyngeal muscles also). Neuromotor
prosthetics comprise a class of brain-computer or brain-machine
interfaces that seek to overcome this paralysis by recording
directly from the brain and decoding this recorded activity to
control devices in the environment, trigger robotic actuators, or
drive implanted neuromuscular stimulators. While several human
trials have shown the safety and efficacy of this approach,
patients achieve control purely by visual feedback. While the
sensory arc may be retained in certain patients with motor neuron
or muscular disease, it is lost in complete spinal cord transection
and is unavailable in all patients when using external robotics.
Several groups have attempted to provide haptic feedback by linking
tactile signals to electrical stimulation provided by macro- and
micro-electrodes implanted into primary sensory cortex. This type
of artificial haptic feedback appears to be effective in non-human
primates and has not yet been tested in humans. As with children
and adults with Friedrich's ataxia, living electrodes offer the
promise of recapitulating and expanding the sensory arc by being
implanted directly into the sensory cortex (see FIG. 20, panel B).
In addition to embodiments driven by externally worn sensors, in
other embodiments, living electrode activity could be triggered by
sensors mounted on robotic arms, powered robotic exoskeletal
braces, wheelchair components and other assistive devices. In this
way, a paralyzed patient could literally "feel" their own limbs and
the "limbs" of these devices to facilitate enhanced voluntary
control. Additionally, by providing a bidirectional interface to
both motor and sensory cortex that is routed through internally
implanted microprocessors, in various embodiments the living
electrodes could modulate inter-cortical communication in a
real-time closed-loop to restore motor function and
sensory/proprioceptive feedback.
Chronic Pain
[0163] In various embodiments, tailored living electrodes may be
useful to modulate inputs to a pain-dampening circuit. In various
embodiments, living electrodes could be created using peptidergic
neurons secreting endorphins or enkephalins and then implanted in
the substantia gelatinosa of the spinal cord, the periaqueductal
gray, ventroposterior thalamus or the anterior cingulate cortex. In
various embodiments, this would replace the non-specific approaches
of spinal and brain electrical stimulators. In various embodiments,
control of this neuromodulation could be user-dependent (e.g.,
analogous to a systemic pharmaceutical pump) and, unlike
microfluidics that would directly inject opiates or other peptides,
and unlike electrodes that would non-specifically modulate a target
volume, living electrodes comprised of neurons would themselves
undergo up- and down-regulation hence providing additional
prophylaxis against the development of tolerance, abuse or
withdrawal.
Alzheimer's Disease and Dementia with Lewy Bodies
[0164] A hallmark of both Alzheimer's disease (an
amyloid-tauopathy) and dementia with Lewy bodies (an
alpha-synucleinopathy), is loss of cholinergic neurons in the basal
forebrain. These neurons are reciprocally linked to medial temporal
lobe structures, including the hippocampal formation, and are
necessary to form episodic memories. In various embodiments, living
electrodes built using cholinergic neurons could be implanted into
the septal nuclei or other adjacent basal forebrain nuclei such as
the nucleus basalis of Meynert or the diagonal band of Broca. In
various embodiments, a living electrode stereotactically implanted
in the basal forebrain and semi-externalized to the brain service
(following the path of the columns of the fornix) could allow
closed-loop control with external computers: different
subpopulations of neurons within the living electrode (cholinergic,
GABAergic, glutamatergic) could be triggered differentially via
optogenetics and intraosseous anchored waveguides, depending on
detection of memory interference local field potential signatures
decoded from the activity of separate living electrodes implanted
into the temporal lobe to enhance episodic encoding. Likewise, in
other embodiments, external cues (e.g., reminders on a smart phone,
and user-triggered push button flagging) could be used to modulate
basal forebrain activity to enhance storage and recall. In various
embodiments, a second living electrode could be implanted into
entorhinal cortex and the hippocampus and then linked, via external
computers, to the living electrode implanted into the basal
forebrain to functionally re-instantiate the bidirectional fornix
septohippocampal pathway.
Frontotemporal Dementia and Autism Spectrum Disorder
[0165] In another aspect, for agrammatic primary progressive
aphasia, a frontotemporal dementia tauopathy affecting the dominant
inferior frontal gyrus, living electrodes could be implanted both
to link Broca's area to premotor and primary motor cortices (to
compensate for aphemia and allow motor substitution gestures) and
to link Broca's area to Wernicke's area as an artificial arcuate
fasciculus. In embodiments directed to behavioral variant
frontotemporal dementia (a TDP-43opathy and sometimes tauopathy),
constructs linking degenerating orbitofrontal cortices to intact
dorsolateral prefrontal, frontopolar, and anterior cingulate
cortices could reinstantiate behavioral inhibition and
self-regulation. In embodiments directed to the semantic dementia
variant of FTD (TDP-43 or tau), degeneration of the fronto-ventral
aspects of the temporal lobe may occur leading to loss of semantic
knowledge stores and a variety of reading and perceptual
disturbances. In various embodiments, an excitatory glutamatergic
living electrode implanted into the visual word form area of the
fusiform gyrus may boost residual function in this area, and the
living electrode could be crafted as an auxiliary axonal bundle
linking primary and secondary visual cortical areas to the ventral
temporal lobe to recreate the lost "ventral-what" pathway and
restore semantic processing. In both autism-spectrum disorder and
behavioral variant frontotemporal dementia, social perception and
interaction are compromised. In various embodiments, a living
electrode built with glutamatergic neurons at the surface and
within left dorsolateral prefrontal cortex and oxytocinergic
neurons apposed to supraoptic and paraventricular nuclei in the
hypothalamus could quench behavioral disinhibition and recover
social behavior; the surface cortical population could be triggered
by external computers tracking social cues decoded from microphones
and micro-cameras mounted unobtrusively in the frames of glasses,
hearing aids, bracelets or other apparel.
Stroke and Cerebral Palsy
[0166] Both ischemic and hemorrhagic stroke result in focal brain
tissue destruction and varying degrees of inflammation. In ischemic
stroke, a surrounding penumbra of tissue may remain functional and
simultaneously metabolically vulnerable to further insult (such as
from decreased blood pressure or hypoxia). When occurring in utero
or in the perinatal period, stroke (e.g., germinal matrix
hemorrhage) can lead to a static insult around which the rest of
the brain attempts to develop normally, in certain cases leading to
cerebral palsy with varying degrees of motor and cognitive
impairment. When an area of the brain is damaged, two aspects of
function are lost: the local gray matter "computation" and also the
axonal (both focal intrinsic and also crossing fibers of passage)
"connectivity." In various embodiments, micro-TENNs could directly
restore both computation and connectivity and serve as "replacement
parts" for the irreversibly damaged piece of the brain and to
metabolically, electrically and functionally revive and support the
surrounding penumbra. In an animal model of stroke with middle
cerebral artery occlusion, optogenetic grafts were shown to restore
functional mobility. Whereas this graft was "driven" by an external
laser, a functionalized living electrode could allow both intact
areas of the brain, and external modulation triggered by body
sensors or computer-driven rehabilitation, to do the "driving" to
restore activity within the penumbra and restore functional
mobility and behavior following stroke.
Refractory Depression
[0167] Severe clinical depression that is refractory to
pharmacotherapy, psychotherapy and electroconvulsive therapy, is
characterized by neurometabolic derangements including disrupted
glucose uptake in limbic structures including the cingulate gyrus.
In various embodiments, micro-TENNs could be implanted to enhance
connectivity between frontopolar cortex and the anterior cingulate,
or to link supragenual to subgenual anterior cingulate cortices so
that the former modulates the latter to restore normal metabolic
activity and relieve symptoms. Likewise, in various embodiments, if
seeded with dopaminergic neurons, living electrodes implanted into
the nucleus accumbens could be deployed to provide dynamic, phasic
alteration of catecholamine tone and hence alter mood salience
labeling of thoughts and perception to relieve depressive symptoms
without causing rebound dysphoria or tolerance post-synaptic
upregulation.
Epilepsy
[0168] The application for epilepsy exhibits two ways in which the
advanced functionality of living electrodes could achieve treatment
goals in a manner impossible with existing approaches. In the
first, living electrodes could be forged such that the population
of neurons closest to the target area secreted the inhibitory
neurotransmitter GABA diffusely to the target region, either
constitutively or evoked from the brain surface based on
measurements of early epileptiform activity (as described below).
In this approach, the living electrode effectively serves as a GABA
reservoir and delivery system (see FIG. 20, panel C). In various
embodiments, the living electrode could be seeded with excitatory
glutamatergic neurons in an extracellular matrix decorated with
neuroligins to coax synaptogenesis with local endogenous GABAergic
neurons. Either approach may achieve disruption of hypersynchronous
activity and hence arrest generation or transmission of
pathological seizures from a target region in the brain. Because
neurons in the epileptiform network within the brain may form
synapses onto dendrites extending out from neurons within the
construct, various embodiments may achieve focal, closed-loop
self-attenuating circuits such that focal epileptiform activity
would quench itself via this autoinhibitory loop mediated by the
inhibitory living electrode. In embodiments directed toward
multi-focal epilepsy, living electrodes could be implanted at two
or more epileptigenic foci (e.g., identified by intracranial
surface and depth recording). In various embodiments, sensors
(e.g., intraosseous or subgaleal leads capturing ongoing local
field potentials) could be used to pick up signatures of
pre-seizure or seizure activity to trigger photostimulation of
optogenetically modified surface externalized micro-TENNs to
pre-emptively arrest seizure propagation in a manner impossible
with conventional electrodes.
Parkinson's Disease
[0169] In a further aspect, the invention provides micro-TENN
compositions and methods for the treatment of Parkinson's disease.
In one aspect, the invention comprises a substantially cylindrical
extracellular matrix core; one or more neurons implanted along or
within the substantially cylindrical extracellular matrix core,
wherein the neurons are dopaminergic neurons. In another aspect,
the invention comprises a method of treating Parkinson's disease
comprising implanting a dopaminergic micro-TENN into the SNpc of a
patient.
[0170] Current treatments for Parkinson's disease, including the
use of dopamine replacement strategies and deep brain stimulation,
are aimed at alleviating motor disabilities rather than correcting
the underlying cause of the motor symptoms. Furthermore, while
dopaminergic neuron and/or fetal graft implants into the striatum
may provide a local source of dopamine, these approaches do not
recreate the nigrostriatal circuit. To address these gaps, a
tissue-engineered solution that could be precisely delivered to
physically restore lost dopaminergic neurons in the SNpc and their
axonal projections to the striatum is sought. To achieve this
objective, in one aspect the invention comprises micro-TENNs
utilizing primary dopaminergic neurons. It was found that
micro-TENNs plated with neuronal aggregates grew more than 6 mm in
length when fabricated with the optimal inner core ECM.
Furthermore, the dopaminergic micro-TENNs exhibit evoked dopamine
release and are capable of synapsing with a population of striatal
cells in vitro, and show survival and maintenance of
cytoarchitecture upon transplant in vivo.
[0171] Dopaminergic micro-TENNs may be fabricated using a
population of ventral mesencephalic neurons that, may be enriched
in dopaminergic neurons, some embodiments are not a pure
dopaminergic population. Other embodiments comprise a pure
dopaminergic population. In various embodiments a higher purity of
dopaminergic neurons may be used for functional efficacy, in other
embodiments a later developmental time point for midbrain isolation
cell sorting, and/or differentiation from stem cell sources may be
used, all of which have been shown to further enrich dopaminergic
populations.
[0172] In another aspect, the invention comprises a method to plate
micro-TENNs with dopaminergic "aggregates" that may alleviate the
lack of separation between the neuronal somata and neurites that
was observed in some cases when micro-TENNs were plated with
dissociated cells. It was particularly important to ensure that the
micro-TENNs demonstrated the desired cytoarchitecture of a discrete
cell body region projecting axons through the length of the inner
core since separate somatic and axonal regions is a key feature of
the nigrostriatal pathway.
[0173] Without wishing to be limited by theory, it is believed that
better approximation of the cytoarchitecture of the nigrostriatal
pathway leads to improved functional outcomes following micro-TENN
implantation to reconstruct the degenerated dopaminergic neurons in
the SNpc and their projections to the striatum. In particular, the
dopaminergic neurons in the SNpc synapse directly onto striatal
neurons without exerting their effects through intermediate
synapses and/or neurons. Therefore, in order for the connectivity
and timing of micro-TENNs to be correct upon integration with the
host, the micro-TENNs will likely need to achieve modulation of
striatal neurons by propagating signals from the SNpc through a
mono-synaptic pathway. Dopaminergic micro-TENNs that approximate
the architecture of the nigrostriatal pathway are disclosed herein,
and these possess an increased the likelihood that functional
integration in vivo will emulate the native mono-synaptic
pathway.
[0174] In some embodiments, the dopaminergic cell aggregates
produced neurite outgrowth that was approximately 10.times. longer
than projections from individual cells. Again, without wishing to
be limited by theory, this may be influenced by the fact that the
cellular density within the neuronal aggregates is more
representative of the density within the brain. This higher cell
density may give the aggregated neurons better control over their
3D microenvironment than dissociated cells, which, in turn,
promoted better cell viability and health and therefore enhanced
axonal extension. Moreover, the gene expression of cells in the
aggregates may also be more representative of cells in the brain
and allows the cells to tap into developmental programs to initiate
axonal outgrowth. Furthermore, many neuronal subtypes are
programmed for either short or long distance communication. In
dissociated micro-TENNs, "long distance" axons likely meet synaptic
partners at intermediate distances along the length of the
micro-column. In contrast, for the aggregate micro-TENNs, the
absence of any intermediate neighbors may have prompted the "long
distance" neurons to up-regulate proteins for long distance
outgrowth and thus project the length of the micro-columns. Lastly,
the aggregates give rise to grouped and fasciculated axonal
projections, which may produce a sustained drive for axonal
extension due to concentrated and persistent progrowth signaling,
and/or physical/structural advantages. The rates and lengths of
dopaminergic axonal extension from the neuronal aggregates were
unprecedented, although clearly further studies are required to
elucidate the mechanisms of ultra-long axonal projections from the
aggregates.
[0175] In some embodiments, the aggregate micro-TENNS comprise
collagen I and a collagen I and laminin cocktail in the inner core.
Without wishing to be bound by theory, it is believed that this
configuration benefits axonal outgrowth because, unlike the laminin
coating and empty cores, the collagen I and collagen I and laminin
cocktail both provide a continuous, 3D scaffold that supports
axonal outgrowth. While crosslinked collagen also provides a
continuous scaffold, it is much stiffer and likely more resistive
to enzymatic degradation upon growth cone extension, however
embodiments comprising cross-linked collagen may possess desirable
structural properties that may be suitable for various
applications.
[0176] In various embodiments, the dopaminergic micro-TENNs release
dopamine both within the neuronal aggregate and at the axon
terminals.
[0177] At one week and one month following injection of GFP+
micro-TENNs to span the nigrostriatal pathway, evidence of
micro-TENN survival and maintenance of cytoarchitecture was found.
Specifically, surviving GFP+ and TH+ neurons were located within
the lumen along the injection trajectory. Out to one month
post-implant, histological sections orthogonal to the implant
trajectory revealed dense GFP+ neurons/axons and TH+ axons in
cross-section. The circuitry of the nigrostriatal pathway is
complex, and while the trajectory of implantation may be refined in
future studies, these findings are sufficient to demonstrate
proof-of-concept for implant and survival in vivo.
[0178] In various embodiments, dopaminergic output of the
micro-TENN may be continuously modulated by striatal feedback and
SNpc input to alleviate potential runaway dopamine excess and
dystonia, a potential side effect from mesencephalic dopaminergic
cell transplants into the striatum. Micro-TENNs are an auxiliary
pathway. This engineered circuit is unique in that it is mimicking
the function of dopaminergic axons projecting from the SNpc to the
striatum and seeks to provide dopaminergic inputs that can be tuned
and controlled. In addition to direct circuit reconstruction, in
various embodiments optogenetically active micro-TENNs may also be
deployed as dopaminergic living electrodes to provide controlled
neuromodulatory input via engineered axonal tracts (see FIG. 20,
panel A). In FIG. 20 the neuronal somata population is left
quasi-externalized on the brain surface to allow for controlled
interface with a sub cranial micro-LED array. The interface beyond
the nigrostriatal tract would provide a mechanism whereby
information from other brain areas (e.g., beta oscillations
recorded from primary motor cortex), external sensors (e.g.,
gyroscopes and accelerometers both within the battery case in the
chest wall or streamed from implanted or externally worn sensors in
the hands or feet), and external computers (e.g., processing 3D
motion capture and force sensors embedded in the shoes, treadmill
and gait analyzer surfaces), could modulate the basal ganglionic
circuitry into a healthier activity pattern.
EXAMPLES
[0179] The following Examples may be useful to a person of skill in
the art in understanding the disclosure but should in no way be
construed as limiting the invention.
[0180] Various materials and methods employed in certain of the
following examples are here presented. All procedures were approved
by the IACUCs at the University of Pennsylvania and The Michael J.
Crescenz Veterans Affairs Medical Center and were carried out in
accordance with Public Health Service Policy on Humane Care and Use
of Laboratory Animals (2015).
Three-Dimensional Micro-TENN Fabrication
[0181] All supplies were from Invitrogen, BD Biosciences, or
Sigma-Aldrich unless otherwise noted. Micro-TENNs included an
agarose ECM hydrogel molded into a cylinder through which axons
could grow. The outer hydrogel structure consisted of 1% agarose in
Dulbecco's phosphate-buffered saline (DPBS). The agarose cylinder,
with an outer diameter of 398 .mu.m, was generated by drawing the
agarose solution into a capillary tube (Drummond Scientific) via
capillary action. An acupuncture needle (diameter: 160 .mu.m)
(Seirin) was inserted into the center of the agarose-filled
capillary tube in order to produce an inner column. Cured
micro-columns were pushed out of the capillary tubes and placed in
DPBS where they were cut to 6-12 mm in length and sterilized under
UV light (1 hour). 5 .mu.L of the appropriate ECM cocktail was
added to each micro-column. ECM cocktails included: rat tail type 1
collagen, 1.0 mg/mL; rat tail type I collagen, 1.0 mg/ml mixed with
mouse laminin, 1.0 mg/ml; mouse laminin, 1.75 mg/ml; and rat tail
type 1 collagen, 1.0 mg/mL in 11.70 mM
N-(3-Dimethylaminopropyl)-N'-ethylcarboiimide hydrochloride, 4.3 mM
N-Hydroxysuccinimide, and 35.6 mM sodium phosphate monobasic. These
micro-columns were then incubated at 37.degree. C. for 15-30
minutes, after which DPBS was added to the petri dish.
Neuronal Cell Culture
[0182] Female Sprague-Dawley rats (Charles River) were the source
for primary ventral mesencephalic neurons, a midbrain region
previously shown to be enriched in dopaminergic neurons (Weinert et
al. 2015, Isolation, culture and long-term maintenance of primary
mesencephalic dopaminergic neurons from embryonic rodent brains,
Journal of visualized experiments: JoVE, (96)). Carbon dioxide was
used to euthanize timed-pregnant rats (embryonic day 14), following
which the uterus was extracted. The brains were removed in Hank's
balanced salt solution (HBSS) and the ventral midbrain was
isolated. The ventral midbrains were dissociated in accutase for 10
minutes at 37.degree. C. The cells were centrifuged at a relative
centrifugal force (RCF) of 200 for 5 minutes and resuspended at 1-2
million cells/mL in standard media consisting of NEUROBASAL.RTM.
medium+2% B27+1% fetal bovine serum (Atlanta Biologicals)+2.0 mM
Lglutamine+100 .mu.M ascorbic acid+4 ng/mL mouse basic fibroblast
growth factor (bFGF)+0.1% penicillin-streptomycin). High
concentration growth media consisted of NEUROBASAL.RTM. medium+2%
B27+1% fetal bovine serum (Atlanta Biologicals)+2.0 mM
Lglutamine+100 .mu.M ascorbic acid+0.1% penicillin-streptomycin+12
ng/mL mouse bFGF+10 ng/mL brain-derived neurotrophic factor
(BDNF)+10 ng/mL glial cell-derived neurotropic factor (GDNF)+10
ng/mL ciliary neurotropic factor (CNTF)+10 ng/mL cardiotrophin.
Dopaminergic neuron aggregates were created based on protocols
adapted from Ungrin M D, Joshi C, Nica A, et al. 2008,
Reproducible, ultra high-throughput formation of multicellular
organization from single cell suspension-derived human embryonic
stem cell aggregates, PloS one, 3 (2): e1565. Custom-built arrays
of inverted pyramidal wells were fabricated using
polydimethylsiloxane (PDMS) (SYLGARD.RTM. 184, Dow Corning) cast
from a 3D printed mold and placed in a 12-well plate. 12 .mu.L of
the cell solution was transferred to each pyramidal well, and the
12-well plate was centrifuged at 1500 rpm for 5 minutes, after
which 2 mL of standard media was placed on top of each array. The
centrifugation resulted in forced aggregation of neurons
(approximately 3,200 cells per aggregate). The wells were then
incubated overnight. At the time of plating, the DPBS was removed
from the dishes containing the micro-columns and replaced with
media. Using forceps, the aggregates were inserted into one
(unidirectional) or both (bidirectional) ends of the micro-columns,
and the cultures were placed in an incubator (total micro-TENNs
created with dopaminergic neurons: n=3 00).
[0183] For micro-TENNs containing dissociated cells with no ECM
core, dopaminergic cells were suspended in standard media at 10
million cells/mL and 5 .mu.L of this cell suspension was added to
each micro-column. The micro-TENNs were incubated for 60 minutes,
after which media was added. For micro-TENNs containing dissociated
cells with an ECM core, dopaminergic cells were suspended in rat
tail type 1 collagen, 1.0 mg/mL (10,000,000 cells/mL) at the time
of plating and 5 .mu.L of this mixture was added to each
micro-column. The micro-TENNs were incubated for 15 minutes, after
which media was added.
[0184] Pre-warmed media was used to replace the culture media every
3-4 days in vitro (DIV). In some instances, micro-TENNs were
transduced with an adeno-associated virus (AAV) vector
(AAV2/1.hSynapsin.EGFP.WPRE.bGH, UPenn Vector Core) to express
green fluorescent protein (GFP) in the neurons. Here, at 3 DIV the
micro-TENNs were incubated overnight in media containing the vector
(3.2.times.1010 genome copies/mL) and the cultures were rinsed with
media the following day.
[0185] Female Sprague-Dawley rats (Charles River, Wilmington,
Mass.) were the source for primary striatal neurons. Carbon dioxide
was used to euthanize timed-pregnant rats (embryonic day 18), after
which the uterus was extracted. To isolate striatal neurons, the
brains were removed in HBSS and striata were isolated. The striata
were dissociated in trypsin (0.25%)+ethylenediaminetetraacetic acid
(EDTA) (1 mM) for 12 minutes at 37.degree. C. The trypsin-EDTA was
then removed and the tissue was triturated in HBSS containing DNase
I (0.15 mg/mL). The cells were centrifuged at 1000 rpm for 3
minutes and resuspended at 1-2 million cells/mL in NEUROBASAL.RTM.
medium+2% B27+0.4 mM Lglutamine. Striatal aggregates were created
and inserted into micro-TENNs as previously described. When testing
if dopaminergic aggregates would form synapses with striatal
aggregates, striatal aggregates were inserted into the vacant ends
of dopaminergic micro-TENNs at 10 DIV. When testing if striatal
aggregates would increase the growth rate of dopaminergic
micro-TENNs, they were inserted at 3 DIV.
Immunocytochemistry
[0186] Micro-TENNs were fixed in 4% formaldehyde for 35 min and
permeabilized using 0.3% Triton X100 plus 4% horse serum for 60
minutes. Primary antibodies were added (in phosphate-buffered
saline (PBS)+4% serum) at 4.degree. C. for 12 hours. The primary
antibodies were the following markers: (1) .beta.-tubulin III
(1:500, Sigma-Aldrich, cat #T8578), a microtubule element expressed
primarily in neurons; (2) tyrosine hydroxylase (TH; 1:500, Abcam,
cat #AB113), an enzyme involved in the production of dopamine; (3)
microtubule-associated protein 2 (MAP-2) (1:500, Millipore, cat
#AB5622), a microtubule-associated protein found in dendrites; (4)
dopamine-and-cAMP-regulated neuronal phosphoprotein (DARPP-32)
(1:250, Abcam, cat #AB40801) a protein found in striatal
medium-sized spiny neurons; and (5) Synapsin 1 (1:1000, Synaptic
Systems, cat #106001), a protein expressed in synaptic vesicles of
the central nervous system. Appropriate fluorescent secondary
antibodies (Alexa-488, -594 and/or -649 at 1:500 in PBS+30 nM
Hoechst+4% serum) were added at 18-24.degree. C. for 2 hours.
Transplantation of Micro-TENNs
[0187] Male Sprague-Dawley rats (325-350 g) were anesthetized with
isoflurane and mounted in a stereotactic frame. The scalp was
cleaned with betadine, bupivacaine was injected along the incision
line, and a midline incision was made to expose the Bregma
landmark. A 5 mm craniectomy was centered at the following
coordinates in relation to Bregma: +4.8 mm (AP), 2.3 mm (ML). The
micro-TENN was loaded into a needle (OD: 534 .mu.m, ID: 420 .mu.m;
Vita Needle, Needham, Mass.) attached to a Hamilton syringe mounted
on a stereotactic arm. The stereotactic arm was positioned at
34.degree. relative to the horizontal plane, the dura was opened,
and the needle lowered into the brain to a depth of 11.2 mm. The
needle was kept in place for 10 seconds, at which time a stationary
arm was positioned to contact the plunger of the Hamilton syringe.
The needle containing the micro-TENN was then withdrawn from the
brain. The scalp was sutured closed and buprenorphine was provided
for postoperative analgesia. Animals receiving micro-TENNs were
survived for either 1 week (n=5) or 1 month (n=5). At the time of
sacrifice, animals were anesthetized and underwent transcardial
perfusion with heparinized saline followed by 10% formalin.
Immunohistochemistry
[0188] After 24 hour post-fix in 4% paraformaldehyde, brains were
prepared for either parafin processing or cryosectioning. Brains
were blocked sagittally and processed through paraffin or put into
30% sucrose until saturated and frozen. Sections were cut at 8
.mu.m (paraffin) or 35 .mu.m (cryosections), mounted on slides, and
processed for immunohistochemistry.
[0189] Paraffin sections were deparaffinized and then rehydrated
Endogenous peroxidase was quenched using 3% hydrogen peroxide in
water (Fisher, cat #S25359) followed by heat-induced epitope
retrieval in TRIS-EDTA. Sections were blocked with horse serum (ABC
Universal Kit, Vector Labs, cat #PK-6200) for 30 min. Rabbit
anti-TH (1:750; Abcam, cat #ab112) was applied in Optimax buffer
overnight at 4.degree. C. The antigen of interest was visualized
using DAB (Vector Labs, cat #SK-4100). Frozen sections were blocked
with 5% normal horse serum in 0.1% Triton-x/PBS for 30-45 minutes.
Primary antibodies (Rabbit anti-TH, 1:750, Abcam AB112; Mouse
anti-Tuj 1, 1:1000, Sigma T8578) were applied to the sections in 2%
horse serum/Optimax.RTM. buffer for two hours at room temperature.
Secondary antibodies (1:1000) were applied in 2% horse serum/PBS
for one hour at room temperature. Sections were counterstained with
Hoechst.
Microscopy and Data Acquisition
[0190] For in vitro analyses, micro-TENNs were imaged using
phase-contrast and fluorescence on a NIKON.RTM. ECLIPSE.RTM. Ti-S
microscope with image acquisition using a QICLICK.RTM. camera
interfaced with NIKON.RTM. ELEMENTS.TM. software. In order to
determine the length of neurite penetration, the longest observable
neurite in each micro-TENN was measured from the proximal end of
the neuronal aggregate after fixation. For in vitro
immunocytochemistry analyses, cultures and micro-TENNs were
fluorescently imaged using a NIKON.RTM. A1RSI Laser Scanning
Confocal microscope. All micro-TENN confocal reconstructions were
from full thickness z-stacks. For analysis of micro-TENNs
post-transplant into the brain, micro-TENNs were fluorescently
imaged using a NIKON.RTM. A1RSI Laser Scanning Confocal microscope.
Each section was analyzed to assess the presence, architecture, and
outgrowth/integration of micro-PENN neurons/neurites.
Statistical Analyses
[0191] No method was used to pre-determine the sample sizes of
groups. Due to obvious visual differences between experimental
groups, in most cases investigators were not blinded to treatment
group during experiments or data assessment. For in vivo transplant
studies, rats were randomly assigned for use in this experiment.
The normality of all data was examined, and adjustments were made
for non-normal data. An unpaired, parametric two-sided t-test was
performed to determine if there were statistically significant
differences in axonal outgrowth between uni-directional versus
bi-directional micro-TENNs containing a dopaminergic end target.
Unpaired, non-parametric, two sided Mann-Whitney tests were
performed to determine if there were statistically significant
differences in axonal outgrowth between the following treatment
pairs: dissociated versus aggregated cells, high versus regular
growth factor concentration, and uni-directional versus
bi-directional micro-TENNs containing a striatal end target. An
unpaired, nonparametric, two-sided Mann-Whitney test was performed
to determine if there were statistically significant differences
between the lengths of TH+ axons as a percentage of total axonal
length with collagen I versus collagen I-laminin cocktail inner
cores. ANOVA was performed for the extracellular matrix studies.
When differences existed between groups, post-hoc Tukey's pair-wise
comparisons were performed. For all statistical tests, p<0.05
was required for significance. Data are presented as
mean.+-.standard deviation.
Cortical Neuron Isolation and Culture
[0192] Neural cell isolation and culture protocols are similar to
that of published work. Briefly, timed-pregnant rats were
euthanized, and the uterus removed. Embryonic day 18 fetuses were
transferred from the uterus to cold HBSS, wherein the brains were
extracted and the cerebral cortical hemispheres isolated under a
stereoscope via microdissection. Cortical tissue was dissociated in
0.25% trypsin+1 mM EDTA at 37.degree. C., after which the
trypsin/EDTA was removed and replaced with 0.15 mg/ml DNase in
HBSS. Dissociated tissue+DNase was centrifuged for 3 min at 3000
RPM before the DNase was removed and the cells re-suspended in
neuronal culture media, composed of NEUROBASAL.RTM. +B27.RTM.
+Glutamax.TM. (ThermoFisher) and 1% penicillin-streptomycin.
Micro-TENN/Living Electrode Fabrication
[0193] Micro-TENNs were constructed in a three-phase process (FIG.
29). First, agarose micro-columns of a specified geometry (outer
diameter (OD), inner diameter (ID), and length) were formed in a
custom designed acrylic mold (FIG. 29, panel A). The mold is an
array of cylindrical channels that allow for the insertion of
acupuncture needles (Seirin, Weymouth, Mass.) such that the needles
are concentrically aligned within the channels. Molten agarose in
Dulbecco's phosphate buffered saline (DPBS) was poured into the
mold-needle assembly and allowed to cool (agarose: 3%
weight/volume). Once the agarose solidified, the needles were
removed and the mold disassembled, yielding hollow agarose
micro-columns with a specific outer diameter equal to the size of
the channels and inner diameter equal to the outer diameter of the
needles. Micro-columns were sterilized via UV light for 30 min and
stored in DPBS to prevent dehydration until needed. For these
studies, the mold channels were 398 .mu.m in diameter and the
acupuncture needles were 180 .mu.m, resulting in micro-columns with
a 398 .mu.m OD and a 180 .mu.m ID. Micro-columns were cut to either
two or five millimeters in length. Next, primary cortical neurons
were forced into cell aggregates (FIG. 29, panel B). These
aggregates provide the necessary architecture for the growth of
long axonal fascicles spanning the length of the microcolumn. Cells
were transferred to an array of inverted pyramidal wells made in
PDMS (SYLGARD.RTM. 184, Dow Corning) cast from a custom-designed,
3D printed mold (FIG. 29, panel B). Dissociated cortical neurons
were suspended at a density of 1.0-2.0 million cells/ml and
centrifuged in the wells at 200 g for 5 min. This centrifugation
resulted in forced aggregation of neurons (or any other cell type)
with precise control of the number of neurons per aggregate/sphere
(12 .mu.L cell suspension per well). Pyramidal wells and forced
aggregation protocols were adapted from Ungrin et al. Finally,
micro-columns were removed from DPBS and excess DPBS removed from
the micro-column interior via micropipette. Micro-columns were then
filled with extracellular matrix (ECM) comprised of 1.0 mg/ml rat
tail collagen+1.0 mg/ml mouse laminin (Reagent Proteins, San Diego,
Calif.) (FIG. 29, panel C). Unidirectional or bidirectional
micro-TENNs were seeded by carefully placing an aggregate at one or
both ends of the micro-columns, respectively, using fine forceps
under a stereoscope and were allowed to adhere for 45 min at
37.degree. C., 5% CO2. To create dissociated micro-TENNs,
dissociated cortical neurons were transferred via micropipette into
the ECM-filled micro-column as detailed in prior work. Micro-TENNs
were then allowed to grow in neuronal culture media with fresh
media replacements every 2 days in vitro (DIV).
Growth Characterization
[0194] Phase-contrast microscopy images of micro-TENNs in culture
were taken at 1, 3, 5, 8, and 10 DIV at 10.times. magnification
using a NIKON.RTM. Eclipse Ti-S microscope, paired with a
QIClick.RTM. camera and MS Elements BR 4.13.00. Micro-TENNs were
fabricated and classified into one of four groups: dissociated/2 mm
long (LE.sub.DISS,2 mm) (n=7), unidirectional aggregate/2 mm long
(LE.sub.UNI,2 mm) (n=6), bidirectional aggregate/2 mm long
(LE.sub.BI,2 mm) (n=9), or bidirectional aggregate/5 mm long
(LE.sub.BI,5 mm) (n=7). Growth rates for each group at specific
timepoints were quantified as the change in the length of the
longest identifiable neurite divided by the number of days between
the current and preceding timepoint. The longest neurites were
manually identified within each phase image using functions from
the Image Processing Toolbox in MATLAB (MathWorks, MA), and length
was measured from the source aggregate to the neurite tip. The same
starting micro-TENNs and starting points were used across
timepoints for more accurate analysis. Mean growth rates were found
for each group at the specified timepoints and compared with
two-way analysis of variance (ANOVA), with post-hoc analysis
performed where necessary with the Bonferroni procedure (p<0.05
required for significance). All data presented as mean.+-.s.e.m. To
identify aggregate-specific growth across the micro-columns,
cortical neuronal aggregates were labeled with either green
fluorescent protein (GFP) or the red fluorescent protein mCherry
via adenoassociated virus 1 (AAV1) transduction (Penn Vector Core,
Philadelphia, Pa.). Briefly, after centrifuging aggregates in the
pyramid wells, 1 .mu.L of AAV1 packaged with the human Synapsin-1
promoter was added to the aggregate wells (final concentration:
.about.3.times.109 viral copies per aggregate). Aggregates were
incubated at 37.degree. C., 5% CO2 overnight before the media was
replaced twice, after which transduced aggregates were plated in
micro-columns as described above, each with one GFP+ and one
mCherry+ aggregate (n=4 total). Over multiple DIV, images of the
micro-TENNs were taken on a NIKON.RTM. A1RSI Laser Scanning
confocal microscope paired with MS-Elements.RTM. AR 4.50.00
software. Sequential slices of 10-20 .mu.m in the z-plane were
acquired for each fluorescent channel. All confocal images
presented are maximum intensity projections of the confocal
z-slices.
Viability Assessment
[0195] To assess neuronal viability, 5-mm long unidirectional
(LE.sub.UNI) and bidirectional (LE.sub.BI) constructs and
age-matched planar cultures plated on polystyrene were stained with
a calcein-AM/ethidium homodimer-1 (EthD-1) assay (ThermoFisher) at
10 and 28 DIV. Metabolically active cells convert the
membrane-permeable calcein AM to calcein, which fluoresces green
(.lamda..sub.exc .about.495 nm; .lamda..sub.em, .about.515 nm),
while EthD-1 enters membrane-compromised cells and fluoresces red
upon binding to nucleic acids (.lamda..sub.exc .about.495 nm;
.lamda..sub.em .about.635 nm). Briefly, cultures were gently rinsed
in DPBS. A solution of calcein-AM (1:2000 dilution; final
concentration .about.2 .mu.M) and ethidium homodimer-1 (1:500;
.about.4 .mu.M) in DPBS was added to each culture, followed by
incubation at 37.degree. C., 5% CO2 for 30 min. Following
incubation, cultures were rinsed twice in fresh DPBS and imaged at
10.times. magnification on a Nikon.RTM. A1RSI Laser Scanning
confocal microscope paired with MS-Elements.RTM. AR 4.50.00
software. Viability was quantified as the ratio of the total area
of calcein-AM-positive cells to the total area of both
calcein-AM-positive and ethidium homodimer-positive cells using
ImageJ (National Institutes of Health, MD). Sample sizes for each
group were as follows: LE.sub.UNI,5 mm (n=4, 4); LE.sub.BI,5 mm
(n=7, 4); planar cultures (n=9, 5) for 10 and 28 DIV, respectively.
All data presented as mean.+-.s.e.m.
Live Calcium Imaging
[0196] As proof-of-concept for investigating micro-TENN aggregate
connectivity, aggregates were transduced with the genetically
encoded calcium reporters GCaMP6f or RCaMP1b (Penn Vector Core,
Philadelphia, Pa.). After centrifuging aggregates in the pyramid
wells, 1 .mu.L of AAV1 packaged with the human Synapsin-1 promoter
was added to the aggregate wells (final concentration:
.about.3.times.109 viral copies per aggregate). Aggregates were
incubated at 37.degree. C., 5% CO2 overnight before the media was
replaced twice, after which transduced aggregates were plated in
micro-columns as described above. Micro-TENNs were imaged after
7-10 DIV using a NIKON.RTM. Eclipse Ti.RTM. microscope paired with
an ANDOR Neo/Zyla camera.RTM. and Nikon.RTM. Elements AR 4.50.00,
after which calcium transients from the recordings were identified
in NIKON.RTM. Instruments.RTM. Elements AR 4.50.00. The intensities
of selected regions of interest (ROIs) of the micro-TENNs were
plotted over time relative to background (defined as ROIs without
cell bodies or axons).
Functional Analysis
[0197] Fluorescent calcium recordings were collected as described
above to generate .tiff stacks of micro-TENN activity. Each .tiff
stack was composed of 120 seconds of activity recorded at 20 frames
per second (2400 total frames). Functional analyses were performed
using three MATLAB.RTM. software toolboxes--FluoroSNNAP,
MATLAB.RTM. software Statistics Toolbox, and SIFT for EEGLAB.
FluoroSNNAP is an interactive software package designed by Meaney
et al. to perform calcium imaging-based network analysis of neurons
in vitro. Briefly, a time-averaged image was generated from the
.tiff stack, after which ROIs measuring approximately 20 .mu.m in
diameter were manually selected. Using FluoroSNNAP, intensities of
the ROIs were extracted and normalized to assess functional
connectivity patterns within bidirectional micro-TENNs;
specifically, normalized Pearson cross-correlation and normalized
phase synchronization matrices were created from the calcium
transient patterns of the ROIs. Additionally, to assess information
flow across the micro-TENN, the SIFT toolbox was used to fit a
multivariate autoregressive (MVAR) model, which in turn was used to
generate normalized Direct Transfer Function (nDTF) connectivity
matrices. The nDTF has been used in literature to determine
direction and frequency content of EEG activity19,20. Here, it was
applied to detect information flow from one aggregate to the other
in bidirectional micro-TENNs. The nDTF estimates were obtained
using a numerically stable, 10th order MVAR model with an 80-second
sliding window and a 40-second time step. nDTF coefficients were
obtained over 1-9 Hz due to the Nyquist limit.
Immunocytochemistry
[0198] Micro-TENNs were fixed in 4% formaldehyde for 35 min at 4,
10, and 28 DIV. Micro-TENNs were then rinsed in 1.times. PBS and
permeabilized with 0.3% Triton X100+4% horse serum in PBS for 60
min before being incubated with primary antibodies overnight at
4.degree. C. Primary antibodies were Tuj-1/beta-III tubulin (T8578,
1:500, Sigma-Aldrich) to label axons and synapsin-1 (A6448, 1:500,
Invitrogen) to label presynaptic specializations. Following primary
antibody incubation, micro-TENNs were rinsed in PBS and incubated
with fluorescently-labeled secondary antibodies (1:500; sourced
from Life Technologies, Invitrogen, and Jackson ImmunoResearch) for
2h at 18.degree.-24.degree. C. Finally, Hoechst (33342, 1:10,000,
ThermoFisher) was added for 10 min at 18.degree.-24.degree. C.
before rinsing in PBS. Micro-TENNs were imaged on a NIKON.RTM.
A1RSI Laser Scanning confocal microscope paired with MS Elements AR
4.50.00. Sequential slices of 10-20 .mu.m in the z-plane were
acquired for each fluorescent channel. All confocal images
presented are maximum intensity projections of the confocal
z-slices.
Cerebral Cortical and Corticothalamic Implantation
[0199] As proof-of-concept for micro-TENN behavior in vivo,
preformed micro-TENNs with GFP+ neurons/axons were delivered into
the brain via stereotaxic microinjection similar to descriptions in
prior work 12,13. Male Sprague-Dawley rats weighing 325-350 grams
were anesthetized with isoflurane at 1.0-2.0 liters per minute
(induction: 5.0%, maintenance: 1-5-2.0%) and mounted in a
stereotactic frame. Meloxicam (2.0 mg/kg) and bupivacaine (2.0
mg/kg) were given subcutaneously at the base of the neck and along
the incision line, respectively. The area was shaved and cleaned
with betadine solution, after which a small craniotomy over the
sensory cortex was made (coordinates: +4.8 mm AP, .+-.2.3 mm ML).
Bidirectional micro-TENNs (5 mm in length) were loaded into a
needle coupled to a Hamilton syringe, mounted onto a stereotactic
arm for precise placement. To deliver the construct into the brain
without forcible expulsion, the needle was slowly inserted into the
cortex to a depth of 6 mm. The plunger of the Hamilton syringe was
then immobilized, while the needle containing the micro-TENN was
manually raised 5 mm at approximately 1.5 mm/minute. This process
allows for the low-force delivery of micro-TENNs to connect the
whisker barrel cortex with the ventral posteromedial nucleus (VPM),
a deeper thalamic structure.
Tissue Harvest and Histology
[0200] At 7 and 28 days post-implant, rats were euthanized and
perfused with cold heparinized saline and 10% formalin. After
post-fixation of the head overnight, the brain was harvested to
assess micro-TENN survival and host/micro-TENN synaptic
integration. Briefly, brains were sagitally blocked and cut in 40
.mu.m slices for cryosectioning. For frozen sections, slices were
air-dried for 30 minutes, twice-treated with ethanol for three
minutes, and rehydrated in PBS twice for three minutes. Sections
were blocked with 5% normal horse serum (ABC Universal Kit, Vector
Labs, cat #PK-6200) in 0.1% Triton-x/PBS for 30-45 minutes. Primary
antibodies were applied to the sections in 2% normal horse
serum/Optimax buffer for two hours at room temperature. Primary
antibodies were goat anti-GFAP (1:1000), rabbit anti-IBA1 (1:1000),
chicken anti-MAP2 (1:1000), and mouse anti-Tuj 1 (1:1000). Sections
were rinsed with PBS three times for five minutes, after which
secondary antibodies (1:1000) were applied in 2% normal horse
serum/PBS for one hour at room temperature. Sections were
counterstained with DNA-specific fluorescent Hoechst 33342 for ten
minutes and then rinsed with PBS. After immunostaining, slides were
mounted on glass coverslips with Fluoromount-G mounting media.
Micro-TENN In Vitro Development
[0201] Hydrogel micro-columns were optimized in vitro to support
neuronal survival and directed axon growth. Micro-columns were 5-30
mm in length, and consisted of hollow agarose tubes (350-500 .mu.m
outer diameter) to direct axonal outgrowth through a central
extracellular matrix (ECM; 150-400 .mu.m inner diameter).
Dissociated neurons were delivered into the proteinaceous matrix at
one or both ends of the micro-columns, and cultured for 7-42 days
in vitro (DIV) based on the desired length of axonal outgrowth
(FIG. 4). Using electrical stimulation and Ca.sup.2+-sensitive dyes
in bidirectional micro-TENNs, the ability to stimulate one
population of neurons and have the resulting action potentials
travel across the axonal region to the other population was
demonstrated. Micro-TENNs have been generated using multiple
neuronal subtypes, including primary cerebral cortical neurons,
dopaminergic neurons, and dorsal root ganglion neurons (FIG.
5).
[0202] Long micro-TENNs with axon fascicles (.about.1 cm by 14 DIV
and over 2 cm by 28 DIV) were also created as depicted in FIG. 6.
Additionally, smaller micro-TENNs spanning on the order of hundreds
of microns to millimeters were created as depicted in FIG. 5. These
were more appropriately scaled for one of the current application
of living electrodes to penetrate Layer IV or V in the rat
cortex.
[0203] FIG. 17 provides views of immunolabeled long-projecting
unidirectional axonal-based living electrodes. These axon-based
living electrode constructs are on the order of several hundred
microns in diameters--similar to the diameter of a human hair--yet
may extend at least on the order of centimeters to reach deep
layers/nuclei in the brain with a relatively small microinjection
footprint.
Micro-TENN In Vivo Delivery and Survival
[0204] Micro-TENNs were delivered into the brain via stereotaxic
microinjection to provide a bridge of living axons to reconnect
discrete anatomical regions. For in vivo delivery, the hydrogel
casing provided structural support to protect the micro-tissue
during transportation and transplantation. Moreover, the small size
permits minimally invasive implantation into delicate regions of
the nervous system. Micro-TENNs at the desired length were drawn
into a needle, slowly inserted into the cortex, and expelled using
a plunger. Micro-TENNs were stereotaxically injected to connect
thalamic structures with the barrel fields of the cortex to assess
construct survival and integration.
[0205] At 3, 7, and 28 days post-implant, immunohistochemistry and
fluorescent microscopy revealed surviving neurons in the micro-TENN
interior, which maintained a tight cluster with axonal fascicles
extending parallel to the cortical-thalamic axis (FIG. 6). This
demonstrated that micro-TENN neurons survived and maintained their
axonal architecture. Additionally, micro-TENN neurons showed
cortical integration as dendrites from the implanted neurons
penetrated the cortex with structural evidence of synapse formation
between micro-TENN neurons and hist neurons (FIG. 7).
Recording/Stimulation Arrays
[0206] Living electrodes are used as a biological conduit to relay
information to/from neurons deep within the brain to the surface of
the brain. At the surface, arrays of devices are used to either
record or stimulate the superficial end of the micro-TENN. These
arrays could include electrical, optical, magnetic, chemical,
acoustic, or other modality to create a specific and broad array
that is capable of interfacing with the superficial end of the
micro-TENN in a precise, temporal and spatial, manner. These arrays
can be placed at the surface of the brain (subdural), above the
dura (epidural), within the skull defect (intraosseous or
periosseous), outside the skull under the gallea aponeurotica
(subgaleal), or outside the skin (non-invasive on the scalp). These
arrays can be linked to or integrated into microelectronics. They
can include a battery for power, a radiofrequency and/or infrared
induction coil, a light source, multiplex/demultiplex circuitry, a
heat sink leveraging cerebrospinal flow or vascular beds, and/or
embedded waveguides.
Method of Operation
[0207] FIG. 8 describes advantages of living electrodes versus
conventional electrodes and optrodes, highlighting the enhanced
specificity due to synaptic integration of the living
electrodes.
[0208] Preformed 3-D living electrodes provide at least two
clinical deployment advantages: (1) spatial constraint of
transfection agents and cells/axons via transduction in vitro and
(2) avoidance of maturation/attrition issues from cell
suspension.
[0209] The living electrode provides a soft pathway to route
signals to/from deep brain structures versus rigid materials used
in electrodes/optrodes, thus mitigating a chronic foreign body
response, mechanical separation issues, and glial scarring.
[0210] While optrodes can achieve a high level of specificity,
optical methods may have a limited extent due to tissue absorption.
Electrodes inherently stimulate or record from a fixed volume
around the electrode (red zone in FIG. 8). Spread of optogenetic
transduction is illustrated by yellow neurons in multiple layers in
FIG. 8). In contrast, living electrodes offer high specificity, as
the constructs can be designed to synapse with specific neuronal
subtypes, and thus may form many synapses with surrounding neurons
(FIGS. 8 and 9).
[0211] FIGS. 10 and 11 illustrate examples of the living electrode
to be used for recording or stimulation paradigms in the rat
cortex. The operation of the living electrode is not relegated to
just the cortex, but can be employed anywhere in the brain where
nonsuperficial signals need to be recorded or stimulated.
Additionally, the input/stimulation version of the micro-TENN can
be designed to interface with a specific type of neuron in order to
selectively activate excitatory or inhibitory neurons. Although a
rat is pictured here, the technology could be ultimately used in
non-human primates and humans. A further iteration of the living
electrode could include the usage of multiple neural types to
achieve interaction with multiple neural subtypes. For example, one
might want a living electrode that excites one neural population
while inhibiting another population of neural cells (FIG. 1). As
previously mentioned, the living electrode could also be developed
to be a true bidirectional interface modality to enable closed loop
interaction with the brain. This living electrode would be able to
record, process, and then stimulate the brain, in controlled closed
loop manner to provide refined and well-controlled stimulation of
the nervous system to treat disease with a delicate hand instead of
the brute force method using constant application of electrical
stimulation currently.
Tissue Engineered Constructs for Neurosurgical Implantation
[0212] As shown in FIG. 12, living electrodes can be implanted and
interface with the brain to record or stimulate neurons for the
treatment or as a diagnostic of a particular function or
dysfunction of the nervous system (i.e., Parkinson's disease,
obesity, inflammation, migraine, diabetes, epilepsy, etc.). For
example, in a patient with Parkinson's disease dopaminergic living
electrodes restore dopamine inputs to deep brain structures (e.g.
striatum) with brain surface control of activation. For patients
with epilepsy GABAergic living electrodes could be used to inhibit
seizure foci, with activation upon detection of early epileptiform
activity (e.g., inhibitory living electrodes can precisely deliver
copious GABA directly to foci at the earliest sign of pre-seizure
neural activity).
[0213] Implanted living electrodes can also record or stimulate
neurons for the precise and selective interface between biological
and non-biological entities for the restoration or augmentation of
function in persons with or without locked-in syndrome to affect
action via thought or for input of sensory information. For
example, motor control (reading activity of neurons in the motor
cortex or motor output hub in the brain or spinal cord) for device
(e.g., robotic hand/arm) actuation can be monitored. Sensory
feedback (inputting information to the sensory cortex or a sensory
processing hub in the brain) for feel and/or proprioception of
external device (e.g., robotic hand/arm) can also be recorded.
[0214] Living electrodes can also be implanted and interface with
the brain to use the activation as a method to enhance regrowth or
improvement of function. For example, implantable living electrodes
can control local neural activity to facilitate/elicit endogenous
regeneration. In another example, living electrodes can control
local neural activity to enhance healthy network function and/or
attenuate/block deleterious network function (e.g. rectify issues
with circuit timing).
Confocal Reconstruction of Macro-TENN in Vitro: Proof of
Concept
[0215] Applicant grew primary dorsal root ganglia neurons in
macro-TENN constructs over 7 days in vitro (DIV). Following this
period, the cells were fixed and immunolabelled with markers for
cell nuclei (DAPI) and their axons (beta-tubulin-III/Tuj-1). The
constructs were then imaged on a confocal microscope. The images
were reconstructed and stitched to provide a detailed overview of
the construct in all planes (FIG. 14). What the images showed are
best represented by the cartoon in FIG. 14, Panel D, which
represents the reconstruction pictured in FIG. 14, Panel E looking
in they direction of the construct (i.e., looking down the length
of the construct). FIG. 14, Panels D and E depict that the axons
are only growing along the inner surface of the tube and not in the
extracellular matrix that fills the core of the construct. In
smaller constructs, the axons grow through the entire cross-section
of the construct, not just the interface as seen here. Looking from
above the construct, FIG. 14, Panels A-C show sequential slices of
the confocal image, starting from bottommost (A) to topmost (C). In
FIG. 14, Panel C, the axons are only growing along the sidewalls,
while in FIG. 14, Panel B at the bottom of the construct, the axons
are spread out along the entire surface of the bottom.
Proposed Device in PNS
[0216] Based on the findings showing axonal regrowth only at the
interior face of the macro-TENN, Applicant proposes to create a
regenerative electrode as depicted in FIGS. 15A-15C. The deployment
of this device would allow for the growth of axons through an
unimpeded channel (contrary to sieve electrodes). The channel could
be the same size of the nerve or larger to provide further
spreading and defascicularization of a particular nerve and its
fascicles. Electrodes could be placed along the periphery and along
the length of the construct to allow for selective stimulation and
recording (FIGS. 15A and 15B). If the construct were to be sized
larger than the nerve, the construct could taper at the proximal
and distal ends to enable suturing and attachment to the nerve of
choice (FIG. 15C). A larger cross sectional area advantageously
allows the axons/fascicles to spread out further, thereby providing
more selectivity. Embodiments of the invention provide superior
conformation to the interior of the side wall of the tube. Without
being bound by theory, Applicant believes that the conformation is
superior because of a greater surface area due to use of the
interior of the tube versus midplane technology. Again, without
being bound by theory, Applicant believes that the greater surface
area would allow for greater separation of axons/fascicles and
therefore greater selectivity.
Further Micro-TENN Fabrication Techniques
[0217] Further embodiments of the invention provide novel
micro-tissue engineering methodology to more consistently create
micro-TENNs of the desired architecture consisting of discrete
neuronal population(s) spanned by pure axonal tracts. In
particular, embodiments of the invention utilize "forced cell
aggregation" within custom-build pyramidal micro-wells to create
"aggregates" or "spheres" of neurons with precise control of the
number of neurons--and hence diameter--per aggregate/sphere as
depicted in FIG. 16, Panels C-E. To accomplish this, dissociated
neurons were transferred to a chamber containing an array of
inverted pyramid micro-wells made in PDMS (SYLGUARD.RTM. 184, Dow
Corning) cast from a custom-designed 3D-printed mold. The wells
were then centrifuged at 200 g for 5 min. This centrifugation
resulted in forced aggregation of neurons (or any other cell type
if desired) with precise control of the number of neurons per
aggregate/sphere based on the density of neurons used and the
volume added to each well. 12 .mu.L of neuronal suspension per well
at a density of 1-2 million cells/mL was suitable to create
aggregate/spheres of appropriate diameter to fit within
micro-columns. The aggregates/spheres were then carefully placed
within one or both ends of the micro-columns and allowed to adhere
for 45 min at 37.degree. C., 5% CO.sub.2. Seeded micro-columns were
then allowed to grow in neuronal growth media consisting of
NEUROBASAL.RTM. Media, GLUTAMAX.TM. media, and B-27.RTM. media
(ThermoFisher.TM.) over at least several days in vitro to form
micro-TENNs (as described in Laura A. Struzyna et al., "Rebuilding
Brain Circuitry with Living Micro-Tissue Engineered Neural
Networks", 21(21-22): 2744-2756 Tissue Engineering Part A (2015)
and J. P. Harris et al., "Advanced biomaterial strategies to
transplant preformed micro-tissue engineered neural networks into
the brain," 13(1) J. Neural Eng. 016019 (2016). This methodology
resulted in the formation of uni- or bi-directional micro-TENNs
with defined neuronal somatic regions and axonal extension of
several millimeters over a few days in vitro (FIG. 17). Notably,
this methodology consistently produces the ideal micro-TENN
cytoarchitecture consisting of a defined zone with neuronal somata
as aggregates at one or both ends of the micro-column and a defined
zone with axonal projections running longitudinally to span the
central portion of the micro-column (FIG. 17). This ideal
distribution was further verified by immunocytochemistry and
confocal microscopy to label these aggregate micro-TENNs using
antibodies recognizing all axons (beta-tubulin III) and all cell
nuclei (Hoechst), and the hydrogel comprising the micro-column is
non-specifically labeled. Also, this micro-TENN was labeled for a
synaptic marker (synapsin), suggesting functional maturation and
electrochemical activity in the micro-TENNs.
[0218] Applicant advanced micro-tissue engineering methodology to
improve the consistency of the biomaterial construction of the
micro-columns. Here, a commercially available BIOBOTS.TM. 3D
printer designed for tissue engineering applications was used to
print the micro-columns from hyaluronic acid or a similar hydrogel.
Micro-columns were printed at outer diameter C of 200 .mu.m and
inner diameter A of 100-150 .mu.m inner diameter while concurrently
being filled with a bioactive extracellular matrix (generally 1
mg/mL collagen and/or 1 mg/mL laminin), leaving a gap having a
depth G of 100 .mu.m on one or both ends of the micro-column free
for cell aggregate delivery as depicted in FIG. 18. The micro-TENN
can be printed with a central radial axis oriented horizontally or
vertically. Horizontal printing may be preferred for speed and/or
so that any ridges, grooves, or other artifacts from printing run
parallel to the central radial axis and can act as a guide for
neural growth.
Use as Biological Interface
[0219] Embodiments of the invention can be utilized as interfaces
between hosts and various electronic devices. Although embodiments
of the invention have been described in the context of optical or
magnetic sensors, embodiments of the invention can also support
interfacing via electrical impulses applied to neurons within the
living electrodes.
[0220] As discussed herein, tissue engineered "living electrodes"
will allow a stable long-term interface to probe and modulate the
nervous system. "Living electrodes" increase target specificity
while mitigating foreign body response inherent in non-organic
electrodes.
[0221] Without being bound by theory, Applicant believes that the
invention described herein operates via one or more of the
following mechanisms of action. First, embodiments of the invention
provide target specificity by integrating with specific neuronal
subtype(s) while mitigating chronic foreign body response. Second,
embodiments of the invention provide synaptic integration that
offers permanence not possible with prior approaches. Third,
biological multiplexing is possible through the robust effects
elicited by relatively few axons. Integration of tailored "living
electrodes" is synaptic-mediated with specific cells/regions in the
brain. Synaptic integration via engineered axonal tracts offers a
permanence and target specificity not possible with conventional
approaches.
[0222] Synaptic-based interfaces using engineered neurons and/or
axonal tracts can form a biological link between host and
electronics, ultimately enabling prosthetic control,
sensory/proprioceptive feedback, and/or neuromodulation.
[0223] A robust effect, i.e., the recruitment of numerous host
neurons, can be elicited by relatively few axons via a novel
mechanism referred to as "biological multiplexing". For instance,
one micro-TENN axon can (in theory) synapse with hundreds or even
thousands of neurons, creating a significant amplification effect.
Applicant currently builds micro-TENNs with .about.50,000 neurons
within a column approximating the diameter of a human hair--thus
presenting the potential to affect millions of host neurons with a
single construct.
[0224] In various embodiments, neuron phenotypes can be selected to
release/secrete certain specific neurotransmitters to restore
levels relevant to particular disease processes. In various
embodiments, constructs can be tailored to specific lengths to
achieve connectivity at specific anatomical targets and synapse
with specific neuronal subtypes within those regions. In various
embodiments, the proteinacious matrix and co-delivered factors can
be altered to haptotactically and chemotactically attract specific
host neuron-types to be targeted by the modulation. In various
embodiments, the construct neurons can target specific neurons
without any extraneous, artifactual activation (provided that
synaptic specificity is achieved). In various embodiments the
constructs can be seeded with one or more populations of neurons,
these living neural networks can perform multiplexing operations
both within themselves and by achieving high information targeted
output to parenchyma.
Micro-TENNs Plated with Dissociated Neuronal Suspensions
[0225] Dopaminergic neurons were isolated from the ventral
mesencephalon of embryonic rats. In planar culture, these neurons
demonstrated a healthy neuronal morphology, the presence of
dopaminergic neurons (based on TH expression), significant neurite
outgrowth (based on .beta.-tubulin III expression), and network
formation out to 28 DIV. To create dopaminergic micro-TENNs,
initially seeded micro-columns using dissociated neuronal
suspensions were used. These dissociated cells infiltrated the
length of the inner lumen and generally did not produce the desired
cytoarchitecture of a discrete cell body region projecting axons
across the length of the inner core.
[0226] However, the dissociated neurons within the micro-tissue
constructs presented a healthy morphology, and occasionally
self-organized into the desired cytoarchitecture by chance. In
these cases, unidirectional axonal projections achieved lengths of
several millimeters, and, importantly, the health of these
constructs was also maintained out to 28 DIV. In order to see if
the inclusion of additional ECM in the inner core increased the
consistency with which the correct architecture was generated,
dissociated cells were suspended into collagen, and injected the
mixture to gel inside the micro-columns.
[0227] Unfortunately, the presence of the collagen did not aid in
producing the desired cytoarchitecture, and the dissociated cells
continued to spread throughout the length of the inner core (FIG.
23, panels A1-A3). While these results demonstrated the ability to
culture dopaminergic neurons that formed extensive neurite networks
within hydrogel micro-columns, these techniques were not sufficient
to consistently generate the desired cytoarchitecture.
Forced Neuronal Aggregation Method
[0228] As previous micro-TENN fabrication methods did not reliably
generate the desired cytoarchitecture, a method to mechanically
group neurons into aggregates was adapted (Ungrin M D, Joshi C,
Nica A, et al. 2008, Reproducible, ultra high-throughput formation
of multicellular organization from single cell suspension-derived
human embryonic stem cell aggregates, PloS one, 3 (2): e1565.).
After dissociating embryonic tissue into a single cell suspension,
this solution was centrifuged in inverted pyramidal wells in order
to pellet the cells at the bottom of the wells. The wells were left
in the incubator overnight, during which the pelleted cells became
aggregated spheres of neurons. Once formed, the aggregates were
inserted into the ends of the agarose micro-columns. This method
consistently produced micro-TENNs with distinct cell body and
axonal regions. Furthermore, it was found that based upon the depth
and placement of the aggregate within the micro-column, it was
possible to create micro-TENNs that exhibited either an
externalized or internalized cell body region (FIG. 23, panel B-D).
Moreover, this technique produced long-projecting unidirectional
axonal tracts, as demonstrated based on TH and .beta.-tubulin III
immunoreactivity (FIG. 23, panel E-F). Indeed, as measured by the
length of the longest neurite in each micro-TENN, it was determined
that the axons projecting from the aggregates grew approximately
10.times. longer than analogous axons extending within microcolumns
seeded with dissociated neurons (FIG. 23, panel G).
Optimization of Micro-TENN Length
[0229] The nigrostriatal pathway measures approximately 6 mm in the
rat, therefore micro-TENNs at least 6 mm in length are desirable.
The effects of the ECM constituents in the inner lumen, presence of
growth factors, and the micro-TENN directionality on outgrowth were
tested in order to optimize growing conditions for length. It was
found that collagen I and collagen I and laminin resulted in the
longest axonal outgrowth, as measured by the length of the longest
neurite in each micro-TENN (FIG. 24). The average axonal outgrowth
for the collagen I and collagen I and laminin cores was 4892.+-.703
.mu.m and 4686.+-.921 .mu.m respectively. In contrast, it was found
that crosslinked collagen (1227.+-.481 .mu.m), laminin-coated
(205.+-.615 .mu.m), and empty cores (.about.0 .mu.m) resulted in
significantly reduced neurite outgrowth. For the two highest
performing groups (lumen comprised of collagen or collagen and
laminin), it was determined that TH+ dopaminergic axonal
projections attained at least 60% of the maximal axonal length
(FIG. 24, panel F). The effect of the media growth factor
concentration on axonal outgrowth within the micro-columns was also
tested. A media containing a relatively low concentration of bFGF
(4 ng/mL) was compared to media containing high concentrations of
growth factors previously shown to increase dopaminergic neuron
outgrowth and survival. At 14 DIV, it was found that the high
growth factor concentration media did not result in denser or
longer axonal outgrowth compared to the low concentration media
(n=14 micro-TENNs in each group). Lastly, it was investigated
whether the use of a target population of dopaminergic cells would
increase axonal outgrowth. Bidirectional dopaminergic micro-TENNs
were plated by inserting dopaminergic aggregates into both ends of
the micro-columns. While the two dopaminergic neuron populations
were separated by 1.2 cm, in order to determine if chemotactic
signaling between the populations would increase outgrowth. At 14
DIV, it was determined that the axonal outgrowth in bidirectional
micro-TENNs was not greater than axonal outgrowth in unidirectional
micro-TENNs (n=14 micro-TENNs in each group). Thus, the use of
engineered neuronal aggregates and specific ECM constituents were
critical factors in axonal extension, while high growth factor
media and the presence of a target neuron population did not affect
axonal outgrowth. Of note, the mean neuronal aggregate length at 14
DIV was 1165.+-.212 .mu.m; therefore, the total micro-TENN length
(neuronal aggregate+axon length) attained using dopaminergic
aggregates in collagen was >6 mm by 14 DIV--suitable to span the
nigrostriatal pathway in rats. Following optimization studies,
dopaminergic micro-TENNs with an inner core of collagen I were
fabricated and allowed to grow over 28 DIV to ascertain if axonal
extension progressed further within the micro-columns. Continued
axonal extension out to 28 DIV was found, with lengths of
6046.+-.670 .mu.m for dopaminergic axons, 7697.+-.1085 .mu.m for
all axons, and 8914.+-.1187 .mu.m for total aggregate+axon lengths.
In some cases, maximum total micro-TENN lengths at this time point
were over 10 mm, well beyond what would be required to span the
nigrostriatal pathway in rats (FIG. 25).
Formation of Synapses with Striatal Population
[0230] As the dopaminergic axons comprising the nigrostriatal
pathway synapse with striatal neurons in the brain, the ability of
tissue engineered nigrostriatal pathways to synapse with a
population of striatal neurons in vitro was probed. Dopaminergic
micro-TENNs were generated and, after 10 DIV, embryonic rat
striatal aggregates were inserted into the vacant ends of the
micro-columns. After 4 more DIV, immunocytochemistry was performed
in order to assess potential synaptic integration between the two
populations. This analysis confirmed the presence of the
appropriate neuronal sub-types in the two aggregate populations,
specifically TH+ dopaminergic neurons and DARPP-32+ medium spiny
striatal neurons (FIG. 26). Moreover, confocal microscopy revealed
extensive axonaldendritic integration and putative synapse
formation involving the dopaminergic axons and striatal neurons
(FIG. 26, panels D,E,G,H). Also, immunocytochemistry confirmed that
the majority of the striatal (DARPP-32+) neurites were also MAP-2+,
suggesting that these were dendrites (data not shown). In order to
determine if chemotactic cues generated by the striatal population
influenced axonal outgrowth from the dopaminergic neuron
aggregates, the length of axonal outgrowth was quantified with and
without the striatal end target. It was found that the axonal
outgrowth in dopaminergic micro-TENNs containing a target
population of striatal neurons was not statistically greater than
axonal outgrowth in unidirectional dopaminergic micro-TENNs (n=9
micro-TENNs in each group; FIG. 26, panel F).
Transplant and Survival of Preformed Dopaminergic Micro-TENNs In
Vivo
[0231] In order to demonstrate the ability to precisely deliver
preformed dopaminergic micro-TENNs into the brain as well as their
survival and architecture at various time points post-implant,
dopaminergic aggregate micro-TENNs with an inner lumen containing
collagen I were transduced to express GFP and grown for 14 DIV,
after which time they were drawn into a custom needle and
stereotaxically micro-injected to approximate the nigrostriatal
pathway in adult male Sprague-Dawley rats. Animals were sacrificed
at 1 week and 1 month time points (n=5 each), revealing surviving
GFP+ neurons and axons within the micro-TENN lumen, which was
easily identified spanning the nigrostriatal pathway since the
agarose micro-column had only partially degraded at these time
point (FIG. 27). Histological sections were co-labeled for the
axonal marker .beta.-tubulin III and the dopaminergic marker TH,
revealing the preservation of a robust neuronal and dopaminergic
axonal population. In particular, longitudinally projecting TH+
axons were present, which confirmed that the micro-TENNs were able
to maintain their cytoarchitecture following transplantation into
the brain (FIG. 27).
Quantification of Living Electrode Growth In Vitro
[0232] In earlier work, micro-TENNs were seeded with single cell
suspensions of primary cortical neurons, which in many cases formed
clusters at random sites throughout the micro-TENN interior (FIG.
29, panel C-D). Current-generation micro-TENNs were formed with
cortical aggregates that have been preformed prior to plating in
the micro-columns, allowing for greater control and reproducibility
of the desired cytoarchitecture of discrete somatic and axonal
zones (FIG. 29, panel F-H). This reproducibility lends itself to
robust analysis of micro-TENNs in vitro, a necessary step in
applying them as living electrodes. Aggregate micro-TENNs were
plated with approximately 8,000-10,000 neurons per aggregate, with
micro-column lengths of 2 mm and 5 mm. Both unidirectional (with
one aggregate) and bidirectional (with two aggregates) 2 mm-long
micro-TENNs were plated (LE.sub.UNI,2 mm and LE.sub.BI,2 mm), while
all 5 mm-long LEs (LE.sub.BI,5 mm) were plated as bidirectional
constructs. Growth characteristics of prior dissociated micro-TENNs
were compared to current aggregate-based constructs by plating 2
mm-long dissociated micro-TENNs (LE.sub.DISS,2 mm) (Table 1, FIG.
30). Healthy axonal outgrowth was found across all aggregate LEs
along the ECM core within the first few days in vitro through
analysis of phase microscopy images (FIG. 30). Aggregate LE neurons
within the LE.sub.BI,5 mm group displayed rapid axonal growth rates
peaking at 1087.7.+-.84.3 microns/day across all measured DIV.
Neurons within the LE.sub.UNI,2 mm group exhibited an initial peak
axonal growth rate of 358.+-.19.8 microns/day on day 1 that
declined steadily over time. Within the LE.sub.BI,2 mm group,
neuronal processes had crossed the length of the micro-column and
synapsed with the opposing population by 5 DIV (FIG. 30, panel A),
with a concomitant decrease in growth rate (FIG. 30, panel D).
Although neurons from the LE.sub.BI,5 mm group grew at a similar
rate to LE.sub.BI,2 mm neurons for the first three DIV, they
exhibited a significant increase in growth rate by 5 DIV and
subsequent slowing of axonal growth as the synapses were formed
across aggregates (FIG. 30, panel C-D). Growth rates in all
aggregate micro-TENN groups surpassed those of dissociated
micro-TENNs, which reached a maximum growth rate of 61.7.+-.5
microns/day at 1 DIV (Table 1).
[0233] Two-way ANOVA revealed significant main effects from the DIV
(F-statistic=15.97) and LE group (Fstatistic=27.4), as well as
their interaction (F-statistic=5.92), all at p<0.0001. As such,
Bonferroni analysis was used for subsequent pairwise comparisons,
revealing several statistical differences both within and between
LE groups at different DIV. In general, the growth rates for
bidirectional micro-TENNs were greater at earlier timepoints than
later timepoints, while the growth rates for dissociated and
unidirectional micro-TENNs did not vary significantly over time.
Notably, the growth rate of LE.sub.BI,5 mm at 5 DIV was greater
than all growth rates for LE.sub.DISS,5 mm<0.0001), LE.sub.BI,2
mm (p<0.0001), and LE.sub.BI,2 mm (p<0.001). Moreover, within
the LE.sub.BI,5 mm group itself, the growth rate at 5 DIV was
statistically greater than that of all other DIV (p<0.01). The
growth rate of LE.sub.BI,2 mm at 3 DIV was also greater than all
growth rates for LE.sub.DISS,2 mm (p<0.01). The growth rate at 1
DIV was greater than that at 10 DIV for both LE.sub.BI,5 mm
(p<0.001) and LE.sub.BI,2 mm<0.01).
TABLE-US-00001 TABLE 1 Micro-TENN Growth Rates. Data presented as
mean .+-. s.e.m. in units of microns/day. LE.sub.UNI, 2 mm
LE.sub.BI, 2 mm LE.sub.BI, 5 mm LE.sub.DISS, 2 mm 1 DIV 358.548
.+-. 462.720 .+-. 585.328 .+-. 61.724 .+-. 19.839 14.118 43.337
5.009 3 DIV 312.800 .+-. 532.548 .+-. 453.618 .+-. 23.244 .+-.
27.483 39.930 30.928 3.406 5 DIV 185.425 .+-. 262.589 .+-. 1087.715
.+-. 9.480 .+-. 19.793 23.375 84.269 1.491 8 DIV 97.423 .+-. 40.819
.+-. 373.588 .+-. 29.757 .+-. 14.406 13.606 64.096 4.348 10 DIV
29.376 .+-. 0 0 -5.377 .+-. 7.595 7.501
Bidirectional micro-TENNs labeled with GFP and mCherry were imaged
over time to observe interactions between axonal projections from
each aggregate (FIG. 31). Confocal images revealed that upon making
contact with opposing axons, projections continued to grow along
each other towards the opposing aggregate, confirming physical
interaction between the two neuronal populations (FIG. 31).
Acute and Chronic Viability of Living Electrodes
[0234] Survival was quantified via live/dead staining and confocal
microscopy for short unidirectional and short bidirectional LEs at
10 and 28 DIV (FIG. 32). Age-matched planar cultures served as
controls. Viability was defined as the ratio of the summed area of
calcein-AM-positive cells to that of all stained cells (i.e. both
calcein-AM+ and ethidium homodimer+ cells). Neuronal survival in
living electrodes was observed to persist up to at least 28 DIV,
with evidence of survival out to 40 DIV (FIG. 32). ANOVA showed
that although the DIV was a significant main effect
(F-statistic=32.21, p<0.0001), the LE/culture group was not
(p>0.84). The interaction effect was also found significant
(p<0.01), so Bonferroni analysis was used to compare groups at
each time point (FIG. 32, panel C). Survival of planar cultures at
28 DIV was found statistically lower than that of LE.sub.UNI
(p<0.05), LE.sub.BI (p<0.001), and planar cultures
(p<0.0001) at 10 DIV. Moreover, planar culture viability at 10
DIV surpassed those of both LE.sub.UNI and LE.sub.BI at 28 DIV
(p<0.01).
Architecture and Synaptogenesis in Living Electrodes Over Time
[0235] To characterize LE architecture over time, bidirectional LEs
were fixed at 4, 10, and 28 DIV and immunolabeled to identify cell
nuclei, axons, and synapses (FIG. 33). Neuronal somata were
localized almost exclusively to the aggregates, which were spanned
by long axons, as indicated with Tuj-1 (FIG. 33); axons and
dendrites were also found within the aggregates from
intra-aggregate connections, presumably formed upon or shortly
after plating. Synapse presence was qualitatively assessed using
the sum area of synapsin 1-positive puncta across the specified
timepoints. A modest distribution of synapsin within micro-TENN
aggregates was observed, as well as an increase in synapsin
expression within the lumen of the micro-columns, suggesting that
neurons within bidirectional micro-TENNs may have the capacity to
communicate across aggregates.
Corticothalamic Implantation
[0236] Preformed micro-TENNs--fabricated as described above to
consist of aligned axonal tracts projecting from a neuronal
aggregate encased in a tubular hydrogel micro-column--were
implanted to replicate corticothalamic pathways by connecting the
whisker barrel cortex with the VPM. One month-post injection in the
rodent brain, GFP+ micro-TENNs were found to have survived and
maintained the preformed architecture of somatic-axonal
distribution (FIG. 34). Large, dense clusters of GFP+ cell bodies
(aggregates) were found at the dorsal and ventral regions of
implantation, with axons and dendrites within the lumen spanning
the two locations (FIG. 34).
Equivalents
[0237] Although preferred embodiments of the invention have been
described using specific terms, such description is for
illustrative purposes only, and it is to be understood that changes
and variations may be made without departing from the spirit or
scope of the following claims.
INCORPORATION BY REFERENCE
[0238] The entire contents of all patents, published patent
applications, and other references cited herein are hereby
expressly incorporated herein in their entireties by reference.
* * * * *