WO2012166719A1

WO2012166719A1 - Strategies and motifs for practicing the micropatterning of cells

Info

Publication number: WO2012166719A1
Application number: PCT/US2012/039839
Authority: WO
Inventors: Wesley C. Chang
Original assignee: Aperys, Inc.
Priority date: 2011-05-27
Filing date: 2012-05-29
Publication date: 2012-12-06

Abstract

Apparatus, patterns, micropatterns and methods cultivate, grow, align and/or organize cells and/or control, guide or direct cell growth and cell interactions. Methods, patterns and apparatus for organizing or patterning cells are provided which may include at least one node configured to receive a cell body and at least one lane extending from the node. Growth of a cell process of the cell body may be directed from the node into the lane in a predetermined manner or direction, which may be determined by a configuration of the node or lane.

Description

STRATEGIES AND MOTIFS FOR PRACTICING THE MiCROPATTERNINC OF

CROSS-REFERENCE TO RELATED APPLICATIONS jOQtilJ The present application claims priority to U.S. Provisional Application Serial No. 61 /491 ,159 filed May 27, 201 1 , the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

10 021 This application is directed to various methods, devices and patterns for cultivating srowing, auidin¾ and/or organizing cells under various conditions, e.g., in vitro conditions,

BACKGROUND

| t>03| Neuroscience fundamentally deals with cell-cell communication involving highly precise spatial relationships between neurons, glia, axonai, and dendritic processes.

Phenomena ranging from cell, migration and axon pathflnding, to synaptic function/plasticity and disease, all involve interactions between specific cell populations at many levels. These advances have been aided by experimental approaches involving defined, in vitro culture systems, which scientists are increasingly turning to as they seek insights into the mechanisms of action of individual genes/proteins identified by in vivo analysis.

jtM HJ A key limitation of current neuronal cell culture is the inherent random

organization of cell bodies and their processes in vitro. Although many different neuronal and giia cell types can be isolated and placed in culture simply by pipetting, conventional approaches do not allow a researcher to dictate the precise location of cells and their processes, nor specify particular cell-cell relationships that are critical for biological phenomena of interest.. As a result, researchers utilizing in vitro approaches fe.ee obstacles when attempting for example, to resolve individual axons from cell bodies and other processes, follow individual neurons/axons over days or weeks, or identify interconnected

Page I of 37 pre-aad postsynaptic neurons for electrophysiological study. The ability to resolve and study the connections of nerve cells in vitro is a fundamental requirement of broad areas of neuroscience research and therapeutic discovery. Scientists engaged in these and other studies requiring high cellular resolution struggle to obtain sufficient sample she, must contend with variability inherent in random systems, and often resort to complex image processing algorithms for data, analysis.

0 5 Furthermore, without the ability to reproduce specific cell-cell relationships in large formats, a broad range of questions involving direct neuron-neuron orneuron-glia interactions in basic biology and disease is difficult to approach, and the potential application of screening paradigms is curtailed. Therefore, there remains a need for a more effective and efficient method, apparatus and/ r design for organizing and directing ceil growth and cell interactions.

SUMMARY

{ 01) 1 Various apparatus, patterns and methods for cultivating, growing, aligning and/or organizing cells and/or controlling, guiding or directing cell growth and cell interactions are described herein.

[00071 In certain variations, a method for organizing or patterning cells may include one or more of the following steps. At least one node configured to receive a cell body may be provided. At least one lane extending from the node may be provided. Growth of a cell process of the cell body may be directed from the node into the lane i a predetermined manner or direction, which may be determined by a configuration of the node leading into the lane.

(90O8| An apparatus for organizing or patterning cells may include a surface or substrate having at least one node configured to receive a cell body. A lane may extend or emanate from the node. At least a portion of the node may be configured to direct a cell process of a cell body torn the node into the lane in a predetermined manner or direction, which maybe determined by a configuration of the node leading into the lane.

}000 j In certain variations, the predetermined manner or direction in which the cell process is directed or guided may be based on or determined b a configuration of the node, a configuration of a transition from the node to the l ne, and/or a configuration of the lane. Optionally, the node raay be tapered, curved or asymmetric to direct or guide the growth of a ceil process into from a node into a desired lane.

J0O1 Θ] In certain variations, a method for organizing or patterning cells may include depositing at least one cell on or near at least one node. The node may have at least one km extending therefrom. Growth of a cell process of the cell may be directed, or guided from the node into the lane in a predetermined manner or direction, which may be determined by a configuration of the node, a configuration of a transition from the node to the lane and/or a configuration of the lane.

[00111 In certain variations, a method for organizing or patterning cells may include providing a network of nodes and lanes. At least one lane may extend or emanate front at least one node, and at least $ome of the nodes and lanes may be interconnected. Growth of a celt pr cess of a cell positioned in the network may be directed in a desired or predetermined direction to isolate a single cell process on a lane, while allowing the ceil to remain connected to other cells in a manner that mimics the naturally occurring connectivity of the cell,

[0012 j In certain variations, an apparatus for organizing or patterning cells may include a network of nodes and lanes, wherein at least one lane extends from at least one node, and at least some of the nodes and lanes are interconnected. The network of nodes and lanes may be configured to direct growth of a cell process of a cell which may be positioned therein in a desired or predetermmed direction to isolate a single cell process on a lane, while allowing the cell to remain connected to other cells in a manner that mimics the naturally occurring connectivity of the cell

BRIEF DESCRIPTION OF THE DRAWINGS

[O013J Figure 1 A illustrates an example of cell migration on a substrate having a node and lane arrangement

[0Ot4j Figure I 8-lC illustrate variations of a node and lane micropatterns.

10015) Figures 1 D- i f illustrate neuronal cells seeded on a lattice micropattern.

jOOl 61 .Figure 2A illustrates a variation, of a node and lane arrangement including lanes of various length. [0017J Figure 2B illustrates a Purkinje neuron in the cerebellura and a granule eel! .

[00181 Figures 3A-3B illustrate variations of a node and lane micropattern.

[00191 Figure illustrates a variation of a node and lane array,

J 0020 J Figures 5A-5B illustrate variations of micropattems having cell body domain regions having.

[00211 Figure illustrates a variation of a method of seeding ceils into distinct regions on culture substrates.

[00221 Figure 7 illustrates a variation of a method tor guiding cell processes into desired lanes.

[00231 Figure 8 A illustrates a variation of a lattice micropattern.

[0024[ Figures 8B-8C illustrate variations of lattice micropatie s.

[0025_.1 Figures 8D-8E illustrate variations of lattice micropattems having cell body domain regions and & paralle l array of lanes.

[00261 Figures 9 A illustrates a variation of a lattice micropattern having a parallel array of lanes.

[00271 Figures 9.B illustrates neurons projecting axons on the lattice micropattern of Figure 9A.

[00281 Figures i OA- 1.0B illustrate variations of synapse biology micropattems and arrays.

[0029j Figures i 1 A-l IB illustrate variations o synap.se biology arrays including cell body domains,

[00301 Figure 11 C illustrates a variation of a micropattern on which three or more neuronal populations may be deposited.

100311 Figures I2A-I2B illustrate variations of a method for overlaying micropattems on a microelectrode array.

[00321 Figures 12C illustrates a variation of a node and lane lattice raicropaitera overlaid on lop of a pair of planar raicroelectrodes.

[00331 Figure 12D illustrates that electrical stimulation by the electrodes of Figure- 12C was focused on neurons on node 1. DETAILED DESCRIPTION

[6034] Various apparatus, patterns and methods for cultivating, growing, aligning and/or organizing cells and/or controlling, guiding or directing eel! growth, behavior and cell interactions are described herein.

10035} In certain variations, various, patterns, micropatterns, customized micropatterns, apparatus, and methods for utilizing the micropatterns to organize various types of cells and to dictate the manner and/or direction in which cell and/or cell process may grow are described herein.

[8036} In certain variations, customized .micro patterns, apparatus, methods, motifs and strategies or templates or substrates for micropattem dessgn for any eel! type, for example, for addressing the unique properties of nerve cells and their morphological, differentiation and. elaboration of long specialized subcellular processes called axons and dendrites are described herein. In certain variations, an application for various customized .micropatterns and patterning motifs is to address the need of investigators for organizing axon and synaptically connected nerve cells into neat arrays for increased experimental throughput, new research paradigms, and to facilitate the use of screening modalities in neuroseience research, in addition to the strategies described, micropatteming can also be used to align neurons and other cells with mtcroe!ectnxle arrays.

[8037} In certain variations, the growth and organization of neurons may be controlled utilizing various micropatterns described herein. Geometric sizing of adhesive micropatterns may be used to segregate single neurons and their axons (Chang & Sreiavan Langmuir, 2008,

24 (22), pp 13048-13057). Single axons may be grown and patterned, e.g., onto 3 -2 micron wide lanes. Neuronal cell bodies, however, do not differentiate on such narrow lanes and prefer to attach to nodes or regions that are, e.g., 20-30 microns or more, in diameter. As a result, customized micropatterns may be developed to organize the locations of neuronal ceil bodies and their axons to suit experimental convenience, throughput, and novel research applications,

18038} When cells are deposited on narrow lanes, e.g., lanes having widths less than the diameter of a cell ^'body, locomotion and migration behavior of the cells may be observed. The lanes may include a cell adhesi ve material Optionally, an array of such lanes may be utilized to recapitulate this behavior in large numbers of cells, e.g., on a single substrate.

[00391 Figure I A shows an example of cell body migration. The figure shoes how cell bodies deposited on a lane can undergo locomotion and migrate from the lanes to the nodes. 10040} Such migration lanes can also contain larger diameter nodes at periodic locations along lanes. In certain variations, the placement of nodes at periodic intervals of no more than about 300 microns can have the effect of stimulating and accelerating the translocation of cell bodies along the lanes and thus better recapitulating the migration phenomenon observed m vivo. Upon reaching such node regions, migrating cells cease locomotive behavior, mimicking the arrest of cell migration upon reaching a. target.

[0041 [ When seeded directly on narrow micropatterned lanes, dissociated embryonic neurons typically exhibit migratory behavior, beginning soon after landing on the lanes. These lane-bound iieurons exhibit back-and-forth locomotion while projcciing nascent processes that extend along the lanes and facilitate the locomotion. This behavior may be induced by the contact guidance from the narrow geometries of the underlying micropatterned lanes. Thus, a high density array of micropatterned lanes can serve as a format for recapitulating or repeating the dynamics of neuronal cell migration using dissociated cells in vitro. Such a format can be used to facilitate imaging and to study the intracellular dynamics associated with cell locomotion and can also provide large populations of migrating cells suitable for screening applications. (See Fig. 1 A)

[00421 A single cell, e.g., a neuronal cell, including a cell body and its axon, may be patterned using a node-lane principal or design. Figure I B shows one example of a node-lane micropattera design or motif suitable tor receivin single cell, e.g., a neuron. The node 2 may accommodate the eel! body and the process or axon, of the ceil may be positioned or extend into a lane 4, e.g., an adhesive lane. The lane may guide the process or axon of the celt, The diameter arid shape of the node 2 can vary but should be large enough to

accommodate a neuronal cell body or other cell, for example, the node may have a diameter of about 10-20 microns or bigger. The node size can be modified to have any desired shape or size to suit the cell or neuronal population of interest. A lane 4, may have a width of about -1- 5 microns, e.g., to accommodate a single axon, A lane may be tens of centimeters in length or may be millimeters to tens of microns in lengt depending on the intended use of the lane and the ceil type. An exemplary length of a lane for receiving axons from a neuronal cell may be from about !OO microns to several millimeters. A lane may have any desired, length or shape to suit a cell population of interest. A lane may be straight or wavy. In certain variations as shown in Figure 1 C, a lane may be curvilinear. The node and lane .may include a cell adhesive wh le the surrounding area may include a cell repellant.

0043 Figure I D shows an example of how a node and lane mieropattern or motif may be used to form a repeating lattice network, with regularly spaced nodes tor neuronal cell bodies and interconnecting lanes for axons and dendrites. Among other functions, such lattices may serve to create an ordered and simplified eel! or neuronal network compared to networks in unpatterned cultures.

jO044j in particular, Figure ID shows neuronal cells seeded on a lattice mieropattern 10, The lattice mieropattern 10 includes interconnected narrow lanes, with enlarged nodes positioned on the vertices of the lattices. Neurons are initially seeded randomly on these lattice and cell bodies landing on the lanes will eventually migrate to the nodes while the lanes may accommodate the neural processes that project from the cell bodies. Figure i E shows an example, where the cell bodies and processes are stained for imaging. The Cell bodies 1.1 are stained orange ( AP2) while the processes 1.3 are stained green. (Tan).

10045 j The various lanes descried herein may be configured to accommodate axons and/or dendrites or other cell processes, Figure 2A shows an example of a node 1.2 and lane 14 arrangement where shorter lanes 16 emanate from the node 12. These shorter lanes ! 6 may accommodate dendrites . In certain variations, the design of a node with dendritic and axon lanes can be made to resemb le the natural branching morphologies of specific types of neurons found in vivo. The length and width and other dimensions of dendritic lanes can vary depending on the particular use or cell type. For example, the length of a dendritic lane may be from about tens of microns to millimeters. The width of dendritic ianes may be from about ~-5 microns to less than 0.5 microns. Branch points 18 may also be provided on the dendritic lanes 16 to duplicate the branching patterns of neurons observed in vivo. Dendritic lanes may be straight or curvilinear. Illustrations of exemplary neuronal cells, e.g., a Purleinje neuron in the cerebellum and a granule cell, are depicted in Figure 2B. Such neuronal cells and other cells may be deposited and organized, where their growth is controlled and directed in a predetemiined or predefined manner and/or direction utilizing the various node-lane patterns described herein,

j0046] in certain variations, an apparatus for organizing cells and/or biasing or dictating the direction of growth of a cell process or dictating ceil behavior is provided, The apparatus may include a surface having one or more nodes or cell body regions. The nodes may receive a eel! or a ceil may be positioned on or migrate to the node. One or more lanes may extend or emanate from the node. At least a portion of the node may have a shape or configuration which, funnels or directs a cell process (growing off of a ceil body) from the node into the desired lane in a predetermined or predefined manner or direction. The node and/or Lhe connection or transition from the node to the lane and/or the lane may be shaped or

configured to facilitate the controlled growth of a cell or eel! process and or to dictate the direction of growth of the ceil process from a node along or into the desired lane with a high degree of certainty or high degree of success.

[ 0471 hi certain variations, a node may include a curved or a tapered portion or side which tapers toward the lane to direct a ceil process into the lane, A given node can have a single tapered portion or side which results in an asymmetrically-shaped node, or multiple tapered portions to direct two different processes in two different directions as desired, which would result once again in an asymmetric node or a node with a form of mirror symmetry. .Asy mmetry may be used to bias the directionality of process outgrowth from cells located in nodes. In certain variations, at least a portion of the node may include a tear drop or elliptical or similar shape to guide or direct growth of a cell process. Various node and lane

configurations described herein were unexpectedly found to direct,, control and/or guide the growth or direction, of growth of eel! processes. Such configurations may stimulate cell movement in a particular direction.

j 048j In certain variations, a method for organizing cells and/or biasing or dictating the direction of growth, of a cel l process or dictating cell behavior may include one or more of the following steps. One or more nodes or cell body regions may be provided. The nodes may receive a cell or a cell may be positioned on or migrate to the node. One or more lanes may emanate or extend from the node. At least a portion of the node or lane may be shaped or

Page S of 37 configured to fuane! or direct a cell process of a ceil body -from the node into the lane in a predetermined manner or direction. One or more cells may be deposited on or near the node. Growth of a cell process may be directed in the predetermined or predefined manner or direction where the node and/or connection or transition from the node to the lane and/or the lane may be shaped or configured to facilitate the controlled growth of a cell or cell process and/or to dictate the direction of gro wth of the ceil process from a node along or into the desired lane with a high degree of certainty or high degree of success.

[00491 in certain variations, at least a portion of the node and/or the connection or transition from the node to the lane and/or the fane may be curved to direct growth of a cell process into the lane. The node may include a iapered portion or side which tapers toward the lane to direct a eel! proces into the lane. certain variations, at least a portion of the node may include a tear drop or elliptical or similar shape to guide or direct growth of a cell process. Various asymmetrically shaped nodes or nodes having mirror symmetry as described above may be utilized. The various node and Sane configurations described herein were unexpectedly found to direct, control and/or guide the growth or direction of growth of cell processes. Such configurations may stimulate cell movement in a particular direction.

|9050j Figures 3A and 3B show variations of a node and lane micropattern or pattern motif or design. At least a portion of the node 22 has a tear drop or elliptical shape. Such a shape helps control or guide a cell or cell process into a predetermined direction by directing or tunneling the ceil process, e.g., an axon outgrowth, into the lane 24 which extends or emanates from the node 22. The tear drop or ell iptical shaped portion of the node 22 leads into the lane 24, providing a curved, sloped or rounded transition from, the node 22 into the lane 24 (e.g., at an angle greater than ninety degrees), which directs the growth of a cell process from a cell deposited on the node 22, from the node 22 into the desired lane 24, As shown in Figure 3B, dendritic lanes 26 may emanate or extend from the node 22. The node and lane micropattern may be positioned on a surface or substrate.

j OSl J A teardrop or elliptical node lane pattern as described supra may control the directionality of a cell process, such an axon growth, within a micropattern. This node and lane arrangement is beneficial for designing micropattern s in which directionality of axons or other cell processes is an important parameter. For example, axon directionality may be important^' in synaptic circuitry, where a first neuron sends neuronal information to a second neuron, but not v ce versa. As such, the axon growth may be contra lied to grow from the first neuron to the second neuron but not from the second neuron to the first neuron.

|6052] I n certain variations, at least a side of the node may be asymmetrically-shaped, and or tapered or tunneled on one or more sides leading into the iane. Optionally, the portion of the lane connecting to the node may include a reverse taper or funnel to facilitate guidance of a cdi process from the node .into the lane. Optionally, the portion, transition or connection bet ween a node and lane, which may incl ude portions of the node and/or lane and/or may include an addition or separate segment may he configured or shaped to guide or direct the growth of a cell process from the node to the cell in a predetermined manner or direction. Optionally, any of the elements described above may be asymmetrically-shaped.

19053_.) in contrast, where a neuronal ceil is deposited on a circular node having an orthogonal lane extending radially therefrom, (i.e., a ninety degree angle between the side of the circle and the entry point of the lane off that side), an axon extending from such a cell body will have a difficult lime finding the orthogonal lane. The present inventor discovered that, when a circular node having an orthogonal lane extending radially therefrom was used as a micropaitern, axons extending from a neuronal cell deposited on the node would grow in any direction with there being no control over the direction of the axon growth. Indeed, the particular direction of axon growth on such a node lane arrangement was random and one would have to merely rely on chance that the axon would in fact find the lane opening and grow into the lane. A cell deposited on a circular node having an orthogonal lane extending radially therefrom would not simply follow and grow in the direction of the particular pattern and into a lane,

j^'O 5 | in certain variations, the node and lane micropattern designs described herei may be utilized as building blocks to create various node and lane arrays.

|9055] Figure 4 shows one variation of a series of node-lane motifs which may be positioned close together so that the lanes 34 can from a geometric array 30 to aid in, e.g., the identification of axons, tire analysis of characteristics of axons, and experimental

manipulation of axons. The lanes 34 may be organi ed into columns or rows or any other pattern. In certain variations, a large number of node-lane motifs may be arrayed in a small footprint. For example, about ~ eighty 20 micron diameter nodes wi th i mm long lanes cm be laid out within a 2 X 2.1 mm footprint. The arrays described herein may increase experimental throughput and may form the basis for screening applications.

10056 J In certain variations, arrays of cell process or axon lanes may be combined with or emanate from one or more cell body domains to form a micropattera. For example, rather than emanating from individual nodes, a larger adhesive domain may be provided to accommodate multiple neurons and from which multiple lanes may emanate or extend.

[0O57J Figure 5A shows one variation of a cell body domain region 40 having fanes 44 (e.g., axon lanes) suitable for single axons or process emanating from the cell body domain region 40. A cell body domain can be of any geometry or shape, e.g., the domains may be irregular in shape, and may be sized according to the number of neurons or cells to be accommodated thereon. The number of lanes can be varied and can emanate from a single side of the cell body domain or radiate away from more than 1 side. Figure SB shows a variation of a cell body domain 50 having lanes 54 radiating away from the domain 50 around the perimeter of the domain 50. Lanes may emanate perpendicular or at an angle away from the domains. The length of the lanes can be varied from tens of microns to millimeters in length, e.g., an exemplary length may he from about 10 microns to several millimeters, furthermore, the lanes can be straight or curved. Lanes emanating from cell body domains may accommodate a single axon or process per lane or may be wider to accommodate more than one axon or process. Cell body domains may provide an tmpa te ned region or domain on which cell bodies ma be positioned or deposited. In certain variations, target cells may be positioned on a ceil body domain where such target cells don't have to be patterned and can may h positioned anywhere on the domain region.

[00581 In certain variations, various strategies and techniques for guiding axons or other cell process into arrays, e.g., parallel arrays are described herein. It may be desirable to have axons arranged in neat parallel arrays for convenient observation and high throughput analysis.

1805 1 Figure 6 shows a variation of a method of seeding cells into distinct regions on culture substrates. In this example, two populations of cells (neurons) were seeded apart, on the left and on the right, separated by a spacer in the middle. The spacer initially was

Page 1 i of 37 positioned over a set of micropattemed lanes to prevent cell bodies from landing there and allows the two populations of cells to e spatially segregated during cell plating. After the ceils take hold, cells front ihe two populations project neural processes or axons into the micropattemed lanes to reach the opposite side and to bridge the two populations.

1 601 However, in practice, only a tew of the lanes making up the lane array in the tnicropattem configuration of Figure 6 are occupied by axons. Axons originate from the ceil bodies and have a strong tendency to meander in the vicinity of cell bodies and other axons and thus tend not to project into the lanes. To increase the occupancy rate of such a lane array, it is often necessary to use or design micropatterns such that they can guide or direct axonal outgrowths into desired lanes.

[0061[ Figure 7 shows one variation of a method, pattern or apparatus for guiding cell processes, e.g., axons, into desired lanes. One or more or a series of widened micropatterned parallel strips are provided for guiding and directing eel! processes into a series of narrow parallel lanes. The strips may increase the chance that that cell processes will project into the lanes. The strips may be oriented parallel with the lanes. Cell bodies may initially be deposited on the wide strips. The widths of these strips may be sufficient to accommodate multiple cell bodies and allow the outgrowth of cell processes, e.g., axons. Over time, axons will encounter an edge of a strip and then extend along that edge. The edges of the wide strips may run along the direction of the micropattemed lanes and converge into one or more of the narrow lanes. Axons that extend along the edge of these strips may be directed into the micropattemed lanes.

( 0621 A basic difficultly of effectively using inicropatterns in organizing many neuronal cell types is that the cells themselves extend processes that are highly branched and interconnected with neighboring cells. However, in many experimental formats, there is a need for single neuron nodes that allow easy identification of the neuronal cell body giving rise to specific axons under study while at the same time also having the benefits of larger cell adhesive areas containing more neuronal cell bodies that can provide beneficial trophic, viability support for each other. Randomly depositing neurons into a larger domain results in intermixed cell bodies and ceil processes making it difficult to resol ve the origin of an axon. [0063] One micropaiteramg strategy for reducing complexity of the inter otiiiectivtty of cells is to put. neurons into a two-dimensional lattice type pattern- Such a lattice may aiso guide axons to more elongated axon lanes. In certain variations, eel! process or axon lane arrays may emanate or extend from various ^'lattice type patterns. "Lattice" may include but not be limited to any configuration i nvolvi ng repeated arrangements of lanes and node combinations and are not limited to the examples described herein.

06 1 Lattices may include narrow lanes for axons and dendrites while enlarged node areas are present at the vertices of the lattices, The nodes may have any desired shape. These nodes are wide enough to accommodate neuronal cell bodies, while the narrow lanes, which may be, e.g., about 1-5 microns wide, are narrower than the cell bodies but can comfortably accommodate axons! and dendritic processes. The lattice configurations are convenient, because cell bodies landing on lanes will automatically migrate to the nodes and take hold in the nodes. Within a few days of seeding cells, regardless of where the cells initially land on a raicropattemed lattice, a network of neuronal cells in which cell bodies are confined to nodes, and axons and dendrites run along lanes, is formed.

0 65| Cells may be initiall seeded and confined to lattice micropatterns, which are configured, to guide the axons into a series of parallel micropatterned lanes to form the desired axon arrays. Neuronal cells may initially be seeded on the lattice micropattems and organize along the micropatterned lattices. Axons may extend from the cell bodies on the nodes, where they are guided along the lattice lanes, which eventually lead to an adjacent series of longer, parallel axons lanes.

[00661 The various lattice micropattern designs or motifs may provide both a reduced complexity culture while more readily providing a convenient array of axons for high throughput experiments. Using a lattice to guide axons into the elongated axon array results in higher chances that axons will occupy each axon lane. In certain variations, various types of lattice micropattems may be combined or used together,

(0067 j Figure A shows a variation of a lattice micropattern SO including tear drop or elliptical shaped nodes 82. The nodes 82 include a tapered side which directs, controls or guides axonal or other cell processes outgrowth towards and in the direction of the long lanes 84, which make up a lane array e.g., an axon lane array. The lattice 80 may include narrow and/or shorter lanes 86, e.g., for axons and dendrites, which facilitate connections between the enlarged nodes 82, which are present at the vertices of the lattice.

[00681 Figure 8.8 shows a variation of a lattice mieropatter 90 which includes nodes 92,

The nodes 92 are interconnected by lanes 96 which receive, e.g., axons and dendrites. A longer lane or axon lane 94 extends from at least one of the nodes, where the lattice may have reduced connectivity.

[0069| Figure 8C shows a variation of a lattice mieropattern 100 design that gradually reduces in its degrees of lattice connectivity between the nodes 102, and finally terminating in one node 102 from which a longer lane or axon lane 104 emanates. The nodes 1.02 may be interconnected by lanes 106 which receive, e.g., axons and dendrites.

[0070[ Within the lattice pattern, the degree of connectivity for nodes can differ and nodes and ceil bodies may connect to one or more other nodes or cell bodies. For example, some nodes may connect to up to 8 or 9 other nodes or neurons positioned therein, whi le others may connect only up to four other nodes or neurons. In certain variations, as shown, in Figures 8D and 8E, the lattices or reduced lattices may have adjoining larger unpaitcrned cell body domain regions or adhesive areas 98, 108 coupled or connected thereto for ceils, to provide additional connectivity or trophic support. Figures 8D and 8E also show multiple lanes or axon lanes 94 and 104 emanating front the lattices 90 and 100 to form & parallel array of lanes or axon lanes.

[00711 Figure 9A shows a variation of a lattice micropattern 110 which may receive neurons and guide axonal outgrowths into long parallel lanes 1 14. Figure 9B shows cortical neurons projecting individual axons (identified by the black arrows) into the lanes 114 (Scale- ΙΟΟμίπ). By using a lattice mieropattera at the seeding area and limiting cell seeding density,, the occupancy rate of lanes may be maximized while isolating individual axons,

[00721 Hippoeampal or cortical neurons from rat and mice have been shown to achieve over 90% (e.g., close to 99%) compliance (i.e., confinement to a ceil adhesive zone) on various lattice (node-and-lane) configurations.

[00731 Various moti fs, designs and or design algorithms for micropaiterns, apparatus and methods described herein, transform a purely randomly distributed neuronal or other cell culture, e.g., resulting from simply using circular nodes and radially extending orthogonal lanes, to cellular circuitry that more readil conforms to the configuration of cell-ceil connectivity; according to the user's intent. An example of user intent is specifying the directionality of the xon, which is a key parameter for determining function in neuronal circuits. The precise configuration and dimensions of the lattices and axon lane arrays may be varied as needed,

[007 | In certain variations, a node and lane lattice pattern may include nodes that are spaced no more than 300 microns apart. As such, a lane and node combination may be 300 microns or shorter. When neurons are deposited on such a lattice pattern, this configuration allows for effective recapitulation of neuronal ceil migration, in certain variations, various micropatterns and designs described herein may be utilized to create synapse biology arrays. Figure I OA shows an example of interconnected neuronal nodes 120 where two cell body nodes 122 are connected by an axon lane 124. This arrangement may serve as a basic motif for synapse bioiogy, Variations of the node and axon lane designs described herein also apply to the synapse interconnected node motif. Figure 10B shows a synapse biology array 130 which is formed by arranging a number of synapse bioiogy motifs or interconnected nodes 132 into a geometric array pattern.

[0075} In certain variations, synapse biolog patterns using two connected domains may be provided. Figure 1.1 A shows a raic.ropattern configuration where two interconnected cell body domains 140 may be used instead of numerous individual cell body nodes. Axon lanes 144 span the two cell body domains 1.40 and can be straight or curved, with ~1 or greater than 1 micron width depending on the number of axons desired in each lane. In certain variations, the same types of neurons can be deposited onto these micropatterns in which case synapses may be formed between neurons of the same type. Optionally, mixed neuronal populations can be seeded or deposited onto these micropatterns, in which case the user may choose synaptic pairings between, specific neuronal populations of interest for analysis. As with the various arrays described above involving reduced lattice patterns that allow for identification of the neuronal cell body of origin, synapse bioiogy arrays may also be created which incorporate such lattices as shown in figure 1 1 B. Figure 1 IB includes two interconnected cell body domains 1.46 connected to lattices 147 which are interconnected by axon lanes 148. [0076} In certain variation, micropatterns involving three or more neuronal populations may be utilized, where it may be advantageous to have three or more neuronal populations for study, This may be useful, e.g., in the study of synaptic competition or in the study of circuit behavior. As described above, each neuronal population may be arranged either in individual nodes tor single neurons, or in larger domains where all neurons of the same ex e iment type may be grouped together. Figure 1 1 C shows an example of a micropattern 150 on which three or more neuronal populations may be deposited. Groups of neurons deposited into domains 1 ,2 may send axons to innervate a common sei of target, neurons located in domain 3.

[8877} For various node and lane array and lattice micropattern configurations described herein, the nodes may include a variety of shapes. For example, in any of the figures which show an exemplary circular node, nodes of other shapes, such as tear-drop, elliptical, tapered nodes or asymmetrically shaped nodes may be used instead. In certain variations, any of the various nodes and lanes described herein may have an adhesive material while the area around the nodes and lanes may have a .repeHani material. In certain variations, any of the nodes and lanes, arrays and lattice oiicropatteras described here may be placed, on a desired, substrate or surface that supports such patterns, e.g., electrodes, therapeutic, and/or diagnostic devices.

18078} In certain variation, various mtcropattems described herein may be integrated with microelectrode arrays. To perform electrical stimulation and electrical measurements of ceils in culture, it is often necessary to position cells in certain proximity to electrode and electrical, devices. Cell measuring or stimulating electrodes can come in a variety of forms and can be fabricated directly on a cell cultur substrate, e.g., as planar strips or shaped areas of conductive material or as conductive material that protrude from the surface of the culture substrate, Micropatte ning can be used to align the position of cultured ceils relative to these electrodes and can be also be used to position cells directly in contact with electrodes. T his may be accomplished by aligning a microfabrication process for surface micropatterning to the electrodes.

18079} For example, Figures 1.2.A and 128 show how a finished planar microelectrode array may serve as a starting substrate for overlaying micropattems via the pPLACeR micropatterning process (Chang and Sretavan (2008)_.). Alternatively, any cell micropatterning method or process for overlaying a icrppattens on a microeleetrode or other substrate or surface may be utilized, A culture of neurons may be micropatiemed in conjunction with underlying gold mieroelecirades. The micropatiemed cells can be precisely aligned over specific stimulating microelectrodes. Figures S 2A nd 12B show a glass substrate 160. Electrodes 161 are positioned on the glass substrate. The μΡΙΑ€¾Ι or other micropaiteming process may be applied such that a FEO-like film 162 is deposited over the eSecirodes 161. and glass substrate 160. Optionally, a po!yiysine 163 m be deposited on the PEO-like film 162 and/or the electrode 161. Any of the mic-ropattem designs or motifs described herein may overlay or be positioned over electrodes.

(0080} Figure 12C shows an example of a substrate wit h pre-fabricated, planar microelectrodes 171. The substrate can be used as a starting substrate for the pPLACeR. or other micropatterning process. Micropatterns of cell adhesive material may be aligned with specific microelectrodes. As shown in Figure I2C , a node and lane lattice mtcropattero (showing nodes 1. 2, and 3) was overlaid on top of a pair of planar microelectrodes 171 and neurons were cultured on this substrate. Figure 121) shows that electrical stimulation delivered by the electrodes was focused on the cluster of neurons on node I . Using highspeed voltage-dye imaging, the selectively perturbation of the neurons in this node due to a single pulse electrical signal delivered by the microelectrodes, while neurons on nearby nodes remained undisturbed, was observed.

[0081 j In certain variations, the node and lane shapes described herein, e.g., the teardrop or tapered node, funnels or directs a cell process, e.g., an axon, into the lane, thereby dictating or biasing the direction of cell process growth and or their positioning or alignment.

10082} in certain variations of node and lane networks, a lane may emanate from a first node and connect to or enter the second node at a non-orthogonal angle (e.g,, connecting to the second node at an angle of less than ninety degrees). This may allow control of axon growth from a first eel I in the first node to a second ceil in the second node, but prevent or reduce the occurrence of growth of an axon from the second cell back to the first cell. The various node and lane shapes and/or the angled (e.g., nono.rthago.nal) take offs or entry points of laties into nodes may create a network that can direct cells to set up a desired cell circuitry. [0083] l.n certain variations, the specific network and/or node and lane designs or patterns described herein may be based on or .mimic the naturally occurring connectivity of a specific ceil type to be studied or its activity as observed in research and/or based on an understanding of a specific culture or behavior of a specific ceil e.g., a neuronal cell, as it may exist and function in vivo {e.g.. serve as biomimeiic representations). In certain, variations, various networks and patterns described herein may dictate the growth, behavior and connections of cells (e.g., neuronal cells) positioned thereon, thereby dictating information flow along the pattern of cells. In certain variations, multiple nodes may provide input onto a single node leading to a single lane on. which a single axon may be isolated. Such a configuration provides a network of converging and diverging cell inputs, providing connectivity between cells such that the cells can. communicate and receive the support and input similar to the connections and support the cell receives in vivo, while still allowing for the isolation of a single cell process or axon. This may allow for an accurate assessment of cell behavior and. functionality. In certain variations, various micropatiems or designs described herein may be combined to simultaneously limit the density of cell, e.g., neuronal, cultures and promote the outgrowth and isolation of individual cell processes or axons on narrow lanes. The lanes may direct axons into arrays, e.g., parallel arrays, on which multiple but isolated axons or process may be lined up together for efficient, high-throughput observation and manipulation,

[0084] In certain variations, various patterns, micropattems, apparatus and methods described herein may have a variety of applications. For example, they may be utilized for maintaining any biological cell type in culture including primary cell types, ceil lines, populations with stem cell potential, which can be human or non-human in origin. Uses range from, but are not limited to, fundamental scientific and biomedical research, to therapeutic discovery, toxicity analysts, diagnostic uses, or use within therapeutic devices,

[00851 Three examples areas are described below where certain of the various patterns, micropatteros, apparatus and method variations described herein may be utilized in the field of neuroscience.

[0086] Axon Biology - Axons form the cellular subunii that conveys information across the nervous system. Beyond advancing the field's understanding of fundamental axon bi ology, the development and growth of axons have implications in furthering therapeutic advance to treat neural trauma. Advances in this area will be accelerated by new technologies such as various patterns, mtcropatterns, apparatus and methods described herein, that enable researchers to investigate fundamental a onal biology at the single axon level and to do so in high-throughput experimental paradigms. Axons are subcellular extensions from the neuronal cell body and can range from about 0,2 to 5 or more microns in diameter, in the body, axons range in length from tens or hundreds of microns to well over a meter in length in some eases. In conventional cell culture, researchers do not have the ability to control the trajectory of axons, and they typically meander in random directions overlapping with processes from, neighboring cells making individual axons difficult to identify, obscure the neuronal cell body of origin, and hinder the analysis of cell biological events occurring within axons. Axon research is important in areas such as paralysis research including spinal cord injury, glaucoma, neurodegenerative diseases, and other C S pathologies such as multiple sclerosis. 10087| Synapse Biology - Synapses are the cellular substrate for the transmission of signals between nerve cells and are thought to be the anatomical substrate for learning and memory. Synapses are formed at the end of axons where they contact typically the dendrites and ceil soma of target post-synaptic neurons. Perturbations in synaptic function and maintenance are now thought to be amongst the earliest events in neurodegenerative entities including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Prion diseases. Thus technological advances such as various patterns, mtcropatterns, apparatus and method described herein that enable highly precise dissection of synaptic events, easy identificati on of pre and posi-synaptic elements, and present synaptic biology in high-throughput formats will greatly benefit the field. In addition, synaptic function is a target for pharmacologic

intervention in behavior, addiction, and mental illness.

[0088] Cell migration - The locomotion and migration of ceils (including but not limited to neurons) is a key behavior during tissue development wound healing, cancer metastasis, as wet! as other physiological events, icropatterned node and lane substrates as described herein can be used effectivel to stimulate and guide locomotion and migration activities in dissociated cells in vitro.

[0089) Another area of significant therapeutic interest is the development of compounds with activity against angiogenesis for use in disease entities such as cancer and diabetes. A comerstotie of research and drug High Throughput Screening (HTS) in these areas utilize tube formation assays by endothelial cell types such as HUVECs. The tubes are formed randomly in culture with substantial effort to resolve tube length, branch points etc. Furthermore,, although known as tube assays, the assays as currently performed in the art do not result in tubes with lumens thai resemble capillaries in vivo. Thus there s a need for more efficient angiogenesis assays which may be performed utilizing various patterns, mic opattems, apparatus and methods described herein.

10090] As described supra, various patterns, micropattems, apparatus and methods described herein relate to customized cell niicropafterns or array micropattems, the principles and motifs of their design, and related systems thai enable researchers to specify the layout of individual cells, e.g., neurons, and their process in culture, and to arrange cell or neuronal elements into whatever desired patterns to suit a particular experimental need. Neurons, glia, and axonal processes may be laid out in pre-defined formats to facilitate analysis of a specific type of cell-cell interaction, increase the convenience and speed of data capture / analysis, maximize the use of rare cell populations, and greatl expand sample size to improve statistical comparisons.

|00911 The variations described herein can be formed fay and used with a variety of cell micropatterning technologies such as micro-contact printing, ion beam etching, among other techniques. Ceil micropattem techniques can. include methods to imprint or deposit, specifically shaped cell adhesive material, or surface modification along any substrate used to receive cells for culture. There are a wide variety of methods for cell micropatterning. One variation of a method of micropatterning for these purposes has been described in

PCT/US20O9/O59I94; WO 20J0/039933A3, "METHODS AND COMPOSITIONS FOR HiGH- ESOLUTlON MICROPA1 ERNING FOR CELL CUITURE," which is incorporated by reference herein in its entirety. This patterning method involves a robust method for microscale (1 micron scale) rendering cells and cellular siractures into any kind of shape or configuration, including arrays on centimeter-scale glass substrates (Chang & Sretavan 2008).

[9092^'] Any of the various patterns, micropattems, apparatus, methods and tnteropatterned substrates described herein may be provided or performed on a substrate or surface and/or utilized in standard eultureware formats, conventional dishes and/or microplates for ease of use and compatibility with imaging and experimental techniques as well as existing ceil culture protocols. The various patterns and mtcropatterns, e.g. , node and lane arrangements, described herein may be placed on any surface that can support a micropattem. for example on a diagnostic or therapeutic device. The various patterns, mieropatterns, apparatus, methods and/or mieropatterned surfaces or substrates described herein may be utilised to pattern and/or study or investigate any cell type or other tissue, e.g., in vitro.

[9993] Variations of the devices, patterns, designs and methods described herein may be best understood from the detailed description when read in conjunction with the

accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings may not be to-scale. On the contrary, the dimensions of the variou features may be arbitrarily expanded or reduced for clarity. The drawings are taken for illustrative purposes only and are not intended to define or limit the scope of the claims to that which is shown.

{9094} AH publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. To the extent there is a conflict in a meaning of a term, or otherwise, the present application will control. Although variations of the foregoing invention has been described in some detail by way of illustratio and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may he made thereto without departing from the spirit or scope of the appended claims. It is also contemplated that combinations of the above described embodimefHs/variations or combinations of the specific aspects of the above described emboditnents variations are within the scope of this disclosure,

{90951 Each of the individual variations described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other variations. Modifications may be made to adapt a particular situation, material, composition of matter, process, process act(s) or step(s) to the objective(s), spirit or scope of the present invention.

19096_.) Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events. Furthermore, where a range of values is provided, every intervening value between the upper and lower limit of that range ami any other stated or intervening value hi thai stated range is encompassed within the invention. Also, any optiona l feature of the inventive variations described may be set forth and claimed independently, or in combination with any one or more of the features described herein.

[00 7j All existing subject matter mentioned herein (e.g., publications, patents, patent applications and hardware) is incorporaied by reference herein in its entirety except insofar as the subject matter may conflict with that of the present invention (in which case what is present herein shall prevail). The referenced stems are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as a admission that the present invention is not entitled to antedate such material by virtue of prior invention.

{ΘΘ98] Reference to a singular item, includes the possibility that there are plural of the same items present. More specifically; as used herein and in the appended claims, the singular forms "a," " n," "said^'" and "the" include plural referents unless the context clearly dictates otherwise. It is further noted, that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely " "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation, Unless defined otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

10 9 1 This disclosure is not intended to be limited to the scope of the particular forms set forth, bin is intended to cover alternatives, modifications, and equivalents of the variations described herein, further, the scope of the disclosure fully encompasses other variations that may become obvious to those skilled in the art in view of this disclosure. The scope of the present invention is limited only by the appended claims.

Claims

CLAIMS We claim:

1. A method for otgsnkmg or patterning cells comprising:

providing at least one node configured to receive a cell body;

providing at lea t one lane extending from the node; and

directing growth of a celt process of the cell body from the node into the lane in a predetermined manner or direction, which is determined by a configuration of the node leading into the lane

2. The method of claim 1 , further comprising depositing at least one cell on or near the node and directing growth of a ee!! process in a predetermined direction, whereby a curved or tapered portion of the node directs t he cell proc ess into the lane.

3. The method as claimed in any one of the preceding claims, wherein a transition or connection from the node to the lane tunnels or directs a celt process of the cell body from the node into the lane in a predetermined manner or direction

4. The method as claimed in any one of the preceding claims wherein a transition or connection from the node to the lane is curved or rounded.

5. The method as claimed in any one of the preceding claims, wherein at least a portion of the node comprises a shape which funnels or directs growth of the cell process from the node into the lane,

6. The method as claimed in any one of the precedin claims, wherein at least a portion of the node comprises a tear drop o elliptical shape for directing or guiding the cell process,

7. The method as claimed in any one of the precedin claims, wherein the node is asymmetric and the asymmetric configuration biases the directionality of a cell process outgrowth from a cell body.

8. The method as claimed in any one o the preceding claims, wherein the lane is connected to the node at a non-orthogonal angle,

9. ^'The method as claimed in any of one of the preceding claims, wherein multiple nodes and lanes are in a lattice pattern, wherein the nodes are spaced no more thars 300 microns apart.

10. The method as claimed in any one of the preceding claims, wherein the lane is

connected to a second node at a non-orthogonal angle and growth of the ceil process is directed to the second node, along the lane.

1 1. The method as claimed in any one of the preceding claims, wherein the lane has a width thai is less than the diameter of a cell body posi tioned in the node.

12. The method as claimed in any one of the preceding claims, wherein multiple nodes and lanes are provided to create an array of parallel lanes; and further comprising isolating single cell process on each of the lanes making up the array of parallel lanes.

13. The method as claimed in any (me of the preceding claims, wherein multiple nodes and lanes are interconnected to form a lattice, wherein the lattice gradually reduces in its degrees of connectivity between the nodes and terminates in one node from which a lane emanates; and further comprising isolating a single cell process on the lane.

1.4. The method as claimed in any one of the preceding claims, wherein an rapattenied cell body domain region is connected to a node or lane to provide additional connectivity or trophic support to a cell body positioned in the node.

15. The method as claimed in any one of the precedin claims, wherein the cell is a neuron and the cell process is an axon projecting from the neuron.

16. The raeihod as claimed in any one of ihe preceding claims, wherein the node and lane are provided on a surface or substrate.

17. Hie method of claim 16. wherein the surface or substrate are on a dish or raicroplate.

18. The method as claimed in any one of the preceding claims, wherein the node and lane are deposited over an electrode or on the surface of a diagnostic or therapeutic device.

19. An apparatus for organizing or patterning cells comprising:

a surface having at least one node configured to receive a ceil body and at least one lane extending from the node;

wherein at least a portion of the node is configured to direct a ceil process of a cell body from, the node into the lane in a predetermined manner or direction, which is determined by a configuration of the node leading into the lane.

20. The apparatus of claim 1.9, wherein the node comprises a tapered portion which tapers toward the lane to direct the cell process into the lane.

21. The apparatus of claim 19 or 20, wherein a transition or connection irom the node to the lane is configured to funnel or direct a ceil process of the cell body from the node into the lane in a predetermined manner or direction

22. The apparatus as claimed m any one of claims 1 -21, wherein a transition or

connection, irorn the node to the lane is curved or rounded,

23. The apparatus as clai med in an one of claims 19-22, wherein at least a portion of the node comprises a shape configured to funnel or direct growth of the cell process from the node into the lane.

24. The apparatus as claimed in any one of claims 19-23 , wherein at least a portion of the node comprises a tear drop or elliptical shape for directing or guiding the cell process in to the lane.

25. The apparatus as claimed in any one of claims 1 -24, wherein the node is

asymmetric and the asymmetric configuration biases the directionality of a cell process outgrowth from a ceil body,

26. ^'Hie apparatus as claimed in any one of claims 1 -25. wherein the lane is connected to the node at a non -orthogonal angle,

27. The apparatus as claimed in any one of claims 19-26, further comprising a second node, wherein the lane is connected to the second node at a non-orthogonal angle.

28. The apparatus as claimed in any of one of claims 1 -27, wherein multiple nodes and lanes are in a lattice pattern, wherein the nodes are spaced no more than 300 microns apart..

29. The apparatus as claimed in any one of claims 1 -28, wherein the lane lias a width that is less than the diameter of a cell body positioned in the node.

30. The apparatus as claimed in any one of claims 19-29, further comprising multiple nodes and lanes configured to create an array of parallel lanes, wherein a single cell process may be isolated on each of the lanes making up the array of parallel lanes.

31. The apparatus as claimed in any one of claims 19-30, further comprising multiple nodes and lanes interconnected to form a lattice, wherei the lattice gradually reduces in its degrees of connectivity between the nodes and terminates in one node from which a lane emanates, wherein a single cell process may be isolated on the lane,

32. The apparatus as claimed in any one of claims 19-31 , further comprising an iinpatteraed cell body domain region connected to a node or lane to provide additional connectivit or trophic support to a eel! body positioned in the node,

33. Hie apparatus as claimed in any one of claims 1 -32, wherein the cell is a neuron and the ceil process is an axon projecting from the neuron.

34. The apparatus as claimed in any one of claims 19-33, wherein the surface is on a dish or micropiate.

35. The apparatus as claimed in any one of claims 1 -34. wherein the surface is deposited over an electrode of on a diagnostic or therapeutic device.

36. A method for organizing or patterning cells comprising;

providing at least one node configured to receive a cell body;

providing at least one lane extending from the node; and

directing growth of a cell process of the cell body from the node into the lane in a predetermined manner or direction, which is determined by a configuration of die node, a configuration of a transition from the node to the lane, and/or a configuration of the lane.

37. The method of claim 36, further comprising depositing at least one cell on or near the node and directing growth of a cell process in a predetermined direction, whereby a curved tapered portion of the node directs the cell process into the lane.

38. The method as claimed in claims 36 or 37, wherein a transition or connection from the node to the lane funnels or directs a cell process of the cell body from the node into the lane in a predetermined manner or direction

39. The method as claimed in any one of claims 36-38. wherein a transition or connection from the node to the lane is curved or rounded.

40. The method as claimed in any one of claims 36-39, wherein at least a portion

of the node comprises a shape which funnels or directs growth of the cell process from the node into the lane.

41. The method as claimed in any one of claims 36-40, wherein at least a portion of the node comprises a tear drop or elliptical shape for directing or guiding the cell process into the lane.

42. The method as claimed in any one of claims 36-4 i , wherein the node is asymmetric and the asymmetric configuration biases the directionality of a cell process outgrowth from a cell body,

43. The method as claimed in any one of claims 36-42, wherein the lane is connected to the node at a no -orthogonal angle.

44. The method as claimed in any one f claims 36-43, wherein multiple nodes and lanes are in a lattice pattern, wherein the nodes are spaced no more than 300 microns apart.

45. The method as claimed in any erne of claims 36-44, wherein the lane is connected to second node at a non-orthogonal angle and growth of the ceil process is directed to the second node, along the lane.

46. The method as claimed in any one of claims 36-45, wherein the lane has a width that is less than the diameter of a cell body positioned in the node.

47. The method as claimed i any one of claims 36-46, wherein multiple nodes and lanes are provided to create an array of parallel lanes; and further comprising isolating a single cell process on each of the lanes making up the array of parallel lanes.

48. The method as claimed in any one of claims 36-47, wherein multiple nodes and lanes are interconnected to form a lattice, wherein the lattice gradually reduces in its degrees of connectivity between the nodes and terminates in one node from which a lane emanates; and further comprising isolating s single ceil process on the lane.

49. The method as claimed m any one of claims 36-48, wherein an unpatterned ceil body domain region is comiecied to a node or lane to provide additional connectivity or trophic support to a ceil body positioned in the node.

50. T he method as claimed in any one of claims 36-49, wherein, the cell is a neuron and the cell process is an axon projecting from the neuron.

51. The method as claimed in any one of claims 36-50, wherein the node and lane are provided on a surface or substrate.

52. The method of claim 51, wherein the surface or substrate are on a dish or microplate.

53. The method as claimed in any one of claims 36-52, wherein the node and lane are deposited over an electrode or on the surface of a diagnostic or therapeutic device.

54. An apparatus for organizing or patterning cells comprising:

a surface having at least one node configured to receive a cell body and at least one lane extending from the node;

wherein at least a portion of the node, a transition from the node to the lane or the lan is configured to direct a eel! process of the cell body from the node into the lane in a predetermined manner or direction, which is determined by a configuration of the node, a configuration of the transition from the node So the lane, and/or a configuration of the lane.

55. The apparatus of claim 54, wherein the node comprises a tapered portion which tapers toward the lane to direct the cell process into the lane.

56. The apparatus of claim 54 or 55, wherein a transition or connection from the node to the lane is configured to funnel or direct a eeii process of the ceil body from the node into the lane in a predetermined manner or direction

57. The apparatus as claimed m any one of claims 54-56, wherein a transition or

connection from the node to the lane is curved or rounded.

58. The apparatus as claimed in an one of claims 54-57. wherein at least a portion of the node comprises a shape configured to funnel or direct growth of the cell process from the node into the lane.

59. The apparatus as claimed in any one of claims 54-58, wherein at least a portion of the node comprises a tear drop or elliptical shape for directing or guiding the cell process into the Sane.

60. The apparatus as claimed in an oae of claims 54-59, wherein the node is

asymmetric and the asymmetric configuration biases the directionality of a cell process outgrowth from a cell body.

61. The apparatus as c laimed in any one of claims 54-60, wherein the lane is connected to the node at a non-orthogonal angle.

62. The apparatus as claimed in any one of claims 54-61, wherein multiple nodes and lanes are in a lattice pattern, wherein, the nodes are spaced no more than 300 microns apart.

63. The apparatus as claimed in any one of claims 54-62, further comprising a second node, wherein the Sane is connected to the second node at a non -orthogonal angle.

64. The apparatus as claimed in an one of claims 54-63 , wherein the lane has a width that is less than the diameter of a cell body positioned in the node.

65. The apparatus as claimed in any one of claims 54-64, further comprising multiple nodes and lanes configured to create an array of parallel lanes, wheretB a single cell process may be isolated on each of the lanes making up the array of parallel lanes,

66. The apparatus as claimed in any one of claims 54-65, further comprising multiple nodes and lanes interconnected to form a lattice, wherein the laitice gradually reduces in its degrees of connectivity between the nodes and terminates in one node from which a Sane emanates, wherein a single cell process may be isolated on the lane.

67. The apparatus as claimed in an one of claims 54-66, further comprising an unpatterned eel! body domain region connected to a node or lane to provide additional connectivity or trophic support to a cell body positioned in the node.

68. The apparatus as cl aimed in any one of claims 54-67, wherein the cell is neuron and the cell process is an axon projecting from the neuron.

69. The apparatus as claimed in any one of claims 68, wherein the surface is on a dish or micropkte,

70. The apparatus as claimed in any one of claims 54-69, wherein the surface is deposited over an electrode or on a diagnostic or therapeutic device.

71. A method for organizing or patterning cells comprising:

depositing at least one cell on or near at least one node, the node having at least one lane extending therefrom; and

directing growth of a eel! process of the cell from the node into the lane in a predetermined manner or direction, which is determined by a configuration of the node, a configuration of a transition from the node to the iane and/or a configuration of the lane.

72. The .method of claim 71 , wherein a curved or tapered portion of the node directs the ceil process into the lane.

73. The method as claimed in claims 71 or 72, wherein a transition or connection rora the node to the lane funnels or directs a cell process of the eel! body from the node into the lane in a predetermined manner or direction.

74. The method as claimedm any one of claims 71-73, wherein a transition or connection from the node to the lane is curved or rounded.

75. The method as claimed in any one of claims 71-74, wherein at least a portion of the node comprises a shape which funnels or directs growth of the cell process from the node into the lane.

76. The method as claimed in any one o f claims 7 -75, wherein at least a portion of the node comprises a tea drop or eliiptical shape for directing or guiding the cell process into the lane.

77. The method as claimed in any one of claims 71-76, wherein the node is asymmetric and the asymmetric configuration biases the directionality of a cell process outgrowth, from a ceil.

78. The method as claimed in any one of claims 71-77, wherein the lane is connected to the node at a non-orthogonal angle,

79. The method as claimed in any one of claims 71 -78, wherein multiple nodes and lanes are in a lattice pattern, wherein the nodes are spaced no more than 300 microns apart.

80. The method as claimed in any one of claims 71-79, wherein the lane is connected to a second node at a non-orthogonal angle and growth of the ceil process is direc ted to the second node, along the lane.

81. The method as claimed in any one of claims ? 1-80, wherein the lane has a width that is less than the diameter of a cell body positioned in the node,

82. The method as claimed in any one of claims 71 -8 I , further comprising providing multiple nodes and lanes to create an array of parallel lanes; and isolating a single cel l process on each of the lanes making tip the array of parallel lanes.

83. The method as claimed in any one of claims 71 -82, further comprising providing multiple interconnected nodes and lanes to form a lattice, wherein the lattice gradually reduces in its degrees of connectivity between the nodes and terminates in one node from which a lane emanates; and isolating a single cell process on the lane.

84. The method as claimed in any one of claims 71 -83, further comprising providing an unpatterned cell body domain region connected to a node or lane to provide additional connectivity or trophic support to a cell body positioned in the node.

85. The method as claimed in any one of claims 71 -84, wherein, the cell is a neuron and the cell process is an axon projecting from the neuron.

86. The method as claimed in any one of claims 71-85 wherein the node and lane are provided on a surface or substrate.

87. The method of claim 86, wherein the surface or substrate are on a dish or niicrop!ate,

88. The method as claimed in any one of claims 71 -86, wherein the node and lane are deposited over an electrode or on the surface of a diagnostic or therapeutic device.

89. A method for organizing or patterning cells comprising: providing a network of nodes and lanes, wherein at l east one lane extends from at least one node, wherein the nodes and lanes are interconnected; and

directing growth of a cell process of a cell positioned in the network in a desired or predetermined direction to isolate a single cell process on a lane, while allowing the cell to remain connected to other cells in a manner that mimics the naturally occurring connectivity of the cell.

90. ^'Hie method of claim 89, wherein an array of parallel lanes extend from the network of nodes and lanes, and wherein a single cell process is isolated on each of the lanes making up the array of parallel lanes,

91. The method of claim 89 or 90, wherein the nodes and lanes are connected at non- orthogonal angles,

92. The method as claimed in any one of claims 89- 1 , wherein the node, a transition from the node to the lane, or the iane directs a cell process of the cell body from the node into the lane in a predetermined manner or direction, which is determined by a configuration of the node, a configuration of a transition from the node to the lane, or a configuration of the lane.

93. The method as claimed in any one of claims 89-92, wherein the node comprises a tapered portion which tapers toward the lane to direct the cell process into the lane.

94. The method as claimed in any one of claims 89-93, wherein at least a portion of the node comprises a tear drop or elliptical shape for directing or guiding the cell process into the lane.

95. Hie method as claimed in any one of claims 89-94, wherein the nodes and

lanes are in a lattice pattern, wherein the nodes are spaced no more than 300 microns apart.

96. The method as claimed in any one of claims 89-95, wherein the nodes are asymmetric and the asymmetric coKfiguration biases the directionality of a cell process outgrowth from a ceii located in a node.

97. Hie method as claimed in any one of claims 89-96, wherein the nodes and lanes are deposite ! over an electrode or on the surface of a diagnostic or therapeutic device,

98. A apparatus for organizing or patterning cells comprising;

a network of nodes and lanes, wherein at least one lane extends from at least one node, wherein the nodes and lanes are interconnected, wherein, the network of nodes and lanes is configured to direct growth of a cell process of a cell which may be positioned therein in a desired or predetermined direction to isolate & single cell process on a lane, while allowing the cell to remain connected to other cells in a manner that mimics the naturally occurring connectivity of the cell.

99. The apparatus of claim 98, wherein an array of parallel lanes extend from the network of nodes and lanes, wherein a single cell process may be isolated on each of the lanes making up the array of parallel lanes,

100. The apparatus of claim 98 or 99, wherein the nodes and lanes are connected at non- orthogonal angles,

101. The apparatus as claimed in any one of claims 98- 100, wherein the node, a transition from the node to the lane, or the lane is configured to direct a cell process of the ceii body from the node into the lane in a predetermined manner or direction, which is determined by a configuration of the node, a configuration of a transition from the node to the lane, or a confi uration of the lane,

102. The apparatus as claimed in any one of claims 98- 101, wherein the node comprises a tapered portion which tapers toward the lane to direct the cell process into the lane.

103. The apparatus as claimed in any one of claims 98- 1 2, wherein at least a portion of the node comprises a tear drop or elliptical shape for directing or guiding the cell process into the lane.

104. The apparatus as claimed in any one of claims 98-103, wherein the nodes and lanes are in a latiice pattern, wherein the nodes are spaced no more than 300 microns apart.

105. The apparatus as claimed in any one of claims 98- i 04, wherein the nodes are asymmetric and the asymmetric configuration biases the directionality of a cell process outgrowth from a cell located in a node.

106. The apparatus as claimed in any one of claims 98- i 05, wherein the nodes and lanes are deposited over an electrode or on the surface of a diagnostic or therapeutic device.

107. An apparatus for organizing or patterning cells comprising:

at least one node configured to receive a cell body; and

at least one lane extending from the node;

wherein a transition from the node to the lane is curved, tapered or rounded to funnel or direct a process of the cell body from the node into the lane in a predetermined direction during growth of the cell process, wherein the predetermined direction is dictated by an asymmetric confi guration of the node and/or the configuration of the transition from the node to the lane,

108. A method lor organizing or patterning cells comprising:

depositing a cell on or near at least one node, the node having at least one lane extending therefrom; and

directing growth of a cel l process in a predetermined direction by directing the cell process along a curved, tapered or rounded portion of the node and into the lane.