Brain-on-a-chip designs and layouts and methods for using same

Abstract

A BoC includes one or more chambers configured to house a population of neurons to be cultured, and one or more cellular-communication platforms placed between at least one pair of chambers from the one or more chambers. The cellular- communication platforms include one or more features / structures configured, shaped, sized, and arranged to promote axonal growth of the population of neurons in a preferred direction and impede axonal growth in directions opposite to the preferred direction, thereby providing control over at least one of, directionality, transmittance, and / or lateral spread between the populations of neurons being cultured. The cellular-communication platforms generate a neuronal circuit between the population of neurons in each pair of the chambers.

Description

BRAIN-ON-A-CHIP DESIGNS AND LAYOUTSAND METHODS FOR USING SAMERELATED APPLICATION

[0001] This application is a NON-PROVISIONAL of, and claims priority to, U.S. Provisional Patent Application Number 63 / 542,889 filed on 06 October 2023, and entitled “DESIGNS AND LAYOUTS FOR BRAIN-ON-CHIP COMPONENTS AND METHODS FOR MAKING AND USING SAME,” which is incorporated in its entirety herein.TECHNICAL FIELD

[0002] The present disclosure relates to the field of neural / neuronal circuits. More particularly, the present disclosure relates to a BoC having cellular-communication platforms configured to facilitate production / culturing of neuronal circuits, and a method for making and using neuronal circuits with the BoC.BACKGROUND

[0003] Neural / neuronal circuits are populations of neurons interconnected by synapses. Such circuits, whether naturally occurring or artificially cultured, carry out a specific function when activated, by virtue of the ability of the neurons to transmit signals from one part of the circuit to another. The signals are transmitted through electrical or chemical processes.

[0004] Neurons can be cultured to form one or more neuronal circuits (sometimes referred to herein as “desired neuronal circuits”) in controlled environments. For example, populations of neurons can be cultured on a petri dish. However, such solutions provide limited control over the design of the neural circuits formed, and hence are unsuitable for situations where neuronal circuits are required that perform or mimic a specific function. For example, certain applications may require an operator to make copies of naturally occurring neuronal circuits, such as for the purposes of experimentation or research, diagnosing diseases, determining efficacy of different treatment options, and the like.

[0005] Some known solutions or BoC designs utilize circular obstacles or pillars to design the neuronal circuits. While these generate symmetrical connectivity and widelateral spread among axons, such obstacles do not sufficiently guide axonal edges of the neurons in desired directions. As a result, axons of one, or both, populations of neurons tend to get lost within the array of pillars. The absence of axonal edge guidance limits transmittance, especially when the two populations of neurons are far apart, and also limits the accuracy and granularity / complexity with which the neuronal circuits can be built or copied.

[0006] Other known designs incorporate horizontal channels that guide axons to the other side of a substrate or an enclosure, resulting in high transmittance. However, these structures introduce strong biases in the connectivity between the population of neurons due to limited lateral spread. Horizontal channels also do not reflect real-world physiological or anatomical tissues, and hence existing solutions are incapable of accurately mirroring or mimicking naturally occurring neuronal circuits.SUMMARY

[0007] The present disclosure pertains to a plurality of BoC and / or BoC platforms, layouts, and designs configured to facilitate the growth of two or more cultured neuronal populations and promote axonal interconnection of the two or more populations of cultured neurons, and a method for culturing and using neuronal circuits with the BoC. The BoC, layouts, and designs disclosed herein are configured to direct axonal growth so that connections between cultured neurons are established with, or without lateral constraints, so that asymmetry for cultured neuronal circuits may be fostered and / or directionality may be recapitulated in ways that, for example, mimic and / or mirror one or more naturally occurring neuronal circuits between two or more types of neurons in communication with one another.

[0008] The BoC includes one or more chambers configured to house a population of cells / neurons to be cultured, and one or more cellular-communication platforms placed between at least one pair of chambers from the one or more chambers. The cellular- communication platforms include one or more features / structures configured (and shaped, sized, and / or arranged) to promote axonal / neuronal growth of the population of neurons in a preferred direction (and / or impede axonal / neuronal growth in directionsopposite to the preferred direction). The cellular-communication platforms culture a neuronal circuit between the population of neurons in the at least one pair of chambers.

[0009] The chambers may be enclosures formed between a base, a body and one or more side walls (e.g., through gaps formed by cavities or protrusions on the body, the base, or the side walls when they are joined together). The side walls may have an opening, which may be defined (such as sized and / or shaped to be large enough) to allow the population of neurons from the at least one pair of chambers to propagate through the opening into the communication platforms. However, the openings may also be defined (such as sized and / or shaped to be small enough) to prevent the population of neurons from moving (e.g., out of the chambers).

[0010] The chambers may house the population of neurons. At times, the population of neurons housed in each of the chambers may be derived from a single neuron. At other times, the population of neurons housed in each of the chambers may be heterogenous.

[0011] The chambers may include one or more wells connected thereto. The wells may be configured to provide a passage for a solution to be added, extracted, and / or replaced from the chambers. Multiple wells may be in fluid communication with each other through the chambers. The solution may be at least one of, a cellular media (e.g., water, nutrients, etc.), or a chemical substance (e.g., pharmaceuticals, chemical dyes, toxins, etc.).

[0012] The structures may be configured to either impede or promote at least one of: transmittance, directionality, or lateral spread of neuronal connections between the population of neurons of the at least one pair of chambers in at least one direction (e.g., the preferred direction). The structures may be configured, shaped, sized, and / or arranged to promote growth of axons / neurons in the preferred direction, and recapitulate directionality and / or symmetry of growth of axons / neurons associated with the population of neurons in the chambers in a direction opposite to the preferred direction, whereby the neuronal circuit is formed with the desired directionality, transmittance, and / or lateral spread. The structures may be defined in an arrangement corresponding to at least one of, a physiological architecture or an anatomical architecture of tissue (e.g., to mimicand / or mirror one or more naturally occurring neuronal circuits between two or more types of neurons in communication with one another).

[0013] The structures may include at least one of, one or more posts (which may have either a triangular shape or a teardrop shape), one or more polygonal structures (which may have a ‘C’-shaped contour), and / or one or more irregular obstacles (having a curved notch and a curved edge). The posts, polygonal structures, and / or irregular obstacles are sized and arranged to form a dwelling area and one or more microchannels. The dwelling area may be configured to impede growth of neurons / axons from the population of neurons in a first direction and redirect the growth of neurons / axons to a second direction. The microchannels may be configured to promote the growth of the neurons / axons in the second direction. For example, the curved notch of the irregular obstacle may define a first curved microchannel between the curved notch and a first post having a teardrop shape, and / or the curved end positioned on an end of the irregular obstacle opposing the curved notch may define a second curved microchannel between the curved end and a second post having a triangular shape, through which neurons / axons may propagate unimpededly. The microchannels may be defined to be either diagonally oriented, linearly oriented, or curved with respect to the cellular- communication platforms. The structures may be either protrusions extending from a body towards a base of the BoC, or pillars defined between the body and the base through the cellular-communication platforms.

[0014] In some embodiments, the population of neurons in a first chamber may be sensory neuronal cells (e.g., olfactory sensory neurons), and the population of neurons in a second chamber may be read-out cells (e.g., olfactory bulb cells). The structures may be configured to synaptically connect (such as by controlling / facilitating direction of growth) the read-out cells to the sensory neuronal cells through the cellular- communication platforms between the first chamber and the second chamber to form the neuronal circuit. The neuronal circuit includes multiple ones of the read-out cells connected to each of the sensory neuronal cells, such as for amplification of signals. The read-out neurons of the neuronal circuit may reinforce incoming synapses that may be activated in pre-defined and / or with recurring temporal patterns and, at times may learn to respond preferentially to the pre-defined and / or recurring temporal patterns.

[0015] A BoC platform may include a neuronal circuit cultured by connecting a population of neurons in each of one or more chambers through one or more corresponding cellular-communication platforms. The neuronal circuits in the BoC platform may be cultured, and used for applications, such as for research, diagnosing diseases, determining treatment plans, and the like. A disk may include one or more BoC platforms placed on a substrate.

[0016] Methods for making neuronal circuits include placing a population of neurons (which may be heterogenous) in a BoC, and either adding, extracting, and / or replacing (through wells connected to chambers of the BoC) a solution in one or more chambers for promoting growth of the population of neurons through the cellular- communication platforms therein, to form the neuronal circuit. The solutions may be added, extracted, and / or replaced using a suction means, such as a pump and / or a dropper. The solution facilitates / promotes the growth of the population of neurons while structures in the cellular-communication platform either impede or facilitate axonal / neuronal growth in a preferred direction.

[0017] Methods of testing impact of chemical substances and / or environmental conditions on neuronal circuits, include culturing (e.g., through housing a population of neurons in chambers and providing a solution to the population of neurons) a neuronal circuit on a BoC. At times, the population of neurons are derived from a patient. The method further includes exposing (e.g., either through wells, or through body and / or base of the BoC) the neuronal circuit to any one or a combination of, one or more chemical substances (e.g., pharmaceuticals, toxins, nutrients, etc.) or one or more environmental conditions (e.g., lighting, temperature, etc.). The methods include determining one or more parameters indicating impact of the chemical substances and / or the environmental conditions on the neuronal circuits. The parameters may correspond to at least one of, rates of communication, types of communication, connectivity, a growth rate, a toxicity response, a morphology, a survival rate, florescence, biomarkers, or omics, between and / or within the population of neurons of each of the one or more chambers. The parameters may be analyzed for determining a presence of a disease in the neuronal circuit, and / or for determining efficacy of the chemical substances and / or theenvironmental conditions for treatment of the neuronal circuit based on analysis of the parameters.

[0018] Optionally, or in addition, the methods include monitoring the growth of the populations of neurons in the BoC, and re-determining the parameters, such as for observing disease progression or drug development.

[0019] Methods for stimulus pattern analysis using neuronal circuits disclosed herein may include culturing a neuronal circuit on a BoC, where the neuronal circuit includes a set of receptor neurons and a set of read-out neurons. The method may further include exposing the neuronal circuit to one or more stimuli to teach the read-out neurons to activate one or more clusters comprising a unique subset of read-out neurons in response to a corresponding stimulus from the one or more stimuli. The method also includes measuring activations at the clusters in response to the stimuli.

[0020] Optionally, the set of receptor neurons may include olfactory receptor neurons and the set of read-out neurons may comprise olfactory bulb neurons. Further, the stimuli may correspond to odorants. Additionally, each of the clusters may be spatially distinct.BRIEF DESCRIPTION OF THE FIGURES

[0021] The present invention is illustrated by way of example, and not limitation, in the figures of the accompanying drawings in which:

[0022] FIG. 1 A is a schematic diagram that illustrates a top view of an exemplary BoC with a vertical bisecting line 1 F-1 F, a horizontal bisecting line 1 D-1 D, and a crosssectioning line 1 E-1 E superimposed thereon, in accordance with one or more embodiments of the present invention;

[0023] FIG. 1 B is a schematic diagram of a side view of the BoC of FIG. 1 A with a horizontal bisecting line 1C-1C superimposed thereon, in accordance with one or more embodiments of the present invention;

[0024] FIG. 1C is a schematic diagram of a horizontal cross-section view of the BoC of FIG. 1A taken along horizontal bisecting line 1C-1C as shown in FIG. 1 B, in accordance with one or more embodiments of the present invention;

[0025] FIG. 1 D is a schematic diagram of a horizontal cross-section view of the BoC of FIG. 1A taken along horizontal bisecting line 1 D-1 D as shown in FIG. 1A, in accordance with one or more embodiments of the present invention;

[0026] FIG. 1 E is schematic diagram of a horizontal cross-section view of the BoC of FIG. 1A taken along horizontal cross-sectioning line 1 E-1E as shown in FIG. 1A, in accordance with one or more embodiments of the present invention;

[0027] FIG. 1 F is the schematic diagram of a vertical cross-section view of the BoC of FIG. 1A taken along vertical bisecting line 1 F-1 F as shown in FIG. 1A, in accordance with one or more embodiments of the present invention;

[0028] FIG. 2A is a schematic diagram of a portion of a platform of the BoC layout shown in FIG. 1C showing a first exemplary layout, or design, for the platform, in accordance with one or more embodiments of the present invention;

[0029] FIG. 2B is a magnified view of a portion of the second BoC of FIG. 2B, in accordance with one or more embodiments of the present invention;

[0030] FIG. 2C is a magnified view of a portion of the second BoC FIG. 2B with indications of directions in which the neurons grow from corresponding chambers of the BoC, in accordance with one or more embodiments of the present invention;

[0031] FIG. 2D is an image that shows a simulation of axonal growth between two neuronal populations via the second BoC of FIG. 2A, in accordance with one or more embodiments of the present invention;

[0032] FIG. 2E is an image that shows a simulation of axonal growth between two neuronal populations using a second exemplary layout, or design, for the second BoC, in accordance with one or more embodiments of the present invention;

[0033] FIG. 2F is an image that shows a simulation of axonal growth between two neuronal populations using a third exemplary layout, or design, for the second BoC, in accordance with one or more embodiments of the present invention;

[0034] FIG. 3A is a schematic diagram of a portion of the BoC shown in FIG. 1C using a fourth exemplary layout, or design, for the platform, in accordance with one or more embodiments of the present invention;

[0035] FIG. 3B provides a detailed view of a portion of the fourth exemplary layout for second platform of FIG. 3A, in accordance with one or more embodiments of the present invention;

[0036] FIG. 4 is a schematic diagram of a disk that includes nine BoC layouts thereon, in accordance with one or more embodiments of the present invention;

[0037] FIG. 5 is a flowchart of a method for making a neuronal circuit using the BoC, in accordance with one or more embodiments of the present invention;

[0038] FIG. 6 illustrates a flowchart of a method for using BoC, in accordance with one or more embodiments of the present invention;

[0039] FIG. 7 illustrates a magnified view of a BoC supporting olfactory bulb neurons connected to different types of olfactory sensory / receptor neurons, in accordance with one or more embodiments of the present invention; and

[0040] FIG. 8 is a flowchart of method for stimulus pattern analysis using neuronal circuits cultivated in a BoC, in accordance with one or more embodiments of the present invention.

[0041] Throughout the drawings, the same reference numerals, and characters, unless otherwise stated, are used to denote like features, elements, components, or portions of the illustrated embodiments. Moreover, while the subject invention will now be described in detail with reference to the drawings, the description is done in connection with the illustrative embodiments. It is intended that changes and modifications can be made to the described embodiments without departing from the true scope and spirit of the subject invention as defined by the appended claims.DETAILED DESCRIPTION

[0042] The brain-on-a-chip (BoC) layouts and / or designs disclosed herein are configured to facilitate the culturing of neuronal circuits while providing control over directionality, transmittance, and / or lateral spread of the populations of neurons forming neuronal circuits. The BoC disclosed herein may include one or more chambers in which populations of neurons may be cultured. The chambers may be positioned on opposing sides of a cellular-communication platform that includes a plurality of features sized, shaped, arranged, and / or configured to encourage axonal growth in one or morepreferred directions so that a neuronal circuit between the one or more populations of neurons may be established and / or generated. In some embodiments, one or more of the neuronal populations may be neurons and / or a set of neurons derived from a single neuron (or a single type of neuron). Additionally, or alternatively, the populations of neurons may include a plurality of different neurons of the same (e.g., a colony of neurons), or a different, type(s).

[0043] Connectivity between two or more populations of neurons and / or the axons of neurons may be characterized by three factors: transmittance, directionality, and lateral spread of connections. Transmittance refers to the total number of axonal projections, or interconnections between axons of the two or more neuronal populations, which is crucial for proper functional transmission of electrical activity between the axons of the two or more neuronal populations. Directionality represents the ratio of connections in one direction compared to the total transmittance. For example, directionality may be represented by the following relationship:Directionality A -> B = transmittance A->B / (transmittance A->B + transmittance B->A) Wherein:A is a first population of neurons;B is a second population of neurons; wherein represents a direction of communication between A neurons and B neurons Directionality may allow for the recreation of specific brain circuits where neurons project in a particular direction between tissues. Lateral spread is necessary to achieve connectivity (e.g., "all-to-all" connectivity or otherwise) between the axons of the two or more neuronal populations, thereby ensuring that synapses can form / connect between all, or most, of the neurons in each of the two or more neuronal populations, which assists with avoiding biases where only neurons facing each other can establish connections.

[0044] As stated, the existing solutions do not allow different populations of neurons to propagate and form neuronal circuits with predetermined properties, such as neuronal circuits having desired directionality, transmittance, and / or lateral spread. The BoC layouts shown and described herein overcome these problems by incorporating features / structures configured, sized, shaped, and arranged to encourage growth of axons in a preferred direction (e.g. toward the opposing chamber and / or neurons includedin the opposing chamber) and / or recapitulate the directionality and symmetry of the neuronal circuits. The features / structures of the BoC disclosed herein may also impede the growth of axons / neurons in directions different from (e.g., opposite to) the preferred direction, whereby the neuronal circuit and / or desired neuronal circuit is formed with the desired directionality, transmittance, and / or lateral spread. Additionally, or alternatively, the features / structures of the BoC disclosed herein facilitate axonal growth in the preferred direction(s) to obtain neuronal circuits with desired geometries, lateral spread, directionality, and / or transmittance. Additionally, or alternatively, BoC layouts shown and described herein may allow for the free growth of axons between features without the use of well-defined / pre-defined tunnels / channels for the axons.

[0045] At times, the features / structures of one or more of the BoCs disclosed herein may be configured, sized, shaped, and / or arranged in a fashion that approximates and / or replicates a physiological architecture found between naturally occurring neuronal populations, which may assist with, for example, synaptic formation, signal transduction between neurons, axonal edge guidance, and / or viability of neuronal populations. For example, the physiological architecture between naturally occurring olfactory receptor neurons and olfactory bulb neurons may include glomeruli. When communication between olfactory receptor neurons and olfactory bulb neurons is to be studied, the feature / structures of a BoCs disclosed herein may be configured, sized, shaped, and arranged to mimic or resemble these glomeruli when populations of olfactory receptor neurons and olfactory bulb neurons are being cultured on a BoC, as described further in reference to FIG. 7.

[0046] Turning now to the figures, FIGs. 1A-1 F provide various views of an exemplary BoC 100, wherein FIG. 1A is top view of BoC 100 with a vertical bisecting line 1 F-1 F and a horizontal bisecting line 1 D-1 D, and a cross-sectioning line 1 E-1 E superimposed thereon; FIG. 1 B is a side view of BoC 100 with a horizontal bisecting line 1C-1C superimposed thereon; FIG. 1C is a horizontal cross-section view of BoC 100 taken along horizontal bisecting line 1C-1C as shown in FIG. 1 B; FIG. 1 D is a horizontal cross-section view of BoC 100 taken along horizontal bisecting line 1 D-1 D as shown in FIG. 1A; FIG. 1 E is a horizontal cross-section view of BoC 100 taken along horizontalcross-sectioning line 1 E-1 E as shown in FIG. 1A; and FIG. 1 F is a vertical cross-section view of BoC 100 taken along vertical bisecting line 1 F-1 F as shown in FIG. 1A.

[0047] As may be seen in FIGs. 1A and 1 B, BoC 100 includes a body 105. with a plurality of wells (in this example, six), that sits on a base 106. First, second, third, fourth, fifth, and / or sixth well 111 , 112, 113, 114, 115, and / or 116, respectively, may be configured to accept and hold a volume of cells, cellular media (e.g., water, nutrients, etc.), and, on some occasions, one or more chemical and / or pharmaceutical substances therein. Exemplary chemicals and / or pharmaceuticals include, but are not limited to, neurological agents, enzymes, anti-neurodegeneration compounds, olfactory stimulating compounds, acetylcholine esterase inhibitors such as donepezil, rivastigmine, and galantamine, dopamine, memantine, a combination drug comprising memantine, donepezil, and / or dopamine, L-Dopa, lecanemab, aducanumab, and / or a biologic product that targets the amyloid-b protein. Additionally, the chemicals and / or pharmaceuticals may include growth factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophic growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), or vascular endothelial growth factor (VEGF), and the like, which may promote cell proliferation, differentiation, and tissue regeneration within the wells. The chemicals and / or pharmaceuticals may be utilized either individually or in combination, to optimize neuronal growth based on experimental and / or therapeutic requirements.

[0048] The plurality of wells (such as the wells 111 , 112, 113, 114, 115, and / or 116) may correspond to hollowed provisions defined in body 105. The plurality of wells may provide a passage for supplying and extracting fluids and / or suspension (e.g., cells, cell media, solutions, etc.) to and from corresponding chambers (such as the first, second, and / or third chambers 130, 140, and 150). At least one end of the wells terminates at (or sits over) the corresponding chamber, and at least one other end terminates at an outer surface of body 105, thereby exposing the chambers to the external environment. In some embodiments, the plurality of wells may include and / or be fitted with a cap (not shown), which may be configured to reversibly fit over the end of a well that is exposed to the external environment. The cap, when fit, may prevent contaminants from the external environment from entering the chamber(s). In some embodiments, at least one end of thewells may terminate at (or sit over) the first chamber 130, and at least one other end of the wells may terminate at the third chamber 150, for example, thereby providing fluid communication between the first and third chambers 130 and 150. In some embodiments, the plurality of wells may be characterized by a cylindrical or ovoid geometry. In other embodiments, the wells may be characterized by a polygonal or irregular geometry. The number, dimensions, and sizes of wells defined in body 105 may be suitably adapted based on the number of chambers defined in BoC 100.

[0049] One or more pairs of the wells (such as first and second wells 111 and 112, second and third wells 113 and 114, and / or third and fourth wells 115 and 116) may be in liquid / fluid communication with each other via a corresponding chamber. As may be seen in the cross-section of FIG. 1C, the first well 111 and the second well 112 are in liquid / fluid communication with one another via a first chamber 130 defined by a first sidewall 135A and a second sidewall 135B; the third well 113 and the fourth well 114 are in liquid / fluid communication with a second chamber 140 defined by a third sidewall 145A and a fourth sidewall 145B; and the fifth well 115 and the sixth well 116 are in liquid / fluid communication with a third chamber 150 defined by a fifth sidewall 155A and a sixth sidewall 155B. First, second, and third chambers 130, 140, and 150 are enclosures formed between body 105, base 106, and corresponding sidewalls 135A and 135B, 145A and 145B, and 155A and 155B, respectively. In some embodiments, body 105 includes cavities / dimples and / or protrusions on and / or extending from one surface thereof. Gaps defined by the cavities / dimples and / or protrusions when body 105 is placed over base 106, forms the first, second, and third chambers 130, 140, and 150 therebetween. Alternatively, or in addition, cavities / dimples and / or protrusions may be defined on at least one surface of base 106, which, when placed beneath body 105, forms first, second, and third chambers 130, 140, and 150 therebetween. In some embodiments, sidewalls 135A and 135B, 145A and 145B, and 155A and 155B may correspond to portions of either body 105, base 106, or both, that define the lateral surfaces of chambers 130, 140, and 150, respectively.

[0050] First chamber 130, second chamber 140, and / or third chamber 150 may be configured to house a population of cells (such as neurons) and / or facilitate the growth and / or culturing of the cells. Exemplary cells that may reside within first chamber 130,second chamber 140, and / or third chamber 150 may include, but not be limited to, somatic cells, neurons, olfactory receptor neurons, and / or olfactory bulb neurons. At times, a type of cell residing within first chamber 130, second chamber 140, and third chamber 150 may be the same and, at other times, the cell types residing within first, second, and / or third chamber 130, 140, and 150 may be different from one another (i.e., cell types in each chamber may be heterogenous). For example, olfactory receptor neurons may reside within first chamber 130 and third chamber 150 and olfactory bulb neurons may reside within second chamber 140. It is appreciated by those skilled in the art that types of cells / neurons used in each of the chambers (such as chambers 130, 140, and 150) may be selected based on requirements of the use case.

[0051] BoC 100 also includes a first cellular-communication platform 120 and a second cellular-communication platform 126, both of which may have a plurality of features, structures, or elements thereon and / or therein that may be configured to facilitate cellular and / or axonal growth with one or more preferred characteristics. In some embodiments, first cellular-communication platform 120 and second cellular- communication platform 126 may be configured to facilitate axonal / neuronal growth of a population of neurons in a preferred direction (and / or impede the axonal / neuronal growth in directions different from and / or opposite to the preferred direction) to generate a neuronal circuit between the population of neurons in each pair of first, second, and / or third chambers 130, 140, 150.

[0052] First cellular-communication platform 120 may be in communication with first chamber 130 via a first chamber-platform interface 122A, and second chamber 140 via a second chamber-platform interface 122B. Second cellular-communication platform 126 may be in communication with second chamber 140 via a third chamber-platform interface 128A, and fourth chamber 150 via a fourth chamber-platform interface 128B. Sidewalls 135B, 145A, 145B, and / or 155A may each have one or more opening(s) configured to allow the population of neurons from each pair of first, second, and / or third chambers 130, 140, and / or 150 to exhibit axonal growth through the respective opening into first and second communication platforms 120 and / or 126, while preventing the population of neurons from moving out of the first, second, and third chambers 130, 140, and 150.

[0053] Cells, cellular media, and other substances (e.g., a solution) introduced into first well 111 and / or second well 112 may travel / flow into first chamber 130 and reside therein. Additionally, or alternatively, cells, cellular media, and other substances introduced into third well 113 and / or fourth well 114 may travel to second chamber 140 and reside therein. Additionally, or alternatively, cells, cellular media, and other substances introduced into fifth well 115 and / or sixth well 116 may travel to third chamber 150 and reside therein. On some occasions, cellular media may be changed, replaced, and / or refreshed via extraction of a volume of old cellular media from first, second, third, fourth, fifth, and / or sixth well 111 , 112, 113, 114, 115, and / or 116, and / or introduction / addition of fresh cellular media into first, second, third, fourth, fifth, and / or sixth well 111 , 112, 113, 114, 115, and / or 116. For example, a volume of fresh cellular media may be added to first chamber 130 by extracting of an (existing) volume cellular media from second well 112 and introducing fresh cellular media to first well 111. The solution may be extracted, added, and / or replaced using a suction means, such as a pump and / or a dropper. Further, since first, second and third chambers 130, 140, and 150 are enclosed, and may be physically isolated, chances of formation of artifacts therein may be reduced. In such cases, BoC 100 may eliminate or minimize the need for analytes.

[0054] The cross-section of FIG. 1 D (taken along line 1 D shown in FIG. 1 A) shows how second well 112, sixth well 116, and second chamber 140 reside within body 105 with a lower edge of BoC 100 being established and / or defined by base 106. FIG. 1 D also shows a population of a first type of neurons 164 (which are represented by circles or dots with cross hatchings) resident within second chamber 140. FIG. 1 D also shows how an upper edge, or roof, of second chamber 140 is defined by body 105. The cross-section of FIG. 1 F (taken along line 1 F shown in FIG. 1A) shows second chamber 140 extending between third well 113 and fourth well 114 and shows a plurality of the first type of neurons 164 positioned along a length of second chamber 140 and a portion of third and fourth wells 113 and 114 as shown.

[0055] As may be seen in the cross-section of FIG. 1 E (taken along line 1 E shown in FIG. 1A), first, second, third, and / or fourth chamber-platform interfaces 122A, 122B, 128A, and / or 128B may be configured as openings to first cellular-communication platform 120 or second cellular-communication platform 126, while allow for liquid / fluidcommunication and / or axonal growth between first chamber 130, second chamber 140, third chamber 150, first cellular-communication platform 120 and / or second cellular- communication platform 126 as shown. In some embodiments, the openings of first, second, third, and / or fourth chamber-platform interfaces 122A, 122B, 128A, and / or 128B may be defined (such as sized and / or shaped) to be large enough to allow for axonal growth through the respective chamber-platform interface but may be defined (such as sized and / or shaped) to be small enough to prevent movement of a cell / neurons through a chamber-platform interface. In this way, the neurons may be held in their respective first, second, or third chambers 130, 140, or 150. At times, chamber-platform interfaces may be implemented as openings on respective sidewalls of first, second, or third chambers 130, 140, or 150 adjoining first and second cellular-communication platforms 120 and 126. For example, as shown in FIG. 1 E, a second type of neurons 166 (represented using circles with cross-hatching) may reside in first chamber 130 and first cellular-communication platform 120 may encourage growth (represented as an arrow between first chamber 130 and second chamber 140) of axons from the second type of neurons 166 in first chamber 130 to second chamber 140 through first cellular- communication platform 120 via first and second chamber-platform interfaces 122A and 122B, where the second type of neurons 166 may connect with an be in communication with the first type of neurons 164, which are resident within second chamber 140. In addition, second cellular-communication platform 126 may encourage growth in a preferred direction (represented as an arrow between second chamber 140 and third chamber 150) of axons from the first type of neurons 164 from second chamber 140 through second cellular-communication platform 126 to third chamber 150 via third and fourth chamber-platform interfaces 128A and 128B so that the first type of neurons 164 may be in communication with a third type of neurons 168, which are resident within third chamber 150.

[0056] First cellular-communication platform 120 and / or second cellular- communication platform 126 may be configured to include a plurality of features / structures such as channels, posts, and / or microstructures sized, shaped, and arranged to facilitate axonal growth of neurons residing within first, second, and / or third chamber 130, 140, and / or 150 with preferred characteristics. At times, the structures maybe sized, shaped and / or arranged to form microchannels providing linear paths through which the population of neurons in first chamber 130, second chamber 140, and / or third chamber 150 may grow, propagate, and form synaptic interconnections therebetween. The microchannels may promote axonal / neuronal growth towards the preferred direction. For example, the features / structures of first and / or second platform(s) 120 and / or 126 may be configured, shaped, sized, and / or arranged to recapitulate the directionality and / or symmetry of the neuronal circuits and / or provide the neurons (or axons thereof) flexibility to form synapses / synaptic connections between the same and / or different populations of neurons in different chambers (such as first, second, and third chambers 130, 140, and 150). Additionally, or alternatively, the features of first and / or second cellular-communication platform(s) 120 and / or 126 may be configured and / or arranged to replicate, or mimic, a physiological and / or anatomical architecture of tissue found between neurons when resident within their natural environment (e.g., olfactory bulb, brain, etc.), which may play a crucial role in signal transduction between axons and the survival of neurons.

[0057] In one embodiment, the features / structures of first and / or second cellular- communication platform(s) 120 and / or 126 may be configured and / or arranged to replicate and / or mimic physiological and / or anatomical structures positioned between olfactory receptor neurons and olfactory bulb neurons, which may be known as glomeruli. These features, which mimic glomeruli, may then foster signal transduction between axons of the olfactory receptor neurons and olfactory bulb neurons and increase viability and / or survival rates of the olfactory receptor neurons and / or the olfactory bulb neurons.

[0058] A first exemplary layout, or design, for second cellular-communication platform 126A is shown in the detailed view of FIG. 2A and further magnified / detailed view of FIG. 2B, in which a portion of second cellular-communication platform 126A is magnified to show an array of a plurality of features / structures in the form of posts 215 with a triangular shape and polygonal structures 210 with a shape approximating a “C” as shown. Posts 215 and polygonal structures 210 are shaped, sized, and / or arranged within second cellular-communication platform 126A to form at least one of, microchannels 220 and / or dwelling areas 225 therebetween. Posts 215 and polygonal structures 210 are configured, shaped, sized, and / or arranged to provide axonal edge guidance and / orfacilitate axonal growth between two populations of neurons (such as those positioned in second and third chambers 140 and 150). Such structures may facilitate growth of neurons from third chamber-platform interface 128A to fourth chamber-platform interface 128B as shown by arrows 180 in FIG. 2C, where outer (convex) edges of the C-like shape of polygonal structures 210 divert the neurons to grow around polygonal structures 210, but not back to the third chamber -platform interface 128A. For example, the C-like shape of polygonal structures 210 may be sized, shaped, and / or configured, to encourage redirection of axons growing toward dwelling areas 225 back in the direction from which they came, as shown by arrows 182 in FIG. 2C. For example, axons growing from fourth chamber-platform interface 128B toward third chamber-platform interface 128A (indicated by arrows 182) are redirected back toward fourth chamber-platform interface 128B by inner (concave) edges of C-like shape of polygonal structures 210. The concave edges provide an arduous path for neurons growing into second cellular-communication platform 126A from fourth chamber-platform interface 128B. In this way, second cellular- communication platform 126 and / or features / structures thereon may encourage growth of axons from third chamber-platform interface 128A toward fourth chamber-platform interface 128B in the preferred direction (i.e., left to right as oriented in FIG. 2A) but not from fourth chamber-platform interface 128B toward third chamber-platform interface 128A in a direction opposite to the preferred direction (i.e., right to left as oriented in FIG. 2A).

[0059] One example of how second cellular-communication platform 126A may provide axonal edge guidance and / or facilitate axonal growth is shown in an image 201 of FIG. 2D, which shows a simulation of axonal growth between a first neuronal population resident within third chamber 140 and third chamber-platform interface 128A (shown in light grey) and a second neuronal population (shown in dark grey) residing in third chamber 150 and fourth chamber-platform interface 128B. As may be seen in image 201 , axons of the first neuronal population extend from third chamber-platform interface 128A through microchannels 220 and dwelling areas 225 of second cellular-communication platform 126A toward fourth chamber-platform interface 128B without the cellular bodies of the first neuronal population neurons passing into second cellular-communication platform 126A. Likewise, axons of the second neuronal population extend from fourthchamber-platform interface 128B through microchannels 220 and dwelling areas 225 of cellular-communication platform 126A toward third chamber-platform interface 128A without the cellular bodies of the second neuronal population neurons passing into second cellular-communication platform 126A.

[0060] A second exemplary layout, or design, for second cellular-communication platform 126B is provided in an image 202 shown in FIG. 2E. As may be seen in image202, second cellular-communication platform 126B includes a plurality of rows of polygonal structures 210 laid out in a manner similar to first exemplary layout, or design, for second cellular-communication platform 126A as shown in FIGs. 2B, 2C, and 2D. However, unlike the first exemplary layout for second cellular-communication platform 126A, second exemplary layout for second cellular-communication platform 126B does not include posts 215. As shown, the lack of posts 215 may cause growth of the neurons / axons to have a substantially normal distribution (or normal distribution with a higher peak along length of cellular-communication platform 126B), as opposed to substantially uniform distribution (or a normal distribution with a fatter tail with respect to length of communication platform 126A) in first exemplary layout where posts 215 are included.

[0061] A third exemplary layout, or design, for second cellular-communication platform 126C is provided in an image 203 shown in FIG. 2F. As may be seen in image203, second cellular-communication platform 126C includes a plurality of rows of polygonal structures 210 and a plurality of rows of posts 215 laid out in a manner similar to first exemplary layout, or design, for second cellular-communication platform 126A as shown in FIGs. 2B, 2C, and 2D. However, polygonal structures 210 and / or posts 215 are smaller in size and / or are more densely packed together in third exemplary layout, or design, for second cellular-communication platform 126C than they are in the first exemplary layout of second cellular-communication platform 126A. The structure of the third exemplary layout may promote greater lateral spread in comparison to the first or the second exemplary layout.

[0062] FIG. 3A provides a schematic diagram of a fourth exemplary layout, or design, for second cellular-communication platform 126D and FIG. 3B provides a detailed view of a portion of the fourth exemplary layout for second cellular-communicationplatform 126D. Fourth exemplary layout for second cellular-communication platform 126D includes a plurality of rows of large teardrop shaped posts 310 interleaved with a plurality of rows of small teardrop shaped posts 315, a plurality of small triangular posts 330, a plurality of large triangular posts 335, a plurality of large irregular obstacles 320 that have a curved notch that allows for a first curved microchannel 345 to reside between the curved notch and small teardrop-shaped post 315, a curved end positioned on an end of irregular obstacle 320 opposing the curved notch that allows for a second curved microchannel 332 to reside between the curved end and small triangular post 330, through which neurons / axons from the population of neurons may grow unimpededly. Second cellular-communication platform 126D also includes a plurality of small irregular obstacles 322 that have a curved notch that allows for a third curved microchannel 350 to reside between the curved notch and large teardrop-shaped post 310, a curved end positioned on an end opposing the curved notch that allows for a fourth curved microchannel 334 to reside between the curved end and large triangular post 335.

[0063] In some embodiments, first, second, third, and / or fourth curved microchannel(s) 345, 332, 350, and / or 334 may have the same, or a similar, degree of concavity, which may be configured and / or designed to redirect axons coming from the right side (as oriented in FIG. 3B) back to their origins through axonal edge guidance. Fourth exemplary layout for second cellular-communication platform 126D further includes a plurality of diagonally oriented (as oriented in the figure) linear microchannels 340 that are formed by the sidewalls of large teardrop shaped posts 310, small teardrop posts 315, large triangular posts 335, small triangular posts 330, large irregular obstacles 320, and small irregular obstacle 322 as shown. While embodiments shown in first, second, third, and fourth layouts have microchannels 220, 340 that are diagonally oriented with respect to first and / or second cellular-communication platforms 120 and / or 126, it is appreciated that shapes, sizes, and / or arrangements of structures may be suitably adapted to provide microchannels which are linear or curved in orientation and shape with respect to first and / or second cellular-communication platforms 120 and / or 126.

[0064] The structures may be defined / implemented as protrusions extending from body 105 towards base 106, or vice versa, where the protrusions may extend part wayup to an opposing surface of base 106 or body 105, respectively. In such implementations, a gap may be provided between the protrusions and the opposing surface of base 106 or body 105. Alternatively, or in addition, some of the structures may be defined / implemented as pillars provided between body 105 and base 106 through the first and second cellular-communication platforms 120 and 126. The pillars may extend up to the opposing surface of body 105 and base 106.

[0065] In some embodiments, a BoC layout may include varying numbers (e.g., 1 , 3, 4, 5, etc.) of first cellular-communication platform 120, second cellular-communication platform 126, and / or other platforms that may have the same and / or different (e.g., different layout design and / or different scale or sized features for the same and / or similar layout designs) from one another. In addition, although FIGs. 2A-2F, 3A and 3B provide exemplary designs for layouts of second cellular-communication platform 126B, this is by way of example and not limitation. The layout designs of FIGs. 2A-2F, 3A and 3B could also be representative of design layouts for first cellular-communication platform 126A.

[0066] The BoCs 100 disclosed herein may be made from any appropriate material (e.g., plastic or silicon, but not limited thereto) via any appropriate method (e.g., casting on a mold, stamping, injection molding, pressing, three-dimensional printing, etc.). For example, in some embodiments, photolithography and / or three-dimensional printing may be used to make a mold for the generation of one or more types of BoCs, and then BoCs 100 may be made by pouring a silicon elastomer (or other appropriate material) onto the mold and allowing it to cure. Once the poured material has cured, the mold may be removed from the cured material to yield the molded BoC 100. In some embodiments, a mold may include a plurality of sections, with each section including a mold for an individual plurality of BoC 100. In these embodiments, the BoC-forming material may be poured on the mold and, once the mold is removed a disk with a plurality of BoC may be formed (see, e.g., FIG. 4). The disk may be made of a substrate, such as glass, ceramic, plastic, or any other substrate made of materials that are biocompatible. In some circumstances, the disk including a plurality of BoCs (such as BoCs 100A-100I) may be divided (e.g., cut apart) to form individual BoCs that may, for example, be placed on a microscopic slide for examination via microscope. In some embodiments, body 105 and base 106 which form chambers of the BoC may be manufactured separately andassembled to form BoC 100. Either body 105 or base 106 may include protrusions or cavities that define chambers, cellular-communication platforms, and / or other structures of a BoC and / or a portion thereof. Body 105 and base 106 may be assembled using a fastening means (e.g., adhesives, nails, screws, rivets, interlocking members, etc.). Components of BoC 100 may be modular, thereby allowing base 106 and body 105 to be manufactured separately and assembled, disassembled, and replaced with other bases and / or bodies having the same or different designs / configurations for the chambers, the cellular-communication platforms, and / or the structures, which may be suitable for different use cases.

[0067] FIG. 4 is a schematic diagram of a BoC platform or a disk 400 that includes nine BoC layouts 100A-100I thereon, which may be the same as, and / or different from, one another in terms of size, layout, and the like. Disk 400 may be used in, for example, a laboratory setting to grow populations of neurons and conduct experiments and / or make observations thereon. For example, once the neuronal populations are grown on platforms like first cellular-communication platform 120 and / or second cellular- communication platform 126 of BoCs like BoC layouts 100A-100I, communication (e.g., through electrical signals and / or chemicals) between them may be observed, measured, or otherwise analyzed to determine, for example, rates and / or types of communication, which may useful when, for example, determining medication dosage and / or responsiveness of one, or both, neuronal populations.

[0068] Different types of cells / neurons may be placed in a corresponding chamber of the BoCs to cultivate / culture desired neuronal circuits. For example, sensory cells (e.g., olfactory sensory neurons) may be placed in one of the chambers (such as second chamber 140), and read-out cells (e.g., olfactory bulb neurons) may be placed in at least one other chamber (such as first chamber 130 or third chamber 150, respectively). Placing the sensory cells and the read-out cells in different chambers may allow sensory activity to be ‘read’ away / in a different location from the sensory cells. The cellular- communication platforms (such as first and second cellular-communication platforms 120 and 126) may be used to facilitate growth of the sensory cells and the read-out cells, and thereby facilitate formation of synaptic connections therebetween. In some embodiments, the structures in the cellular-communication platforms may be defined similarly tophysiological and anatomical architectures in olfactory sensory organs / tissues (such as glomeruli), thereby allowing neuronal circuits to be cultured in a BoC in a manner that closely mimics naturally occurring circuits between olfactory receptor / sensory neurons and olfactory bulb neurons. Such artificially grown neuronal circuits may be used for applications such as scientific research on neuronal circuits, determination of impact of chemical substances and / or environmental parameters on the neuronal circuits, disease diagnosis, determination of efficacy of treatments, and the like, as, for example, further described in reference to FIG. 7. This may be in contrast to basic live-sensing devices, which require placing electrodes or photodetectors (among other instruments) directly on sensory cells to detect electrical signals (or calcium transients) in the neuronal circuits.

[0069] The BoCs disclosed herein may provide flexibility for further adaptation of neuronal circuits for different functionalities. In some embodiments, each sensory cell may be caused to be connected to multiple read-out cells, and / or the read-out cells may be caused to form synaptic connections with themselves, to amplify signals corresponding to sensory activity. Amplification of signals may enable weak signals to be identified / distinguished from stronger signals which may drown and / or obscure the weak signals, for example, thereby identifying otherwise hidden patterns of communication between different populations of neurons.

[0070] The neuronal circuits may also be cultured using one or more BoCs disclosed herein in such a way that they utilize synaptic plasticity occurring in vivo between the sensory cells and the read-out cells to enhance pattern recognition capabilities. For example, while each of the read-out cells may be in synaptic contact with many sensory cells, the read-out cells may only reinforce incoming synapses that are activated in well-defined / pre-defined and recurring (i.e. non-random) temporal patterns, thereby making them impervious / resistant to random fluctuations. The neuronal circuits may also be subjected to contrastive learning mechanisms to prevent the read-out cells from learning patterns redundantly.

[0071] The BoCs disclosed herein may also provide stability to the population of cells / neurons in first, second, and third chambers 130, 140, and 150. Since the sensory cells multiply and replace dying cells (as may occur due to repeated exposure to analytes and / or chemicals), positioning of cells responding to analytes and / or chemicals maycontinually change over a period of time. Meanwhile, the read-out cells, although not replaced, may form new synaptical connections with incoming synapses of new sensory cells, based on activations new incoming sensory cells temporally match with those of afferent sensory cells. Hence the read-out cells may behave in a stable or predictable manner over time. Other applications of the neuronal circuits are explained subsequently in reference to FIGs. 6 and 7.

[0072] The structures of the BoC described herein may be operable to increase lateral spread around the average axonal growth direction by, for example, selectively impeding and / or facilitating growth of the populations of neurons in one or more preferred directions. In some cases, the structures and / or features of the BoC described herein may enable a more distributed and unbiased connectivity between input neurons and output neurons. Further, the structures and / or features of the BoC disclosed herein may facilitate an increase in the number of intersecting axon pairs between different neuron populations. In situations where axonal growth cones find their final targets by selectively following previously established axons based on their common genetic identity and / or electrophysiological behavior, the chances of proper maturation of the culture of the neuronal circuits increase, which is another benefit of the BoC disclosed herein.

[0073] FIG. 5 provides a flowchart of an exemplary process, or method, 500 for using one or more of the BoCs disclosed herein to, for example, culture desired neuronal circuits. At step 505, a first population of neurons may be placed in a first chamber (e.g., first, second, or third chambers 130, 140, or 150) of the BoC and at step 510, a second population of neurons may be placed in a second chamber (e.g., first, second, and third chambers 130, 140, or 150) of the BoC. The first and second neuronal populations may be homogeneous or heterogenous. Over time, axons of the first and second populations of neurons may grow through a cellular-communication platform (e.g., cellular- communication platform 120 or 125) to form a neuronal circuit. The cellular- communication platforms include one or more features / structures that may be configured to facilitate axonal growth of the population of neurons in a preferred direction to generate a desired neuronal circuit between the first and second population of neurons in the at least one pair of chambers. The structures may promote the growth in the preferred direction, and also impede the growth of the neurons in the population of neurons indirections that are not preferred. The structures may include posts (e.g., posts 215, 310 and / or 315), polygonal structures (e.g., polygonal structures 210), and / or irregular structures (e.g. irregular structures 320 and 322), which form dwelling areas (e.g., dwelling areas 225) and multiple microchannels (e.g., microchannels 220, 332, 334, 345 and 350). The dwelling areas may be configured to redirect axonal / neuronal growth of neuron populations back in the direction from which they came (such as the chambers from which they are propagating).

[0074] In step 515, a level and / or volume of a solution (e.g., cellular media, food, saline, etc.) within one or more of the chambers may be adjusted (e.g., added, extracted, and / or replaced) to, for example, promote growth of the corresponding population of neurons through the cellular-communication platforms adjoining the chambers to form the desired neuronal circuit. The solution may be added, extracted, and / or replaced through one or more wells (e.g., first, second, third, fourth, fifth, and sixth wells 111 , 112, 113, 114, 115, and 116) connected to the chambers. The solution may include at least one of, cells, cell media (e.g., water, nutrients, etc.), chemical substances, pharmaceutical substances, and / or the like. The addition of the solution may facilitate the growth of the population of neurons.

[0075] FIG. 6 provides a flowchart of a process, or method, 600 for testing the impact of chemical substances and / or environmental conditions on neuronal circuits grown in a BoC like the BoCs disclosed herein.

[0076] In step 605, a neuronal circuit may be cultured on a BoC (e.g., BoC 100) via, for example, execution of process 500. In some cases, the desired neuronal circuit may correspond to a copy of a naturally occurring neuronal circuit in a physiological or anatomical tissue, such as those of mammals or other species / creatures belonging to the animal kingdom. For culturing the desired neuronal circuit, a population of neurons may be housed in each of one or more chambers (e.g., first, second, and third chambers 130, 140, and 150) of the BoC. The neurons placed in the chambers may be selected based on requirements of the use case / application. For example, to diagnose a disease (such as those of neurological nature), or to determine toxicity or efficacy of a pharmaceutical substance or environmental conditions on neurons of a patient, the population of neurons placed in the chambers may be derived from the patient’s neurons. Further, the desiredneuronal circuits may be cultured by either adding, extracting, and / or replacing a solution in the chambers to promote growth of the population of neurons through one or more cellular-communication platforms (e.g. first and second cellular-communication platforms 120 and 126) of the BoC.

[0077] At step 610, the desired neuronal circuit may be exposed to any one or a combination of, one or more chemical substances (e.g., pharmaceuticals, toxins, nutrients, etc.) and / or one or more environmental conditions (e.g., lighting, temperature, etc.). The chemical substances and / or the environmental conditions may be selected based on the application. For example, in applications requiring diagnosing neurological diseases (such as Alzheimer’s disease), the neuronal circuits in the BoC may be exposed to certain chemical dyes (such as thioflavin T). Since the chemical dyes may bind with proteins or other compounds in the neuronal circuits afflicted with Alzheimer’s disease to produce florescence, changes in fluorescence (or lack thereof) and / or other biomarkers may indicate presence (or absence) of the neurological disease (in this case, Alzheimer’s disease). Similarly, to determine toxicity or efficacy of a pharmaceutical drug to neuronal circuits, the neuronal circuits may be exposed to the pharmaceutical drug. In such examples, the health of the neuronal circuits may be monitored, through which the toxicity and / or the efficacy of the pharmaceutical drug may be determined / inferred.

[0078] At times, the neuronal circuits may be exposed to the chemical substances and / or the environmental conditions through one or more wells (e.g., first, second, third, fourth, fifth, and sixth wells 111 , 112, 113, 114, 115, and 116) connected to the chambers. For example, the chemical substances may be mixed with a solution, and delivered to the population of neurons in the chambers through the wells. At other times, the neuronal circuits may be exposed to the chemical substances and / or the environmental conditions through a body (e.g., body 105) or a base (e.g., base 106) of the BoC. For example, in applications that require the neuronal circuit to be exposed to extreme temperatures, heat may be provided to the base and / or the body, which may conduct the heat to the populations of neurons in the chambers.

[0079] At step 615, one or more parameters indicating impact of the chemical substances and / or the environmental conditions on the neuronal circuit may be determined. The parameters determined, and subsequently analyzed for the givenapplication, may correspond to, for example, at least one of, rates of communication, types of communication, connectivity, a growth rate, a toxicity response, a morphology, a survival rate, florescence, biomarkers, omics, or the like, between and / or within the population of neurons of each of the chambers. At times, the parameters may be determined based on signals “read” from read-out cells / neurons disposed in at least one of the chambers. At other times, the parameters may be determined using instructions such as electrodes or probes inserted into / connected to the neuronal circuits. While the aforementioned techniques may be used for determining parameters associated with connectivity, signal strength, and the like, other parameters may be observed / determined using visual aid tools, such as microscopes or microscopic cameras. Additionally, or alternatively, data may be collected using sensors and / or instruments, which may be processed by a computing device to determine the parameters, such as growth rates, toxicity responses, morphology changes, and survival rates. The data collected may also be used to analyze omics, providing insights into the molecular mechanisms underlying the observed changes in the neuronal circuits. Other techniques to determine and analyze the parameters may include live cell imaging, immunohistochemistry, in situ hybridization, omics, and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis.

[0080] As stated, the parameters may be analyzed for studying behaviour of the neuronal circuits, such as for smell and chemical sensing at olfactory epithelium (OE) and at olfactory bulb (OB) levels. For example, the parameters may be processed and analyzed for identifying olfactory pattern recognition at the OE and OB level for smell sensing. Further, the parameters may be analyzed for determining, among other things, the presence of a disease in the neuronal circuit. The parameters may also be analyzed to determine efficacy and / or toxicity of the chemical substances and / or the environmental conditions as a treatment for the neuronal circuit, which may be afflicted with a disease. Since, in some cases, the neuronal circuits are derived from the neurons of the patient, culturing the neuronal circuits using the BoC of the present disclosure may enable physicians, academics, and / or technicians to determine and curate efficacious treatments which are personalized for each patient.

[0081] Optionally, or in addition, method 600 may include monitoring the growth of the populations of neurons in a BoC and re-determining the parameters. In some cases,the monitoring and / or redetermination step may be performed at predetermined intervals to obtain time-series evaluations of impact of the chemical substances and / or environmental conditions on the neuronal circuits. For example, in applications where development and progression of neurological diseases are to be studied (either with or without the exposure to different the chemical substances and or the environmental conditions), the neuronal circuits may be monitored for a predetermined period of time. Additionally, monitoring neuronal growth and development on the BoC may aid in, for example, disease modeling and drug discovery.

[0082] In some embodiments, process 600 may be executed to improve and / or measure neuronal growth and / or decay for patients with neurological disease. This may be done by, for example, executing process 500 and / or step 605 to culture a sample of an individual patient’s neurons to generate one or more desired neuronal circuits. Then step 610 may be executed using, for example, one or more chemical substances, therapeutics, and / or environmental conditions to determine (step 615) one or more outcomes of execution of step 610 to, for example, monitor neuronal growth and / or development for disease modeling and / or drug discovery and / or quantification of neuronal parameters to, for example, assess disease progression and / or treatment efficacy.

[0083] In some embodiments, the neurons used to grow, or culture, a neuronal circuit using one or more of the systems, methods, and / or BoCs disclosed herein may be from post-mortem tissue and process 600 may be executed to, for example, identify disease biomarkers to determine a cause of death, the presence of one or more diseases, and / or measure a response of the post-mortem neurons to one or more stimuli provided via, for example, execution of process 600.

[0084] Additionally, or alternatively, the neurons used to grow, or culture, a neuronal circuit using one or more of the systems, methods, and / or BoCs disclosed herein may be induced pluripotent stem cell (iPSC)-derived neurons that may be used to, for example, diagnose disease, detect biomarkers, and / or measure a response to one or more stimuli provided via, for example, execution of process 600.

[0085] As stated, the BoCs disclosed herein may be used for several applications including, but not limited to, analyzing / studying behavior (e.g., communication, growth patterns, etc.) of different neuronal circuits and / or neurons, or groups of neurons, withinneuronal circuits. For instance, the BoCs and the methods described herein may be used to recreate parts of neuronal circuits (such as of bilaterians, cnidarians, and / or generally organisms having neurons / brains). To illustrate how this may be done, an example of neuronal circuit that mimics olfactory bulb neuron behavior and / or includes olfactory bulb neurons is herein provided, wherein FIG. 7 shows a magnified view of a BoC 700 supporting a neuronal circuit including olfactory bulb neurons (shown as 704A, 704B, and 704C) connected to different types of olfactory sensory / receptor neurons (shown as 702A and 702B). BoC 700 may be implemented to mimic olfactory neuronal circuits, which may be used for olfactory bulb pattern analysis, for example.

[0086] BoC 700 may include a first chamber (such as first chamber 130) which supports multiple types of olfactory bulb neurons (such as first, second, and / or third olfactory bulb neurons 704A, 704B, and / or 704C, respectively) and a second chamber (such as second chamber 140) which supports multiple types of olfactory receptor neurons (such as first and second olfactory receptor neurons 702A and 702B, respectively). A cellular-communication platform (such as cellular-communication platform 120) may be disposed between first chamber 130 and second chamber 140 and adapted to facilitate neuronal and / or axial growth from first and second olfactory receptor neurons 702A and 702B in first chamber 130 to the olfactory bulb neurons in second chamber 140. The cellular-communication platform is hidden in FIG. 7 for clarity. Neuronal circuits may be formed between any combination of first and second olfactory receptor neurons 702A and 702B to first, second, and third olfactory bulb neurons 704A, 704B, and 704C through the cellular-communication platform. In the example shown in FIG. 7, first olfactory receptor neurons 702A in first chamber 130 may form neuronal connections / circuits with multiple olfactory bulb neurons 704A and 704B in second chamber 140, while second olfactory receptor neurons 702B in first chamber 130 may form neuronal connections / circuits with second and third olfactory bulb neurons 704B and 704C in second chamber 140 through cellular-communication platform therebetween as shown. The neuronal connections / circuits extending from first olfactory receptor neurons 702A are shown using dotted lines, and the neuronal connections / circuits exiting from second factory neurons 702B are shown using dashed lines.

[0087] Multiple ones of first and second olfactory receptor neurons 702A and 702B may be uniformly and / or randomly distributed in first chamber 130, such as to mimic distributions found in naturally occurring neuronal circuits or tissues (for example, in an olfactory epithelium). Further, each of first and second olfactory receptor neurons 702A and 702B may sense a corresponding type of odorant. For example, first olfactory receptor neurons 702A may be activated by a first odorant and second olfactory receptor neurons 702B may be activated by second odorant. A third odorant may activate both first and second olfactory receptor neurons 702A and 702B. On sensing the corresponding odorant, first and second olfactory receptor neurons 702A and 702B may transmit signals to first, second, and / or third olfactory bulb neurons 704A, 704B and / or 704C, using and / or based on neuronal connections / circuits therebetween.

[0088] While olfactory bulb neurons in second chamber 140 may also be initially randomly or uniformly distributed, olfactory bulb neurons may self-organize into different groups (such as a first group of first olfactory bulb neurons 704A, a second group of second olfactory bulb neurons 704B, and / or a third group of third olfactory bulb neurons 704C) corresponding to different predefined and / or temporally recurring patterns of signals received from first and second olfactory receptor neurons 702A and 702B. In such examples, clusters of neurons corresponding first, second, and / or third olfactory bulb neurons 704A, 704B, and 704C learn to respond to unique / respective patterns of signals generated by olfactory receptor neurons 702A, 702B. Different ones of olfactory bulb neurons may learn to extract recurring patterns from first and / or second olfactory receptor neurons 702A and / or 702B as they are (repeatedly) exposed to different stimuli / odorants, which may subsequently group and / or organize themselves as the first group of first olfactory bulb neurons 704A, the second group of second olfactory bulb neurons 704B, and / or the third group of third olfactory bulb neurons 704C.

[0089] The self-organization of “learning” may occur over-time, as the neurons of the BoC 700 are exposed to more and more stimuli. For example, a first subset of olfactory bulb neurons may (learn to) be activated on receiving signals from first olfactory receptor neurons 702A and ignore signals from other olfactory receptor neurons, organizing themselves as first olfactory bulb neurons 704B. Here, first olfactory bulb neurons 704B may be activated by presence of the first odorant. Similarly, a secondsubset of olfactory bulb neurons may (learn to) be activated on receiving signals from second olfactory receptor neurons 702B and ignore signals from other olfactory receptor neurons, organizing themselves as third olfactory bulb neurons 704C. Here, third olfactory bulb neurons 704C may be activated by the presence of the second odorant. Further, a third subset of olfactory bulb neurons may (learn to) be activated on receiving signals from both first and second olfactory receptor neurons 702A and 702B, organizing themselves as second olfactory bulb neurons 704B. Here, third olfactory bulb neurons 704A may be activated by presence of the third odorant.

[0090] In some embodiments, each first, second, and third olfactory bulb neurons 704A, 704B, and 704C may be organized into clusters of similarly activating neurons that are proximate to each other. The clusters may form responsively to mechanisms occurring between each olfactory bulb neuron on activation where each olfactory bulb neuron activates its neighboring olfactory bulb neurons and inhibits other neurons which are further away. Since each olfactory bulb neuron in each of first, second, and third olfactory bulb neurons 704A, 704B, and 704C may be proximally located, as shown in FIG. 7, activations in second chamber 140 may be easily, and more distinctly, identified in comparison to activations in first chamber 130, for the same odorant or stimulus. Since each neuron in first, second, and third olfactory bulb neurons 704A, 704B, and 704C are proximally located, specific regions within second chamber 140 may experience amplified activations in comparison to comparatively uniform or random regional activations in first chamber 130 (where first and second olfactory receptor neurons 702A and 702B are randomly or uniformly distributed). For example, techniques used for detecting neuronal activations (such as electrodes or calcium imaging) may be able to distinctly detect activations in second chamber 140 by observing specific regions where first, second, and third olfactory bulb neurons 704A, 704B, and 704C are concentrated. In contrast, traditional BoCs may require significantly a greater number of instruments (such as electrodes) to be placed at multiple regions in first chamber 130 for signal extraction, and use machine learning algorithms (which further require significant amounts of training data to generate accurate output) to associate each combination of signal patterns with corresponding odorant / stimulus. Such solutions are expensive and impractical / infeasible when, for example, sufficient data is not available.

[0091] Since the olfactory bulb neurons self-learn and organize themselves into clusters, groups, and / or sets, BoC 700 may be used in several applications. For example, first, second, and / or third olfactory bulb neurons 704A, 704B and / or 704C may function as odorant classifiers and the neuronal circuit of BoC 700 and / or communication between the neuronal circuit of BoC 700 may be analyzed (such as through use of instruments), it may be observed that each of first, second, and / or third olfactory bulb neurons 704A, 704B and / or 704C response to specific combination or patterns of odorants, such as only first odorant, only third odorant or both first odorant and second odorant, or only second odorant, respectively. In such examples, BoC 700 and / or neuronal circuits thereon may be used as biological neural networks that are adapted for odorant classification functions, wherein the neuronal circuit of BoC 700 essentially mimics naturally occurring olfactory neuronal circuits.

[0092] While FIG. 7 describes cultivation and analysis of an example olfactory neuronal circuit, it may be appreciated by those skilled in the art that the BoC disclosed herein may be suitably adapted to allow for the growth of different neuronal circuits (either naturally-occurring or artificially generated). For example, the BoCs disclosed herein may be suitably adapted to (re)create any neuronal circuit that correlates different patterns of signals generated from any stimulus (e.g., tactile, optical, olfactory, auditory, etc.) to a specific response. The response may be, for example, the classification of a stimulus and / or the generation of a signal to a different neuronal circuit. Additionally, or alternatively, the BoCs disclosed herein may be used to study the development of neuronal circuits and / or the effects of disease and / or treatment on the behavior of neuronal circuits.

[0093] FIG. 8 provides a flowchart of a process, or method, 800 for stimulus pattern analysis using neuronal circuits cultivated in a BoC, such as BoC 100 and / or 700.

[0094] At step 805, a neuronal circuit may be cultured on a BoC (e.g., BoC 100 and / or 700) via, for example, execution of process 500 and / or process 600 (or step 605 thereof). The neuronal circuit may include a set of receptor neurons in a first chamber (e.g., first chamber 130) connected to a set of read-out neurons in a second chamber (e.g., second chamber 140) through a cellular-communication platform (e.g., cellular- communication platform 120 and / or 126). In an example, the receptor neuronscorrespond to olfactory receptor neurons and the read-out neurons correspond to olfactory bulb neurons.

[0095] At step 810, the neuronal circuit and / or a portion thereof may be exposed to one or more stimuli (e.g., an odorant). In some embodiments, the neuronal circuits may be repeatedly exposed to the stimuli. The read-out neurons in the neuronal circuits may reinforce incoming synapses that are activated in pre-defined and / or recurring temporal patterns on repeated exposure to stimuli, thereby learning to respond preferentially (such as through corresponding activations thereof) on subsequent occurrence of the pattern (such as subsequent exposures to the stimuli). For example, in cases where the neuronal circuit corresponds to an olfactory neuronal circuit, the stimuli may correspond to different odorants. Each of the odorants may activate one or more olfactory receptor neurons in first chamber (e.g., first chamber 130), based on the configuration of the olfactory receptor neurons. For example, a first odorant may activate first olfactory receptor neurons (e.g., first olfactory receptor neuron 702A), a second odorant may activate second olfactory receptor neurons (e.g., second olfactory receptor neuron 702B), and a third odorant may activate both first and second olfactory receptor neurons (e.g., both olfactory receptor neurons 702A and 702B). On activation, the olfactory receptor neurons may transmit signals to olfactory bulb neurons (e.g., olfactory bulb neurons 704A, 704B and 704C) in a second chamber (e.g., second chamber 140) through a cellular-communication platform (e.g., cellular-communication platform 120).

[0096] The olfactory bulb neurons in the second chamber may learn to activate (or generate a response) when a pre-defined pattern and / or recurring temporal pattern of signals is received from the olfactory receptor neurons in the first chamber. Each unique combination of activated olfactory receptor neurons corresponds to a unique pattern of signals, which may be learned by olfactory bulb neurons in the second chamber, as described in reference to FIG. 7.

[0097] At step 815, the activations at the neuronal clusters (e.g., first, second, and third olfactory bulb neurons 704A, 704B, and 704C) in response to the stimuli (e.g., first, second, and third odorants) may be measured. The activations may be measured using any suitable technique, such as calcium imaging or by placing electrodes at the clusters. The activations may be subsequently analyzed to determine a response of the neuronalcircuit to the stimulus. For example, in cases where the neuronal circuit corresponds to an olfactory neuronal circuit, analysis of the activations may reveal that first cluster of olfactory bulb neurons (e.g., first olfactory bulb neurons 704A) are activated only when the first odorant is present, second cluster of olfactory bulb neurons (e.g., second olfactory bulb neurons 704B) are activated only when the third odorant is present, and third cluster of olfactory bulb neurons (e.g., third olfactory bulb neurons 704C) are activated only when the second odorant is present.

[0098] The BoCs, methods, and systems disclosed herein may find use in a variety of applications, including, but not limited to, scientific research on neuronal circuits, determination of impact of chemical substances and / or environmental parameters on the neuronal circuits, disease diagnosis, determination of efficacy of treatments, and stimulus pattern analysis. Specific examples of these applications are described in reference to FIGs. 5-8. However, the present disclosure should not be construed as limited to these examples, as it is appreciated that the BoCs disclosed herein may be adapted to perform a variety of functions relating to neuronal circuits, based on the requirements of the use case.

Claims

CLAIMS1 . A brain-on-a-chip (BoC), comprising: one or more chambers configured to house a population of neurons; and one or more cellular-communication platforms placed between at least one pair of chambers from the one or more clhambers, wherein the one or more cellular-communication platforms comprise one or more features / structures that are configured to facilitate axonal growth of the population of neurons in a preferred direction to culture a neuronal circuit on the one or more cellular-communication platforms between the population of neurons in the at least one pair of chambers.

2. The BoC of claim 1, wherein the one or more chambers are enclosures formed between a base, a body, and one or more side walls of the BoC .

3. The BoC of claim 2, wherein the one or more side walls have an opening, the opening being defined to: allow the population of neurons from the at least one pair of chambers to propagate through the opening into the one or more cellular-communication platforms; and prevent the population of neurons from moving.

4. The BoC of claim 1 , wherein the population of neurons in each of the one or more chambers is derived from a single neuron.

5. The BoC of claim 1 , wherein the population of neurons in each of the one or more chambers is heterogenous.

6. The BoC of claim 1 , wherein the one or more chambers comprise one or more wells connected thereto, the one or more wells being configured to provide a passage for a solution to be added, extracted, and / or replaced from the one or more chambers.

7. The BoC of claim 6, wherein the solution is at least one of: a cellular media or a chemical substance.

8. The BoC of claim 1 , wherein the one or more structures either impede or promote at least one of: transmittance, directionality, or lateral spread of neuronal connections between the population of neurons of each pair of the one or more chambers in at least one direction.

9. The BoC of claim 1 , wherein the one or more structures are defined in an arrangement corresponding to at least one of: a physiological architecture or an anatomical architecture of tissue.

10. The BoC of claim 1 , wherein the one or more structures are at least one of: one or more posts; or one or more polygonal structures, wherein the one or more posts and / or the one or more polygonal structures are arranged to form a dwelling area and one or more microchannels, wherein the dwelling area is configured to impede growth of neurons / axons from the population of neurons in a first direction, and / or redirect the growth of the neurons / axons to a second direction, and wherein the one or more microchannels are configured to promote the growth of the neurons / axons in the second direction.

11. The BoC of claim 10, wherein the one or more posts have at least one of: a triangular shape or a teardrop shape.

12. The BoC of claim 10, wherein the one or more polygonal structures have a ‘C’- shaped contour.

13. The BoC of claim 10, wherein the one or more structures further comprise at least one of: one or more irregularly-shaped obstacles having a curved notch that defines a first curved microchannel between the curved notch and a first post having a teardrop shape; and / or a curved end positioned on an end of the irregularly-shaped obstacle opposing the curved notch that defines a second curved microchannel between the curved end and a second post having a triangular shape.

14. The BoC of claim 10, wherein the one or more microchannels are defined to be either diagonally oriented, linearly oriented, or curved with respect to the one or more cellular-communication platforms.

15. The BoC of claim 1 , wherein the one or more structures are either protrusions extending from a body towards a base of the BoC, or pillars defined between the body and the base through the one or more cellular-communication platforms.

16. The BoC of claim 1 , wherein the population of neurons in a first chamber from the one or more chambers are sensory neuronal cells, and the population of neurons in a second chamber from the one or more chambers are read-out cells, and wherein the one or more structures are configured to synaptically connect the sensory neuronal cells in the first chamber to the read-out cells in the second chamber through the one or more cellular-communication platforms.

17. The BoC of claim 16, wherein the neuronal circuit comprises multiple ones of the read-out cells connected to each of the sensory neuronal cells.

18. The BoC of claim 16, wherein the read-out cells of the neuronal circuit reinforce incoming synapses that are activated in pre-defined and / or recurring temporal patterns and learn to respond preferentially to the pre-defined and / or recurring temporal patterns.

19. The BoC of claim 16, wherein the sensory neuronal cells are olfactory receptor sensory neurons, and the read-out cells are olfactory bulb neurons.

20. A BoC platform, comprising a neuronal circuit cultured by connecting a population of neurons in each of one or more chambers through one or more corresponding cellular-communication platforms.21 .A disk, comprising one or more BoCs of any of claims 1-20 placed on a substrate.

22. A method for culturing neuronal circuits, comprising: placing a population of neurons in a BoC, wherein the BoC comprises: one or more chambers configured to house the corresponding population of neurons to be cultured, and one or more cellular-communication platforms placed between at least one pair of chambers from the one or more chambers, wherein the one or more cellular-communication platforms comprise one or more features / structures that are configured to facilitate axonal growth of the population of neurons in a preferred direction to culture a neuronal circuit between the population of neurons in the at least one pair of chambers; and either adding, extracting, and / or replacing a solution in the one or more chambers to promote growth of the population of neurons through the one or more cellular-communication platforms to form the neuronal circuit.

23. The method of claim 22, wherein the population of neurons placed in each of the one or more chambers is heterogenous.

24. The method of claim 22, wherein the solution is added, extracted, and / or replaced through one or more wells connected to the one or more chambers.

25. A method of testing an impact of chemical substances and / or environmental conditions on neuronal circuits, comprising: culturing a neuronal circuit on a BoC; exposing the neuronal circuit to any one or a combination of: one or more chemical substances or one or more environmental conditions; and determining one or more parameters indicating impact of the one or more chemical substances and / or the one or more environmental conditions on the neuronal circuits.

26. The method of claim 25, wherein for culturing the neuronal circuit, the method comprises: housing a population of neurons in each of one or more chambers of the BoC; and either adding, extracting, and / or replacing a solution in the one or more chambers to promote growth of the population of neurons through one or more cellular-communication platforms of the BoC to form the neuronal circuit.

27. The method of claim 26, wherein the population of neurons is derived from one or more neurons of a patient.

28. The method of claim 26, further comprising: monitoring the growth of the population of neurons in the BoC; and re-determining the one or more parameters.

29. The method of claim 25, wherein the one or more parameters correspond to at least one of: rates of communication, types of communication, connectivity, a growth rate, a toxicity response, a morphology, a survival rate, fluorescence, biomarkers, or omics, between and / or within the population of neurons of each of the one or more chambers.

30. The method of claim 25, wherein the one or more parameters are analyzed for determining a presence of a disease in the neuronal circuit.31 . The method of claim 25, further comprising determining efficacy of the one or more chemical substances and / or the one or more environmental conditions for treatment of the neuronal circuit based on analysis of the one or more parameters.

32. The method of claim 25, wherein the BoC is placed on a disk.

33. A method for stimulus pattern analysis using neuronal circuits, comprising: culturing a neuronal circuit on a brain-on-a-chip (BoC), wherein the neuronal circuit comprises a set of receptor neurons in a first chamber connected to a set of read-out neurons in a second chamber through a cellular-communication platform; exposing the neuronal circuit to one or more stimuli to teach the read-out neurons to activate one or more clusters comprising a unique subset of read-out neurons in response to a corresponding stimulus from the one or more stimuli; and measuring activations at the one or more clusters in response to the one or more stimulus.

34. The method of claim 33, wherein the set of receptor neurons comprise olfactory receptor neurons, and the set of read-out neurons comprise olfactory bulb neurons, and the one or more stimuli correspond to odorants.

35. The method of claim 33, wherein each of the one or more clusters are spatially distinct.

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