Biological assay apparatus

Abstract

A biological assay apparatus (2) for analysing motile biological structures is provided. The biological assay apparatus comprises an input region (12) for receiving a sample of motile biological structures and an assessment structure (6) defining a pathway (14) extending from the input region and along which the sample of motile biological structures can move. The assessment structure comprises one or more obstacles (10) which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

Description

[0001] Biological Assay Apparatus

[0002] The present invention relates to biological assay apparatuses and to methods of using and manufacturing such biological assay apparatuses.

[0003] Biological assays are used to assess physical or chemical properties of a biological sample such as a sample of cells. In particular, it is useful to assess the motility of biological structures such as cells (i.e. their ability to move and manoeuvre) when subject to different stimuli, treatments and / or diseases.

[0004] However, many conventional cell motility assays do not provide a particularly authentic or complete representation of actual biological environments (e.g. organ tissue). For instance, the “scratch assay” (which involves making a thin scratch in a layer of cells and observing how quickly the cells close the scratch) is performed in an effectively two-dimensional cell culture. Other assays can be difficult to observe and image in real-time, which is often important for fully understanding cell behaviour.

[0005] An improved approach may be desired.

[0006] According to a first aspect of the present invention there is provided a biological assay apparatus for analysing motile biological structures, the biological assay apparatus comprising: an input region for receiving a sample of motile biological structures; and an assessment structure defining a pathway extending from the input region and along which the sample of motile biological structures can move; wherein the assessment structure comprises one or more obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

[0007] Thus, it will be appreciated by those skilled in the art that embodiments of the present invention provide an assay apparatus in which motile biological structures (e.g. animal cells) have to navigate around obstacles to move along a pathway. The ability of the motile biological structures to move along the pathway and / or the way in which the motile biological structures move or attempt to move along the pathway may be monitored and used to assess properties of the motile biological structures, such as flexibility, plasticity and mobility.

[0008] Because the obstacles extend at least partially perpendicular to the pathway, the assessment structure presents the motile biological structures with a three-dimensional environment to navigate. This may be more representative of actual biological environments than conventional assay environments. The movement of the motile biological structures along the pathway may thus be physiologically and functionally closer to their actual biological behaviour. The biological assay apparatus can thus provide improved insights into the motile biological structures compared to conventional in vitro assay techniques.

[0009] In some embodiments, one or more obstacles comprises a wall, i.e. which extends outwards from the pathway. A wall may constrain the motile biological structures by physically blocking their movement. In some embodiments, additionally or alternatively, one or more obstacles comprises a pit or well, i.e. which extends inwards from the pathway.

[0010] In a set of embodiments, one or more obstacles extends perpendicularly to the pathway, e.g. comprising a vertical wall extending perpendicularly outward from a horizontal pathway and / or a pit with vertical sides extending perpendicularly inward from a horizontal pathway.

[0011] In a set of embodiments, one or more obstacles comprises a surface with a texture arranged to provide resistance to movement of the motile biological structures. For instance, one or more obstacles may comprise a rough surface texture.

[0012] The biological assay apparatus may be suitable for analysing many different types of motile biological structure. In a set of embodiments, the motile biological structures comprise one or more of the following: cells (prokaryotic or eukaryotic), cell aggregates (e.g. spheroids), organoids, tissue samples. The motile biological structures may be grown in vitro and / or comprise explants or biopsies from humans, animals or plants. In a set of embodiments, the motile biological structures are cancer cells, sperm cells, fibroblasts, immune cells and / or mesenchymal cells. The biological assay apparatus may be tailored for assessing one or more properties of motile biological structures and / or for assessing particular type(s) of motile biological structures. For instance, the biological assay apparatus may be adapted to test particular aspects of individual or collective structure motility.

[0013] The pathway may comprise one or more path elements arranged to assess one or more properties of the motile biological structures, e.g. individual or collective structure motility. Possible path elements include: a straight section, a curved section (e.g. with a radius of curvature less than 1 mm), a gentle corner, a sharp corner, a junction (i.e. an intersection between different sections of a pathway and / or two or more pathways), a dead-end or a U-turn. The pathway may comprise any combination of these features. The shape of the pathway may be designed by including appropriately shaped and positioned obstacles in the assessment structure. A gentle corner may be defined as one that a motile biological structure of interest can navigate without bending or deforming (e.g. a corner with a radius of curvature that is near to or greater than a nominal length of the motile biological structure). A sharp corner may be defined as one that a motile biological structure of interest must bend or deform to navigate (e.g. a corner with a radius of curvature that is less or much less than a nominal length of the motile biological structure).

[0014] Including one or more corners, one or more junctions and / or one or more deadends and / or one or more U-turns in the pathway may test the navigation, flexibility and / or plasticity of the motile biological structures. For instance, the assessment structure may comprise obstacles that form a sharp corner in the pathway to test the flexibility of the motile biological structures.

[0015] Properties of the pathway (e.g. of the one or more obstacles) may be designed for a particular assay. For instance, one or more pathway properties may be arranged to test one or more behaviours of the motile biological structures (e.g. to test the flexibility of a particular type of cell). In a set of embodiments, the pathway comprises a minimum width (e.g. defined as a minimum clearance between two obstacles in the assessment structure) that is based on (e.g. equal to, or a function of) a size of a target motile biological structure. The pathway may comprise one or more narrowing sections in which a width of the pathway decreases (e.g. which decreases past a normal size of the target motile biological structure to test its plasticity and flexibility).

[0016] One or more dimensions of the pathway may be adapted for different assays and / or different types of motile structures. In a set of embodiments, the pathway comprises a minimum width of at least 10 pm, at least 25 pm, at least 50 pm, at least 100 pm, at least 250 pm, at least 500 pm or at least 1 mm. In some embodiments the minimum width of the pathway is 2 mm or less, 1 mm or less, 500 pm or less, 250 pm or less, 100 pm or less, 50 pm or less, 25 pm or less, 10 pm or less, 5 pm or less, 2 pm or less or even 1 pm or less. The pathway may comprise a height (or depth) of 500 pm or less, 200 pm or less, 100 pm or less 50 pm or less, 20 pm or less or even 10pm or less. The pathway may comprise a length (i.e. along its extension away from the input region) of 100 pm or more, 500 pm or more, 1 mm or more, 2mm or more, 5 mm or more, 1 cm or more or even 5 cm or more.

[0017] In a set of embodiments, the pathway is part of a maze. The one or more obstacles may comprise walls of the maze. The maze may be unicursal, i.e. a labyrinth in which the pathway is the only possible route through the maze. Alternatively, there may be multiple pathways within the maze (although not all may be solutions to the maze).

[0018] More generally, the assessment structure may define more than one pathway that the motile biological structures can move along. In such examples, useful information may be gathered by monitoring the pathway(s) taken by motile biological structures.

[0019] In a set of embodiments, the pathway is part of a pattern of pathways (e.g. a repeating pattern). For instance, the assessment structure may comprise a pattern of obstacles (e.g. a repeating pattern) which define a corresponding pattern of pathways between the obstacles. The pathway may be random, e.g. defined by a random distribution of obstacles (e.g. of random size and / or random shape and / or random orientation and / or random position). A random pathway may have nevertheless be characterizable, e.g. by an average and / or variance and / or maximum and / or minimum of one or more pathway parameters (e.g. width, angle of direction changes). The pathway may extend substantially or entirely parallel to a single plane. In other words, the pathway may be substantially planar, with motile structures able to move along the pathway without needing to move up or down relative to the plane (although the obstacles nevertheless provide a three-dimensional environment for the motile biological structures to navigate). For instance, the pathway may have a floor defined by a flat surface of the assessment structure (e.g. including surfaces which have a rough surface texture but are substantially flat overall), with the one or more obstacles extending out of the surface to define sides of the pathway. Whilst in such embodiments the motile biological structures may be able to move towards and away from the flat surface (whilst remaining within the lateral confines of the obstacles), they are not obliged to do so to move along the pathway (i.e. they could move along the pathway without leaving a reference plane). Alternatively, the pathway may extend in three dimensions, e.g. along a sloped, curved or stepped surface. The pathway may pass over, under or around obstacles of the assessment structure. A three-dimensional pathway may more accurately reflect some actual biological environments.

[0020] In use, the pathway may be filled with any medium suitable for supporting the motile biological structures under analysis, e.g. any suitable natural or artificial culture media known in the art perse. The pathway may be accessible, e.g. to enable the application of one or more serums and / or antibiotics to the culture medium.

[0021] In a set of embodiments, additionally or alternatively, one or more mechanical or material properties of the assessment structure are adapted for assessing one or more properties of the motile biological structures.

[0022] In a set of embodiments, the assessment structure may comprise a density and / or a stiffness (e.g. of part or all of one or more obstacles) based on a motile biological structure to be assessed. Additionally or alternatively, the assessment structure may comprise one more surface textures (e.g. of part or all of the pathway and / or one or more obstacles) based on a motile biological structure to be assessed. For instance, a roughness of the pathway and / or one or more obstacles may be selected to assess one or more properties of the motile biological structures. The thickness (e.g. extension in a direction perpendicular to the pathway) and / or shape of the obstacles may be selected based on a motile biological structure to be assessed. One or more obstacles may comprise a thickness (e.g. a height or a depth) of at least 1 pm, at least 5 pm, at least 10 pm, at least 25 pm, at least 50 pm, at least 100 pm, at least 250 pm, at least 500 pm or at least 1 mm. In some embodiments one or more obstacles may comprise a thickness of less than 2 mm, less than 1 mm, less than 500 pm, less than 250 pm, less than 100 pm, less than 50 pm, less than 25 pm, less than 10 pm or less than 5 pm. In a set of embodiments, one or more obstacles has a thickness of approximately 100 pm (e.g. comprising walls with a height comparable to or larger than a size of the motile biological structures). Additionally or alternatively, one or more obstacles may be relatively small relatively to the size of the motile biological structures (e.g. with a thickness of 1-25 pm), e.g. to provide a texture resistant to movement rather than an impassable physical barrier.

[0023] As explained below in more detail, it may be advantageous for the assessment structure to be thin to aid observation of the motile biological structures. In a set of embodiments the assessment structure has a maximum thickness (e.g. measured in a direction perpendicular to the extension of the pathway) that is 5 mm or less, 2 mm or less, 1 mm or less, 500 pm or less or even 250 pm or less (e.g. down to 200 pm or thinner).

[0024] In a set of embodiments, the assessment structure is transparent to one or more wavelengths of light (e.g. visible, IR and / or UV light). This may aid observation of the motile biological structures as they move along the pathway. In a set of embodiments, the assessment structure is transparent to fluorescence from a fluorescent motile biological structure. Additionally or alternatively, the assessment structure may be transparent to light from an external light source (e.g. of an analysis system that uses the biological assay apparatus). It will be recognised that the assessment structure may not need to be perfectly transparent to enable good quality observation. The assessment structure may absorb a reasonable fraction of light whilst still being considered transparent if sufficient light reaches an observer to make meaningful observations of the motile biological structures. In a set of embodiments, the assessment structure is arranged to replicate a particular biological environment (e.g. a particular organ and / or disease-state).

[0025] One or more mechanical or material properties of the assessment structure may vary. For instance, the assessment structure may comprise regions with different densities and / or stiffnesses. There may be a step change between the different areas. Additionally or alternatively in some embodiments there is a gradual change in density and / or stiffness across the assessment structure. In other words, the assessment structure may comprise one or more regions with a density and / or stiffness gradient. As explained below in more detail, a density and / or stiffness gradient may usefully induce the motile biological structures to move by haptotaxis and / or durotaxis.

[0026] In a set of embodiments, the assessment structure comprises hydrogel. The assessment structure may consist of hydrogel. The assessment structure may comprise a polymer. For instance, the assessment structure may comprise a hydrogel comprising a synthetic polymer or a biopolymer. Preferably the hydrogel is a bioactive hydrogel, such as a protein-based hydrogel or a protein-carbohydrate blended hydrogel. The hydrogel may comprise an extracellular matrix (ECM) hydrogel. In a set of embodiments, the assessment structure comprises gelatin methacryloyl (“GelMA”). In a set of embodiments, the assessment structure comprises Polyethylene glycol diacrylate (“PegDA”). Using a bioactive hydrogel helps to improve the biological relevance / authenticity of the assay, because the motile biological structures may not simply adhere to the structure but instead functionally bond to it. The mechanical properties of hydrogels (e.g. stiffness) may be tuned relatively easily, e.g. by controlling the concentration of the hydrogel and / or a level of crosslinking in the hydrogel.

[0027] In a set of embodiments, the assessment structure is monolithic (i.e. formed from a single block of material). In other words, the obstacle(s) may be integral to the rest of the assessment structure. For instance, the assessment structure may comprise a base from which the one or more obstacles extend. The pathway may extend along the base between the one or more obstacles. The input area may be comprised by the assessment structure. In some embodiments, the input area comprises an input container such as a well or a chamber in the assessment structure. The input container may be defined partially or entirely by one or more obstacles of the assessment structure.

[0028] The biological assay apparatus may comprise a support for the assessment structure. The support may provide a substrate for the assessment structure (e.g. a lower layer on which the assessment structure is disposed). The support may partially or entirely enclose the assessment structure, i.e. the support may comprise a housing for the assessment structure. The support may comprise a glass slide, a coverslip, a well-plate, and / or a Petri dish.

[0029] The movement of the motile biological structures along the pathway may occur simply due to random motion of the biological structures and / or due to natural diffusion of the motile biological structures from an area of a high concentration, e.g. in or near to the input region. In other words, the biological assay apparatus may not be arranged to actively induce movement of the motile biological structures along the pathway (e.g. the biological assay apparatus may be free from haptic, chemical or gravitational stimuli).

[0030] However, in some embodiments it may be beneficial to induce movement along the pathway. In a set of embodiments the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway. In some embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by haptotaxis and / or durotaxis and / or topotaxis and / or chemotaxis and / or geotaxis (i.e. gravitational effects) and / or rheotaxis and / or galvanotaxis and / or thermotaxis. The applicant has recognised the useful insight may be gained by monitoring the behaviour of the motile biological structures to navigate around the obstacles in response to a stimulus such as a haptic, chemical or gravitational stimulus. In other words, the obstacles may constrain the inherent movement of the motile biological structures in response to the stimuli, and the way in which they respond to this constraint can be analysed to determine one or more properties of the motile biological structures. In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by haptotaxis and / or durotaxis. In some embodiments, the assessment structure comprises a gradient in mechanical or material properties (e.g. density or stiffness) along the pathway. For instance, one or more coatings may be applied to the assessment structure to produce the desired gradient. Additionally or alternatively, one or more properties of the assessment structure itself (e.g. the material forming the assessment structure, the concentration of the assessment structure and or a degree of cross-linking in the assessment structure) may be varied along the pathway to produce the desired gradient.

[0031] In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by topotaxis (i.e. movement induced by the topography of the pathway). The assessment structure may comprise a gradient in one or more topographical properties (e.g. a roughness of a surface texture) along the pathway.

[0032] In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway using a chemical stimulus. The chemical stimulus may cause motile biological structures to move along the pathway by chemotaxis. In some embodiments, the biological assay apparatus comprises a chemical stimulus holder for receiving a chemical stimulus (e.g. an attractant), such as a well or a chamber. The chemical stimulus holder may be part of the assessment structure (e.g. a well or chamber integrated into the assessment structure). The pathway may be located between the input region and the chemical stimulus holder. The pathway may extend all the way to the chemical stimulus holder, although this is not essential and in some embodiments the chemical stimulus holder is isolated from other parts of the biological assay apparatus, with the chemical stimulant nevertheless diffusing through the assessment structure to cause chemotaxis.

[0033] In some embodiments, in addition to or instead of a dedicated chemical stimulus holder, the biological assay apparatus is arranged to receive directly a chemical stimulus across part or all of the assessment structure. For instance, the chemical stimulus may be applied at different concentrations along some or all of the pathway, e.g. to encourage movement along the pathway.

[0034] In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by geotaxis (i.e. using gravity). For instance, the biological assay apparatus may have a standard orientation in which the pathway is generally inclined, e.g. with a start point and an end point at different elevations. For instance the pathway may extend with a general downward gradient (i.e. such that a starting point of the pathway is higher than other parts of the pathway). In some such embodiments the pathway may have one or more local inclines, but the general downward gradient results in a gravitational force urging the motile biological structures along the pathway. In some embodiments some or all of the pathway may be near or entirely vertical when the biological assay apparatus is in the standard orientation.

[0035] In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by rheotaxis. In some such embodiments the biological assay apparatus may by arranged to induce fluid flow along the pathway, and / or to produce a gradient in fluid viscosity along the pathway.

[0036] In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by galvanotaxis. In some such embodiments the biological assay apparatus may by arranged to generate an electric field over the pathway (e.g. with one or more suitably positioned electrodes). The electric field may be at least partially aligned with the pathway. The electric field strength and / or direction may be uniform along the pathway, or the electric field strength and / or direction may vary along the pathway. The electric field may be fixed, or the electric field may vary over time.

[0037] In a set of embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway by thermotaxis. In some such embodiments the biological assay apparatus may by arranged to generate variations in temperature over the pathway (e.g. a temperature gradient along the pathway). Such embodiments may utilise one or more suitably positioned heating or cooling elements.

[0038] The standard orientation may correspond to a stable orientation, i.e. one in which the biological assay apparatus is in a stable mechanical equilibrium. For instance, the standard orientation may correspond to an orientation where a support or housing of the biological assay apparatus (e.g. a Petri dish) is placed stably onto a flat surface, or when the biological assay apparatus is held in a standard holder (e.g. a glass slide held vertically in a slide holder). The standard orientation may correspond to a standard way to orient a support or housing of the biological assay apparatus (e.g. to orient a Petri dish with its base horizontal). The biological assay apparatus may comprise one or more marking or structures that indicate a standard orientation (e.g. an arrow that should face upward or downward in the standard orientation).

[0039] The biological assay apparatus may be configurable in multiple different orientations, e.g. to induce or exclude gravitational effects on the motile biological structures. For instance, the biological assay apparatus may be configurable into a stable non-inclined orientation in which the pathway is generally non-inclined (i.e. with no overall slope to the pathway), and a stable inclined orientation in which the pathway is generally inclined.

[0040] In some embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structures along the pathway using a combination of different mechanisms. For instance the biological assay apparatus may feature a chemical stimulus holder and be arranged such that the pathway extends at a downward gradient when in use.

[0041] As mentioned above, the biological assay apparatus may facilitate improved analysis of motile biological structures. In some embodiments the biological assay apparatus may be suitable for real-time analysis.

[0042] According to a second aspect of the present invention there is provided a method of analysing motile biological structures, the method comprising: introducing a sample of motile biological structures to an input region of a biological assay apparatus, the biological assay apparatus also comprising an assessment structure defining a pathway extending from the input region; and observing movement of the motile biological structures along the pathway; wherein the assessment structure comprises one or more obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

[0043] In some embodiments, the method comprises performing a motility assay. For instance, the method may comprise determining one or more quantitative or qualitative measures of how well one or more motile biological structures can move, manoeuvre and / or navigate.

[0044] In some embodiments, the method comprises performing a plasticity assay. For instance, the method may comprise determining one or more quantitative or qualitative measures of the plasticity of one or more motile biological structures.

[0045] Embodiments of the present invention may be used for short-term analyses (e.g. comprising observing movement of the motile biological structures over one day or less, twelve hours or less, six hours or less or one hour or less) and long-term analyses e.g. comprising observing movement of the motile biological structures over one or more days, one or more weeks or even over a month or more).

[0046] The movement of the motile biological structures along the pathway may be observed by a human observer, e.g. with the naked eye. For instance, a human user may check and record the progress of the motile structures at regular or irregular intervals.

[0047] However, in some embodiments, imaging apparatus may be used to observe the movement of the motile biological structures along the pathway, such as any suitable microscopy equipment known in the art perse. The method may comprise imaging the assessment structure. The method may comprise repeatedly imaging the assessment structure (e.g. at regular intervals) to observe movement of the motile biological structures. Embodiments of the present invention may be compatible with label-free and labelled imaging techniques. Imaging the assessment structure may be based on detecting scattered light, reflected light and or light from fluorescence in the sample. Additionally or alternatively, imaging the assessment structure may be based on detecting attenuation of light passing through the sample and / or detecting one or more spectra of light passing through or emitted from the sample (e.g. using infrared or Raman spectroscopy).

[0048] In some embodiments the method comprises imaging the assessment structure using microscopy. In a set of embodiments, optical microscopy is used to observe the motile biological structure (e.g. transmission microscopy). Additionally or alternatively, phase microscopy may be used to observe the motile biological structure (e.g. phase contrast microscopy, differential interference contrast microscopy or Quantitative phase-contrast microscopy). Additionally or alternatively, fluorescence microscopy may be used to observe the motile biological structures (e.g. autofluorescence microscopy, epifluorescent microscopy, confocal microscopy, light-sheet microscopy or two-photon microscopy).

[0049] Observing the motile biological structures may comprise tracking the position of one or more motile biological structures.

[0050] Analysing the motile biological structures may comprise classifying or sorting some or all of the motile biological structures into one or more groups. The motile biological structures may be sorted by assigning classifications to the structures based on the observations.

[0051] However, in some embodiments, the structures may physically be sorted as a result of moving through the assessment structure (e.g. where different motile structures travel different distances along a pathway or along different pathways). For instance, some embodiments of the method may comprise sorting the motile biological structures by motility by causing more motile structures to follow a different pathway to less motile structures. The method may comprise collecting sorted structures, i.e. retrieving sorted structures from the assessment structure.

[0052] According to a third aspect of the present invention there is provided a system for analysing motile biological structures, the system comprising: a biological assay apparatus as disclosed herein; and imaging apparatus arranged to image the assessment structure of the biological assay apparatus.

[0053] The imaging apparatus may comprise a microscope, such as an optical microscope (e.g. a transmission microscopy), a phase microscope (e.g. a phase contrast microscope, a differential interference contrast microscope or a Quantitative phasecontrast microscope), or a fluorescence microscope (e.g. an epifluorescence microscope, a confocal microscope, a light-sheet microscope or a two-photon microscope).

[0054] There are various suitable methods for manufacturing the assessment structure. For instance, the assessment structure may be formed using photolithography, i.e. by using suitable masks to form the obstacles. In some embodiments, the assessment structure may be built up in layers.

[0055] The assessment structure may be formed using 3D printing, e.g. with polymer ink. The assessment structure may be formed using light writing, e.g. using a laser to perform accurate and selective crosslinking to form the obstacles.

[0056] In some embodiments, the assessment structure is formed by casting a liquid polymer solution (e.g. a liquid pre-polymer) into the necessary shape. The liquid polymer solution may contain a dissolved pathway-forming substance (e.g. a salt). The liquid polymer solution may then be cured (e.g. by UV light) to form a solid polymer structure in which one or more pieces of (previously-dissolved) pathwayforming substance are trapped. The piece(s) (e.g. salt particles) may then be removed (e.g. by washing the solid polymer structure using water, culture media or a buffer) to leave behind the pathway.

[0057] This approach to forming the assessment structure is believed to be independently inventive and so, according to a fourth aspect of the present invention, there is provided a method of manufacturing an assessment structure for a biological assay apparatus, the method comprising: providing a liquid polymer solution in which a pathway-forming substance is dissolved; casting the liquid polymer solution onto a substrate; curing the liquid polymer solution and causing the pathway-forming substance to come out of solution to form a solid polymer structure in which one or more pieces of pathway-forming substance are trapped; and removing the one or more pieces of pathway-forming substance from the solid polymer structure to form an assessment structure defining a pathway in a space left by the one or more pieces of pathway-forming substance, the assessment structure comprising one or more obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

[0058] Thus, embodiments of the fourth aspect may provide a relatively simple casting and curing technique to be used to produce an assessment structure suitable for analysing motile biological structures, e.g. as described above. The use of the dissolved pathway-forming substance may simplify the production of pathways with very small dimensions (e.g. with a width of 1 pm or less).

[0059] The pathway-forming substance may comprise any substance suitable for dissolving in the liquid polymer solution and forming trapped pieces which can subsequently be removed without adversely affecting the resulting assessment structure. The pathway-forming substance is preferably not harmful to living cells. The pathway-forming substance preferably does not interfere with the curing process. In a set of embodiments, the pathway-forming substance comprises a salt, e.g. NaCI. In such embodiments, the piece(s) of pathway-forming substance trapped in the solid polymer structure may comprise solid salt particles (e.g. salt crystals).

[0060] Providing the liquid polymer solution in which a pathway-forming substance is dissolved may comprise adding the pathway-forming substance to the liquid polymer solution. The pathway-forming substance may be added as a solid (e.g. salt crystals) or as a liquid (e.g. saline solution).

[0061] The piece(s) of the pathway-forming substance formed in the solid polymer structure may be solid (i.e. the pathway-forming substance may be solid when out of solution). The piece(s) of the pathway-forming substance may be crystalline or amorphous. Because the one or more pieces of the pathway-forming substance form by coming out of solution, their size, position and distribution in the solid polymer structure may not be entirely predictable. The pathway may follow a random route. In some embodiments, the assessment structure comprises a pattern of random pathways in the space left by the one or more pieces of the pathway-forming substance.

[0062] However, it may still be possible to exert meaningful control over characteristics of the pathway(s) with appropriate tuning of input materials and / or manufacturing steps. For instance, one or more dimensions of the pathway (e.g. a width) may be determined at least partially by a concentration of the pathway-forming substance dissolved in the liquid polymer solution. For instance, a higher concentration may lead to larger and / or more pieces of the pathway-forming substance trapped in the solid polymer structure and, consequently, a larger pathway. The method may comprise controlling the concentration of pathway-forming substance in the liquid polymer solution. The method may comprise adding the pathway-forming substance to the liquid polymer solution to obtain a substance concentration for producing a target pathway dimension (e.g. adding a particular quantity of the pathway-forming substance to the liquid polymer solution).

[0063] Curing the liquid polymer solution may comprise inducing cross-linking of polymer chains in the liquid polymer solution. Curing the liquid polymer solution may comprise adding a curing agent to liquid polymer solution the and / or exposing the liquid polymer solution to one or more wavelengths of light (e.g. ultraviolet light). In some embodiments, one or more dimensions of the pathway (e.g. a width) may be determined at least partially by a level of curing of the liquid polymer solution. For instance, a greater degree of cross-linking in the solid polymer structure (e.g. a larger number of cross-links) may lead to smaller and / or fewer pieces of the pathway-forming substance and, consequently, a smaller pathway. The method may comprise curing the liquid polymer solution to a level for producing a target pathway dimension (e.g. exposing the liquid polymer solution to UV light for a particular duration).

[0064] Curing the liquid polymer solution may inherently cause the pathway-forming substance to come out of solution to form the trapped piece(s). However, some embodiments comprise one or more additional steps before, after or during curing to cause the pathway-forming substance to come out of solution (e.g. heating or cooling).

[0065] The liquid polymer solution may comprise a liquid pre-polymer such as a hydrogel (e.g. GelMA, PegDA) dissolved in buffered saline (e.g. phosphate-buffered saline (PBS)). The liquid polymer solution may comprise a UV photo-initiator (e.g. LAP or Igracure) to aid curing.

[0066] Removing the one or more pieces of pathway-forming substance may comprise washing the solid polymer structure. Washing the solid polymer structure may comprise re-dissolving the piece(s) or physically removing the piece(s) without dissolving them (or after partial dissolving). The solid polymer structure may be washed with any agent known in the art perse that is suitable for removing the piece(s) of the pathway-forming substance and leaving the pathway behind. In a set of embodiments the method comprises washing the solid polymer structure with one or more of: water, culture medium or a buffer solution.

[0067] Features of any aspect or embodiment described herein may, wherever appropriate, be applied to any other aspect or embodiment described herein. Where reference is made to different embodiments, it should be understood that these are not necessarily distinct but may overlap. It will be appreciated that all of the preferred features of the biological assay apparatus according to the first aspect described above may also apply to the other aspects of the invention.

[0068] One or more non-limiting examples will now be described, by way of example only, and with reference to the accompanying figures in which:

[0069] Figure 1 is a schematic diagram of a biological assay apparatus according to an embodiment of the present invention;

[0070] Figure 2 is a schematic cutaway diagram of the biological assay apparatus;

[0071] Figure 3 is a schematic diagram of a system for analysing motile biological structures, according to an embodiment of the present invention;

[0072] Figure 4 is an example image captured by the system of Figure 3; Figure 5 is schematic diagram of part of a biological assay apparatus according to another embodiment of the present invention; and

[0073] Figures 6-8 are schematic diagrams showing various steps in a method of manufacturing an assessment structure according to an embodiment of the present invention.

[0074] A biological assay apparatus 2 for analysing motile biological structures such as animal cells (e.g. cancer cells, sperm cells, immune cells and / or mesenchymal cells) is shown in Figure 1. Figure 2 provides a cutaway view of the biological assay apparatus 2.

[0075] The biological assay apparatus 2 comprises a Petri dish 4 in which an assessment structure 6 is disposed. The assessment structure 6 is formed from a bioactive hydrogel and comprises a flat base 8 from which a plurality of walls 10 extend to form a maze. The biological assay apparatus 2 has an input region 12 at an outer part of the assessment structure 6 for receiving a sample of motile biological structures.

[0076] The walls 8 act as obstacles to movement of the motile biological structures and thus define several pathways 14 through the maze. Part of one pathway 14 is highlighted with a dashed line in Figure 2.

[0077] The biological assay apparatus 2 comprises a chemical stimulus holder 16 at the centre of the maze. The chemical stimulus holder 16 is a small well for holding a chemical attractant. However, this is not essential and some embodiments of the invention do not use chemical stimuli.

[0078] The assessment structure 6 is thin (e.g. 200 pm) and transparent to visible light.

[0079] An analysis system 100 for analysing motile biological structures is shown in Figure 3. The system 100 comprises a microscope 102, a sample stage 104, a controller 106, a light source 107 and the biological assay apparatus 2. The biological assay apparatus 2 is positioned on the sample stage 104 so that the microscope 102 can be operated to image the biological assay apparatus 2 and its contents. In use, a sample of motile biological structures is added to the input region 12 of the biological assay apparatus 2. A chemical attractant is added to the chemical stimulus holder 16. The motile biological structures begin to move by chemotaxis towards the chemical attractant. To do so the motile biological structures are forced to navigate around the walls 10 and along the pathways 14 of the maze.

[0080] The microscope 102 continuously captures images of the biological assay apparatus 2 as the motile biological structures move along the pathways 14. Figure 4 shows one such image, illustrating a group of motile biological structures 108 (e.g. cells) navigating a U-turn around a wall 10 in the assessment structure 6. The cells 108 are moving by chemotaxis towards the chemical stimulus holder (the arrow below the image shows the direction of increasing chemical attractant)

[0081] The images captured by the microscope 102 may be processed and reviewed to analyse the ability of the cells 108 to navigate the maze. This is used to assess various properties of the cells 108. For instance, the images may be used to assay motility and plasticity of the cells 108.

[0082] Because the maze is formed from three-dimensional obstacles 10, it is more representative of an actual biological environment than many conventional assay environments. The movement of the cells along the pathways 14 may thus enable more accurate insights into real-world cell behaviour.

[0083] Figure 5 shows part of another biological assay apparatus 202. The biological assay apparatus 202 comprises an assessment structure 206 formed from a bioactive hydrogel. Figure 5 is a partial view of the assessment structure 206.

[0084] The assessment structure 206 comprises a flat base from which a plurality of obstacles 208 extend (out of the page in the view of Figure 5) to form a maze of pathways 210 between the obstacles 208. Motile biological structures are input to an input region of the biological assay apparatus 202 (not shown), and move along various of the pathways 210 (e.g. by haptotaxis and / or chemotaxis). The movement of the motile biological structures through the maze may be observed (e.g. with a system such as that shown in Figure 3) to perform motility and / or plasticity assays of the motile biological structures.

[0085] One or more properties of the assessment structure 206 may be tailored to test different motile biological structures (e.g. different cells) and / or to test different properties of the motile biological structures. For instance, the pattern of obstacles 208 may be varied to produce pathways 210 of varying width.

[0086] One approach to manufacturing an assessment structure, e.g. for use in the biological assay apparatuses described above, will now be described with reference to Figures 6-8.

[0087] In a first step, illustrated in Figure 6, a liquid polymer solution 304 is prepared and casted onto a substrate 302 (e.g. a glass slide). The liquid polymer solution 304 comprises a liquid pre-polymer (e.g. comprising GelMA), in which a salt (e.g. NaCI) is dissolved.

[0088] Casting the liquid polymer solution 304 comprises applying the solution 304 to the substrate 302 to form a layer of liquid polymer solution 304 with a relatively uniform thickness.

[0089] In a second step, illustrated in Figure 7, the cast liquid polymer solution 304 is exposed to UV light 305. This causes polymer chains in the solution 304 to crosslink (i.e. it cures the liquid polymer solution), and the salt to come out of the solution. This forms a solid polymer structure 306 in which a plurality of solid salt particles 308 are trapped. The salt particles 308 are randomly distributed within the solid polymer structure 306.

[0090] In a third step, the solid polymer structure 306 is washed (e.g. with water), dissolving and removing the salt particles 308 without affecting the solid polymer structure 306. This produces an assessment structure 310 defining a pattern of pathways in the space 312 left behind by the removed salt particles 308. The upper surface of the assessment structure 310 undulates due to the newly-created empty space 312. The solid polymer of the assessment structure 310 adjacent the space forms obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway. It is to be noted that Figures 7 and 8 are schematic diagrams which in which the salt particles 308 are enlarged to aid clarity. In practice the salt particles 308 may be much smaller than a thickness of the assessment structure 310.

[0091] Various properties of the pathway (e.g. dimensions) are a result of the distribution of the salt particles 308 within the solid polymer structure 306. This may be tuned by controlling the concentration of salt in the liquid polymer solution 304, as well as by controlling a level of cross-linking in the solid polymer structure 306 (e.g. by controlling cure time and / or UV intensity). This process can produce patterns of pathways with widths of 1 pm or less to several hundred pm or more.

[0092] While the invention has been described in detail in connection with only a limited number of embodiments, it should be readily understood that the invention is not limited to such disclosed embodiments. Rather, the invention can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the scope of the invention. Additionally, while various embodiments of the invention have been described, it is to be understood that aspects of the invention may include only some of the described embodiments. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.

Claims

Claims1. A biological assay apparatus for analysing motile biological structures, the biological assay apparatus comprising: an input region for receiving a sample of motile biological structures; and an assessment structure defining a pathway extending from the input region and along which the sample of motile biological structures can move; wherein the assessment structure comprises one or more obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

2. The biological assay apparatus of claim 1 , wherein the pathway comprises one or more path elements arranged to assess one or more properties of the motile biological structures.

3. The biological assay apparatus of claim 1 or 2, wherein the pathway comprises one or more of the following path elements: a straight section, a curved section, a gentle corner, a sharp corner, a junction, a dead-end and / or a U-turn.

4. The biological assay apparatus of any preceding claim, wherein one or more pathway properties are arranged to test one or more behaviours of the motile biological structures.

5. The biological assay apparatus of any preceding claim, wherein the pathway comprises a minimum width that is based on a size of a target motile biological structure.

6. The biological assay apparatus of any preceding claim, wherein the pathway comprises one or more narrowing sections in which a width of the pathway decreases.

7. The biological assay apparatus of any preceding claim, wherein the pathway comprises a minimum width of 100 pm or less.

8. The biological assay apparatus of any preceding claim, wherein the pathway comprises a length of 1 mm or more.

9. The biological assay apparatus of any preceding claim, wherein the assessment structure defines more than one pathway that the motile biological structures can move along.

10. The biological assay apparatus of claim 9, wherein the assessment structure comprises a pattern of obstacles which define a corresponding pattern of pathways between the obstacles.11 . The biological assay apparatus of any preceding claim, wherein the assessment structure has a maximum thickness that is 5 mm or less.

12. The biological assay apparatus of any preceding claim, wherein the assessment structure is transparent to one or more wavelengths of light.

13. The biological assay apparatus of any preceding claim, wherein the assessment structure is arranged to replicate a particular biological environment.

14. The biological assay apparatus of any preceding claim, wherein the assessment structure comprises regions with different densities and / or stiffnesses.

15. The biological assay apparatus of any preceding claim, wherein the assessment structure comprises a bioactive hydrogel.

16. The biological assay apparatus of any preceding claim, arranged to induce movement of the motile biological structures along the pathway by haptotaxis and / or durotaxis and / or topotaxis and / or rheotaxis and / or galvanotaxis and / or thermotaxis.

17. The biological assay apparatus of any preceding claim, arranged to induce movement of the motile biological structures along the pathway using a chemical stimulus.

18. The biological assay apparatus of any preceding claim, arranged to induce movement of the motile biological structures along the pathway by geotaxis.

19. A system for analysing motile biological structures, the system comprising: the biological assay apparatus of any preceding claim; and imaging apparatus arranged to image the assessment structure of the biological assay apparatus.

20. A method of analysing motile biological structures, the method comprising: introducing a sample of motile biological structures to an input region of a biological assay apparatus, the biological assay apparatus also comprising an assessment structure defining a pathway extending from the input region; and observing movement of the motile biological structures along the pathway; wherein the assessment structure comprises one or more obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

21. The method of claim 20, comprising performing a motility assay and / or a plasticity assay22. The method of claim 20 or 21 , comprising repeatedly imaging the assessment structure to observe movement of the motile biological structures.

23. The method of any of claims 20-22, comprising sorting some or all of the motile biological structures into one or more groups.

24. The method of claim 23, arranged to physically sort the motile biological structures as a result of moving through the assessment structure.

25. A method of manufacturing an assessment structure for a biological assay apparatus, the method comprising: providing a liquid polymer solution in which a pathway-forming substance is dissolved; casting the liquid polymer solution onto a substrate;curing the liquid polymer solution and causing the pathway-forming substance to come out of solution to form a solid polymer structure in which one or more pieces of pathway-forming substance are trapped; and removing the one or more pieces of pathway-forming substance from the solid polymer structure to form an assessment structure defining a pathway in a space left by the one or more pieces of pathway-forming substance, the assessment structure comprising one or more obstacles which extend at least partially perpendicular to the pathway and which at least partially define the pathway.

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