Assessing motile structures
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- UNIVET I TROMS NORARKTISKE UNIV
- Filing Date
- 2025-12-18
- Publication Date
- 2026-06-25
Smart Images
Figure EP2025088059_25062026_PF_FP_ABST
Abstract
Description
[0001] 409.201.174315
[0002] Assessing motile structures
[0003] The present invention relates to biological assays and more particularly to methods for assessing motile biological structures such as cells.
[0004] 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.
[0005] 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. Conventional approaches also typically assess entire populations of cells together, and so do not enable quantitative assessment of individual biological structures.
[0006] An improved approach may be desired.
[0007] According to a first aspect of the present invention there is provided a method of assessing a motile biological structure using a biological assay apparatus, the biological assay apparatus comprising: an input region for receiving a sample comprising a motile biological structure; and an assessment structure defining a pathway extending from the input region and along which the motile biological structure can move, the pathway comprising a first section having a first transit difficulty and a second section having a second, different, transit difficulty; wherein the method comprises: introducing a sample comprising a motile biological structure to the input region; allowing the motile biological structure to move along the pathway; determining a location of the motile biological structure; incrementing a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and incrementing a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.
[0008] Thus, it will be recognised by those skilled in the art that, because the first and second sections have different transit difficulties, observing the progress of the motile biological structure through these sections can enable quantitative motility scoring of the structure (i.e. a quantitative assessment of how well one or more biological structures can move, manoeuvre and navigate). The motility score of biological structures that have passed through a more difficult one of the sections will be better than those which have not, e.g., facilitating individual comparisons between structures.
[0009] Moreover, because the method considers directed movement along a specific assessment pathway (e.g. rather than unconstrained movement), the resulting motility score may provide a more authentic evaluation of motility in actual biological environments (e.g. in an animal or human) because the movement of the motile biological structures along the pathway may be physiologically and functionally closer to their actual biological behaviour. The biological assay apparatus can thus provide improved insights into the motility of biological structures compared to conventional in vitro assay techniques.
[0010] The biological assay apparatus may comprise an end region to which the pathway extends. The end region may be accessible, e.g. for introducing a stimulus and / or extracting structures that pass along the entire pathway. As explained below, the pathway may extend to several end regions.
[0011] Whilst in some cases it may be desired to assess a single biological structure at a time, in a set of embodiments the sample comprises multiple motile biological structures and the method comprises: allowing multiple motile biological structures to move along the pathway: determining a location of multiple motile biological structures; and, for each motile biological structure whose location is determined: incrementing a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and incrementing a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.
[0012] Locating and scoring multiple motile biological structures may enable better analysis of the motility of the sample as a whole and / or can enable sorting of the sample by motility (e.g. by retaining only those motile biological structures whose motility is sufficient to reach an end region).
[0013] The transit difficulty of a section of the pathway may depend on various factors.
[0014] In a set of embodiments, the transit difficulty of a section is based at least in part on one or more dimensions of the section. For instance, a transit difficulty may be based on a length of the section (i.e. the distance a structure has to travel to pass through the section). A longer section may have a higher transit difficulty than a shorter section. This reflects the fact that more motile biological structures typically travel further than less motile biological structures. In a set of embodiments, the second section has a different length to the first section.
[0015] In some embodiments, the transit difficulty of a section is based on a minimum, maximum or average width, height or diameter of the section (i.e. in a direction perpendicular to a transition direction along the pathway). In other words, a transit difficulty may be based on how narrow a section is. Narrower sections (i.e. those with a smaller minimum, maximum or average width, height or diameter) may have a higher transit difficulty than wider sections. This reflects the fact that less motile biological structures may be less able to transit through narrower passages. In a set of embodiments, the second section has a different minimum, maximum or average width, height or diameter to the first section.
[0016] In a set of embodiments, the transit difficulty of a section is based on a shape of the section. Possible section shape types include straight (i.e. in which a structure moves in only one direction to pass through the section), corner (in which a structure has to change direction in the section to pass through the section), U-turn (in which a structure has to change from one direction to an opposite direction to pass through the section), curved (in which a structure has to gradually change direction in the section to pass through the section), tapered (in which the width, height or diameter of the section changes gradually), stepped (in which the width, height or diameter of the section changes abruptly). Straight sections may have a lower transit difficulty than more complex shapes. These shapes may be combined (e.g. tapered curved sections). In a set of embodiments, the second section has a different shape to the first section.
[0017] The transit difficulty of a section including a corner may depend on more specific properties of the corner. For instance, a higher angle corner (i.e. which involves a greater change in direction) may have a higher transit corner than a lower angle corner. Additionally or alternatively, the transit difficulty may depend on the sharpness of the corner. For instance, a gentle corner may have a lower transit difficulty to a sharp corner. A gentle corner may be defined as one that a motile biological structure of interest can navigate without bending or deforming, or a corner with a radius 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, or a corner with a radius that is less or much less than a nominal length of the motile biological structure.
[0018] In a set of embodiments, the transit difficulty of a section is based on the presence of one or more obstacles in the section. For instance, a section may comprise a wall, pit or well that physically obstructs structures passing through the section. Sections with obstacles may have a higher transit difficulty than those without. In a set of embodiments, at least one of the first and second sections comprises one or more obstacles, and the first and second sections comprise a different number of obstacles.
[0019] In a set of embodiments, the transit difficulty of a section is based on a material property of the assessment structure in the section such as density or stiffness. A section may have a constant density and / or stiffness, or may comprise a density and / or stiffness gradient. In a set of embodiments, the first and second sections comprise different material properties.
[0020] In a set of embodiments, the transit difficulty of a section is based on a surface texture of the assessment structure in the section. For instance, a rougher section may have a higher transit difficulty to a smoother section. In a set of embodiments, the first and second sections comprise different surface textures.
[0021] In a set of embodiments, the motility score of a motile biological structure may be determined at least in part by how the motile biological structure interacts with the assessment structure. These interactions may vary depending on material property(ies) of the assessment structure and / or surface texture(s) of the assessment structure.
[0022] In a set of embodiments, one or more sections of the pathway (and possibly the entire pathway) extends entirely in a single plane - i.e. a structure can reach all parts of the section with movement in only two orthogonal directions. In other words, a section may be substantially planar, with motile structures able to move along the section without needing to move up or down relative to the plane. For instance, the section 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). Whilst in such embodiments the motile biological structures may be able to move towards and away from the flat surface, they are not obliged to do so to move along the section (i.e. they could move along the section without leaving a reference plane).
[0023] However, in some embodiments one or more sections extend in a plurality of planes, i.e. so that movement in three dimensions is required for a structure to reach all parts of the section. For instance a section may comprise a gradual or abrupt slope, curve or step upwards or downwards from an initial plane. In some sections movement in three dimensions may be required to pass through the section. 3D sections may further increase the authenticity of the assessment structure. As explained above, the transit difficulty of a section is intended to reflect how difficult it is for the biological structure to pass therethrough. In a set of embodiments, the transit difficulty of a section is determined based on one or more properties of the motile biological structure being tested. For instance, the difficulty of a section may be determined based in part on a comparison between ta minimum, maximum or average width, height or diameter of the section and one or more dimensions of the motile biological structure (e.g. a maximum, minimum or average diameter of the structure). For instance, a section with a width that is lower than an average diameter of the structure may have a transit difficulty which is higher than a section with a width that is greater than the average diameter of the structure. This may reflect that more motile structures may be more able to deform and / or manoeuvre through tight spaces.
[0024] In a set of embodiments, the motility score of the motile biological structure may depend at least in part on interactions between the motile biological structure and another motile biological structure, for example, cell-cell interactions. Such interactions may relate to intercellular dynamics, signalling and mechanical contacts.
[0025] In a set of embodiments, the motility score of the motile biological structure may depend at least in part on interactions within the motile biological structures (e.g. intracellular behaviours). Such interactions may relate to morphological changes to the motile biological structure or phenotypical changes to the motile biological structures, as examples. For example, if a motile biological structure changes geometry with pH, the motility score of the motile biological structure may change.
[0026] When a plurality of motile biological structures of a particular type is being tested, the section difficulty may be based on an expected property (e.g. dimension) for that type of structure and / or an average property of the plurality.
[0027] In a set of embodiments, the first and second sections are arranged serially, i.e. the pathway comprises a branch which contains the first and second sections. In such embodiments, to pass through the second section of the pathway a given biological structure must have passed through the first section of the pathway. The first and second sections may be adjacent, i.e. the second section may begin immediately after the first section, although this is not essential: in some embodiments there may be one or more intervening sections located between the first and second sections that do not contribute to a structure’s motility score (e.g. they may have a comparatively negligible transit difficulty). Similarly, the first section may be located at the start of the pathway (i.e. the first section may extend from the input region), although in other embodiments there may be one or more intervening sections located between the input region and the first section that do not contribute to a structure’s motility score.
[0028] It will be recognised that in embodiments where the first and second sections are arranged serially, a structure’s motility score may reflect how far along the pathway the structure has reached when it is located. A structure with poor motility may have been unable to pass through either section or only the first section by the time it is observed, whereas a structure with better motility may have passed through both sections and thus be located further along the pathway.
[0029] In a set of embodiments, the first and second sections are arranged in parallel, i.e. the pathway comprises a first branch containing the first section and a second separate branch containing the second section. In such embodiments, it will be appreciated that a motile biological structure does not have to pass through the first section in order to reach the second section. Instead, the motile biological structure can only reach the first section if it enters the first branch and the motile biological structure can only reach the second section if it enters the second branch. Different branches of the pathway may lead to different end regions.
[0030] In a set of embodiments, the pathway includes one or more further sections (e.g. a third section, a fourth section etc), and the method comprises incrementing a motility score of a motile biological structure by a value corresponding to a transit difficulty of a further section if the motile biological structure has passed through said further section of the pathway. Further sections may have the same transit difficulty as the first or second section, or they may have different transit difficulties (i.e. by appropriate combination of the parameters discussed above). Further sections may be arranged serially with the first and / or second section (i.e. in the same branch as the first and / or second section), or in parallel with the first and or second section (i.e. in a separate branch to at least one of the first and second sections and possibly both).
[0031] In a set of embodiments, the pathway comprises at least five sections, at least ten sections or at least twenty sections. Providing additional sections may enable more precise quantitative analysis of motile biological structures.
[0032] In some embodiments, a final motility score for the / each structure may be determined by determining the location of the motile biological structure only once, e.g. at the end of an experiment duration. In such embodiments a motility score for a given biological structure is determined by simply totalling the values corresponding to the transit difficulties of sections through which the structure has passed. As mentioned above, multiple biological structures may be in the biological assay apparatus at the same time, and motility scores for each may thus be determined by locating the biological structures only once (e.g. at the end of the experiment duration). In a set of embodiments, population-level statistics may also be taken into account when determining a final motility score for multiple motile biological structures, e.g. a number or proportion of motile biological structures which move along the pathway to a milestone or end region.
[0033] In other words, the motility of the motile biological structure(s) may be assessed by determining how which sections the motile biological structure(s) has passed through after an overall experiment duration.
[0034] However, the applicant has recognised that it may be beneficial in some embodiments to locate the motile biological structure(s) at least two different times, i.e. to monitor the progress of structures through the assessment structure. Some such embodiments may comprise updating a motility score for the motile biological structure for the at least two different times.
[0035] Determining a motility score for a given motile biological structure at more than one time may enable more sophisticated motility analysis, e.g. more than a single final motility score. For instance, such embodiments may comprise determining how quickly a motile biological structure passes though sections of different transit difficulty. In other words, the motility of the motile biological structure(s) may be assessed by determining which sections the motile biological structure(s) has passed through and how quickly those sections were passed through.
[0036] In a set of embodiments, the location and motility score of the motile biological structure(s) are updated substantially continuously, e.g. in real time as the structure moves along the pathway.
[0037] The location of the motile biological structure(s) may be determined by a human observer, e.g. with the naked eye or with microscope assistance.
[0038] However, in some embodiments, imaging apparatus may be used to image the biological assay apparatus to locate motile biological structure(s). Accordingly, some embodiments comprise producing an image of the biological assay apparatus (i.e. imaging the biological assay apparatus) and determining the location of the motile biological structure(s) from said image. In embodiments which comprise locating the motile biological structure(s) at least two different times, this may comprise imaging the biological assay apparatus at at least said two different times. Such embodiments may comprise imaging the biological assay apparatus at one or more additional times (e.g. before, between or after the two different times). In some embodiments the biological assay apparatus is imaged substantially continuously, e.g. to aid tracking of individual biological structures, even if their motility score is only updated less frequently.
[0039] Embodiments may utilise any suitable imaging apparatus known in the art per se. Embodiments of the present invention may be compatible with label-free and labelled imaging techniques. Imaging the biological assay apparatus may be based on detecting scattered light, reflected light and or light from fluorescence in the sample. Additionally or alternatively, imaging the biological assay apparatus 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).
[0040] In some embodiments the method comprises imaging the biological assay apparatus using microscopy. In a set of embodiments, optical microscopy is used to image the motile biological structure (e.g. transmission microscopy). Additionally or alternatively, phase microscopy may be used to image the biological assay apparatus (e.g. phase contrast microscopy, differential interference contrast microscopy or Quantitative phase-contrast microscopy). Additionally or alternatively, fluorescence microscopy may be used to image the biological assay apparatus (e.g. autofluorescence microscopy, epifluorescent microscopy, confocal microscopy, light-sheet microscopy or two-photon microscopy). Additionally or alternatively, non-optical methods may be used to image the biological assay apparatus, such as acoustic microscopy (e.g. utilising high frequency ultrasound rather than light).
[0041] The method may comprise illuminating the biological assay apparatus, e.g. with a suitable light source for a desired imaging approach known in the art per se.
[0042] Embodiments of the present invention may be used for short-term analyses (e.g. comprising determining a location of the motile biological structure(s) after one day or less, twelve hours or less, six hours or less or one hour or less following their introduction to the input region) and long-term analyses (e.g. comprising determining a location of the motile biological structure(s) after one or more days, one or more weeks, or one or more months following their introduction to the input region)).
[0043] Embodiments of the present invention may be used to assess many different types of motile biological structure. In a set of embodiments, the sample contains one or more of the following motile biological structures: cells (prokaryotic or eukaryotic), cell aggregates (e.g. spheroids), cell clusters, 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, sample includes one or more cancer cells, sperm cells, fibroblasts, immune cells and / or mesenchymal cells.
[0044] As explained above, one or more dimensions of the sections 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 1 pm, at least 2 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 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 total 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.
[0045] 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 per se. The pathway may be accessible, e.g. to enable the application of one or more serums and / or antibiotics to the culture medium.
[0046] Preferably, the movement of the motile biological structure along the pathway is primarily or entirely due to inherent activity of the structure, e.g. rather than being merely carried along the pathway by flow of a surrounding fluid medium. This may enable more accurate assessment of intrinsic structure motility. In a set of embodiments, the motile biological structure is not driven along the pathway by external interference (e.g. there is no pumping of fluid along the pathway).
[0047] The movement of the motile biological structure(s) 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, some embodiments of the method may not involve actively inducing movement of the motile biological structures along the pathway (e.g. the biological assay apparatus may be free from haptic, chemical or gravitational stimuli).
[0048] However, in some embodiments it may be beneficial to induce movement along the pathway. A set of embodiments includes inducing movement of the motile biological structure(s) along the pathway. In some embodiments, the biological assay apparatus is arranged to induce movement of the motile biological structure(s) 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 that useful insight may be gained into the motility of structures by monitoring their passage through the section(s) in response to a stimulus such as a haptic, chemical or gravitational stimulus.
[0049] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced 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.
[0050] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced 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.
[0051] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced 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, e.g. the chemical stimulus holder may be located in an end region of the assessment structure. 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.
[0052] In some embodiments, in addition to or instead of a dedicated chemical stimulus holder, a chemical stimulus is present 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.
[0053] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced 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 structure(s) 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.
[0054] 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 or multi-well plate) 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).
[0055] The biological assay apparatus may be configurable in multiple different orientations, e.g. to induce or exclude gravitational effects on the motile biological structure(s). 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. In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced by rheotaxis. Fluid flow a gradient in fluid viscosity may be created along the pathway.
[0056] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced by galvanotaxis. In some such embodiments an electric field is generated 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.
[0057] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced by thermotaxis. Variations in temperature over the pathway may be generated (e.g. a temperature gradient along the pathway). Such embodiments may utilise one or more suitably positioned heating or cooling elements.
[0058] In a set of embodiments, movement of the motile biological structure(s) along the pathway is induced 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.
[0059] As explained below in more detail, it may be advantageous for the assessment structure to be thin to aid observation of motile biological structures in the pathway. 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).
[0060] 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.
[0061] In a set of embodiments, the assessment structure replicates a particular biological environment (e.g. a particular organ and / or disease-state).
[0062] 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.
[0063] In a set of embodiments, the assessment structure is monolithic (i.e. formed from a single block of material). The input region may be part of the same block of material as the assessment structure. In some embodiments, the input region comprises a container such as a well or a chamber. The end region may be part of the same block of material as the assessment structure. In some embodiments, the end region comprises a such as a well or a chamber.
[0064] 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. As explained below, a single support (e.g. a single well plate) may support several different assessment structures.
[0065] The applicant has recognised that it may be possible and advantageous to process multiple assays in parallel by including multiple input regions and corresponding assessment structures in the same biological assay apparatus.
[0066] In a set of embodiments the biological assay apparatus comprises a plurality of input regions and corresponding assessment structures. Each of the input regions may be for receiving a sample comprising one or more motile biological structures. Each of the assessment structure may define a pathway extending from the input region and along which the motile biological structure(s) can move. Each pathway may comprise a first section having a first transit difficulty and a second section having a second, different, transit difficulty. It will be recognised that the transit difficulties of respective first and second sections of assessment structures may not be the same (i.e. each assessment structure may have sections of different difficulties).
[0067] In such embodiments, the method may comprise introducing samples comprising one or more motile biological structures to each input region. Different input regions may receive samples comprising different amounts and / or types of motile biological structures. The method may comprise allowing the motile biological structures to move along the pathway of each assessment structure at the same time. The method may comprise determining locations of the motile biological structures and, for each motile biological structure in each assessment structure: incrementing a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and incrementing a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway. Providing multiple assessment structures in the same biological assay apparatus may thus enable multiple assays to be run in parallel. The assessment structures (and optionally the corresponding input regions) may be positioned in close enough proximity to be imaged by a single microscope. For instance, the assessment structures and their corresponding input regions may be provided in respective wells of a multi-well plate.
[0068] In some embodiments the assessment structures (and optionally the corresponding input regions) are all positioned within a field of view of a single microscope. This may allow all of the assessment structures to be imaged at the same time. Additionally or alternatively, the biological assay apparatus is mounted on a movable stage, to allow different assessment structures (and optionally corresponding input regions) to be brought into a field of view of a single microscope as needed.
[0069] As explained above, the transit difficulties of the first and second sections (and potential further sections) may depend on properties of the pathway, i.e. , properties determined in advance of the assessment. In some embodiments, the method comprises obtaining transit difficulty information (e.g. the transit difficulties themselves and / or information about the pathway from which the transit difficulties can be determined such as dimension information, shape information or material information).
[0070] The transit difficulty information may be obtained from the biological assay apparatus. For instance, the transit difficulty information may be encoded in a graphical representation printed, etched or otherwise displayed on the apparatus and / or stored on a memory of the biological assay apparatus (e.g. in an RFID chip). Obtaining the transit difficulty information about the pathway may comprise using a graphical representation reader (e.g. a barcode reader) to read a graphical representation in which transit difficulty information is encoded. Additionally or alternatively, the transit difficulty information may be determined by measuring one or more properties of the assessment structure (e.g. measuring a shape or dimension of the pathway). Additionally or alternatively, the transit difficulty information may be obtained from a source external to the biological assay apparatus itself, e.g. from a local memory of a device used to perform part of the method or from a remote server. In such embodiment’s the biological assay apparatus may include information identifying where and / or how to obtain transit difficulty information (e.g. encoded in a graphical representation printed, etched or otherwise displayed on the apparatus (e.g. a barcode) and / or stored on a memory of the biological assay apparatus (e.g. in an RFID chip)).
[0071] The method may comprise obtaining said information and using the information to obtain transit difficulty information. Obtaining the information may comprise using a graphical representation reader (e.g. a barcode reader) to read a graphical representation in which the information is encoded.
[0072] The biological assay apparatus providing transit difficulty information for a given assessment structure may be particularly useful in embodiments featuring multiple assessment structures because it may allow associated scoring values for each assessment structure to be quickly and reliably obtained and applied automatically, e.g. by suitable computing means.
[0073] In a set of embodiments, the biological assay apparatus comprises a plurality of input regions and corresponding assessment structures, and transit difficulty information for each assessment structures. Additional or alternatively, the biological assay apparatus comprises a plurality of input regions and corresponding assessment structures, and information identifying where and / or how to obtain transit difficulty information for each assessment structures. For instance, a biological assay apparatus may comprise a suitable graphical representation for each assessment structure.
[0074] In a set of embodiments, the biological assay apparatus may be used to generate supplementary motility information of the motile biological structure. The supplementary motility information may reflect or indicate characteristics the of the motile biological structure that are not necessarily captured in the motility score, e.g. allowing for greater understanding of the motility of the motile biological structure. The supplementary motility information may comprise quantitative or qualitative information relating to the motile biological structure (e.g. a quantitative supplementary numeric score and / or a qualitative classification label).
[0075] In a set of embodiments, the supplementary motility information relates to at least one of: interactions between the motile biological structure and the assessment structure, interactions between the motile biological structure and another motile biological structure, and interactions within the motile biological structure. For instance, the supplementary motility information may contain an indication of the nature, strength and / or frequency of such interactions.
[0076] The supplementary motility information may be obtained using an algorithm.
[0077] The algorithm may generate the supplementary motility information. The algorithm may use the image(s) generated by imaging the biological assay apparatus (e.g. using microscopy) to generate the supplementary motility information. The algorithm may use the motility score to generate the supplementary motility information.
[0078] The algorithm may comprise an artificial intelligence algorithm. The algorithm may comprise a machine learning algorithm. The algorithm may comprise a pre-existing artificial intelligence model (e.g. a model that has been pre-trained prior to use in the method, e.g. an off-the-shelf model). The algorithm may be a fine tuned artificial intelligence model (e.g. an off-the-shelf model artificial intelligence model that has been adapted and / or fine-tuned with supplementary motility information). Alternatively, the method may comprise training the artificial intelligence algorithm (e.g. using one or more sets of training data).
[0079] The algorithm may be an artificial intelligence model that has been trained from scratch (e.g. specifically trained for obtaining the supplementary motility information of a motile biological structure moving in the assessment structure). Training the algorithm from scratch may involve using training data comprising training input data (e.g. images of a motile biological structure moving in the assessment structure) and associated supplementary motility information (e.g. provided by human expert analysis). According to a second aspect of the present invention there is provided a system for assessing a motile biological structure comprising: a biological assay apparatus; an imaging apparatus arranged to image the biological assay apparatus; and a processing module; wherein the biological assay apparatus comprises: an input region for receiving a sample comprising a motile biological structure; and an assessment structure defining a pathway extending from the input region and along which the motile biological structure can move, the pathway comprising a first section having a first transit difficulty and a second section having a second, different, transit difficulty; and the processing module is arranged to: cause the imaging apparatus to produce an image of the biological assay apparatus; use said image to determine a location of a motile biological structure moving along the pathway; and increment a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and increment a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.
[0080] The biological assay apparatus may comprise a plurality of input regions and corresponding assessment structures as explained above. The system may be arranged to obtain transit difficulty information for each assessment structure. For instance, the system may comprise a barcode reader arranged to read graphical representations corresponding to each assessment structure in which transit difficulty information or information identifying where and / or how to obtain said transit difficulty information is encoded.
[0081] 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.
[0082] One or more non-limiting examples will now be described, by way of example only, and with reference to the accompanying figures in which:
[0083] Figure 1 is a schematic diagram showing a system according to an embodiment of the present invention;
[0084] Figure 2 is a flow diagram illustrating a method according to an embodiment of the present invention;
[0085] Figures 3A, 3B and 3C are a schematic diagrams of a biological assay apparatus being used in embodiments of the present invention; and
[0086] Figure 4 is schematic diagram of another biological assay apparatus for use in embodiments of the present invention.
[0087] Figure 1 shows a system 100 for assessing a motile biological structure. The system 100 comprises a microscope 102, a barcode reader 103, a sample stage 104, a controller 106 and a biological assay apparatus 108. The biological assay apparatus 108 is positioned on the sample stage 104 so that the microscope 102 can be operated to image the biological assay apparatus 108 and its contents. The system 100 may also include other elements such as a light source to enable imaging of the apparatus and its contents.
[0088] The biological assay apparatus 108 is a multi-well plate which includes several assay wells 110. The sample stage 104 may be controllable to position each of the assay wells 110 in the field of view of the microscope 102. Alternatively, the microscope 102 may have a sufficiently wide field of view to image all assay wells 110 at the same time. Each assay well 110 has a barcode 111 alongside. The barcode 111 encodes transit difficulty information about an assessment structure in the corresponding well 110. The barcode reader 103 reads the barcode 111 and the controller 106 uses the result to obtain transit difficulty information about the corresponding assessment structure.
[0089] In use, as explained in more detail below, a sample comprising motile biological structures is added to an assay well 110 and imaged by the microscope 102 to assess the motility of the motile biological structures. The controller 106 comprises a processing module 112, a memory 114 and a user interface 116. As explained in more detail below, the controller 106 may process images captured by the microscope to automate some or all of the motility assessment steps.
[0090] An example method of assessing motile biological structures using the biological assay apparatus 108 will now be described with reference to Figures 2 and 3A-3C. Figures 3A-3C illustrate the contents of one of the assay wells 110 of the multi-well plate 108.
[0091] The (assay well 110 of the) biological assay apparatus 108 comprises an input region 200, an end region 202 and an assessment structure 204 that extends between the two. The assessment structure 204 comprises a pathway along which motile biological structures in the sample can move. The pathway is made up of a series of sections 206a-206j (indicated by dotted lines in Figures 3A-3C). Each section 206a-206j is assigned a transit difficulty value. The value of each section is an indication of how challenging it is for a given motile biological structure to pass through the section. The transit difficulty values may depend on the type of motile biological structure that is being tested. A set of example difficulty values is given in Table 1 below.
[0092] Table 1
[0093] It can be seen that short straight sections (e.g. 208A, 208C) have the lowest difficulty value, whilst longer sections (e.g. 206e), corners (e.g. 206b) and narrowing sections (e.g. 206j) have higher difficulty values. The sharp U-turn corner 206d has a higher difficulty value than the curved U turn corner 206f.
[0094] In this example, the biological assay apparatus 108 is used to assess a sample of immune cells, to determine motility scores for individual cells of the sample. In other examples, other motile biological structures may be assessed. The controller 106 obtains the transit difficulty values for the pathway by reading the barcode 111 with the barcode reader 103.
[0095] In step 302, the sample of cells 210 is added to the input region 200. A chemical attractant is added to a chemical stimulus holder 208 in the end region 202. Figure 3A shows the biological assay apparatus 108 at this initial stage.
[0096] The cells 210 begin to move by chemotaxis towards the chemical attractant. To do so the cells 210 have to navigate through the assessment structure 204.
[0097] In step 304, the microscope 102 captures an image of the biological assay apparatus 108 as the cells 210 move along the pathway. Figure 3B shows the biological assay apparatus 108 at a time h (e.g. one minute after the cells 210 were added to the input region 200).
[0098] In step 306, the processing module 112 of the controller 106 processes the image from the microscope 102 and determines the locations of cells 210 in the assay apparatus 108 at ti. The controller 206 identifies first and second cells 210a, 210b have progressed into the assessment structure 204.
[0099] In step 308, the controller 106 determines that the first cell 210a has passed through the first section 206a, but that the second cell 210b has not yet passed through any section of the pathway. The controller 106 increments a motility score for the first cell 210a by the difficulty value of the first section 206a, i.e. by 1. The motility score of the second cell 210b remains at it starting value of 0.
[0100] The microscope 102 continues to capture images of the biological assay apparatus 108 as the cells 210 move along the pathway, identifying the location of cells and updating their motility scores. In other words, steps 304, 306 and 308 are repeated.
[0101] Figure 3C shows the biological assay apparatus 108 at a later time t2 (e.g. two minutes after the cells 210 were added to the input region 200). In step 306, the controller 106 identifies the new location of the first and second cells 210a, 210b and the location of a newly entered cell 210c. The controller 106 determines that the first cell 210a has now additionally passed through the second, third and fourth sections 206b, 206c, 206d, and increments its motility score by these sections’ difficulty values (2, 1 and 4). The motility score for the second cell 210b at time t2 is thus 9.
[0102] The controller 106 also determines that the second cell 210b has now passed through the first section 206a of the pathway and increments its motility score to 1. This process of imaging and scoring the cells continues until a desired experiment duration is complete. Final motility scores can then be assigned to each cell. These are output in step 310, e.g. to the user interface 116 of the controller 106. Additional information (e.g. how quickly particular cells passed through various sections) may also be determined from the images and provided to a user. The controller 106 may be configured to execute an artificial intelligence algorithm using the images of the biological assay apparatus to generate supplementary motility information to be provided to the user (e.g. supplementary numeric score(s) and / or descriptive labels). For instance, the supplementary motility information may provide an indication or explanation of a motility score (e.g. highlighting weak or strong cellstructure interactions, cell-cell interactions and / or intracellular behaviour).
[0103] In other examples, the location of cells may be determined only once, e.g. at the end of an experiment duration. This may produce only a motility score for each cell. The maximum motility score that can be achieved (i.e. by reaching the end region 202) is the cumulative total of the sections’ difficulty values. Figure 4 shows another biological assay apparatus 400 that may be used in examples of the present invention. The apparatus 400 comprises an input region 402, a first end region 404, a second end region 406 and an assessment structure 408 that extends between the input region 402 and the end regions 404, 406. The assessment structure 408 comprises a pathway with two branches. The first branch 410 extends to the first end region 404. The second branch 412 extends to the second end region 406.
[0104] Each branch 410, 412 is made up of a series of sections. The first branch 410 features several sharp corner sections 414. In contrast, the second branch 523 features several gentle corner sections 416. The two branches 410, 412 thus have different transit difficulties.
[0105] The movement of motile biological structures along the branches 410, 412 of the pathway may be observed as described above to determine quantitative information about the motility of those motile biological structures.
[0106] 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 method of assessing a motile biological structure using a biological assay apparatus, the biological assay apparatus comprising: an input region for receiving a sample comprising a motile biological structure; and an assessment structure defining a pathway extending from the input region and along which the motile biological structure can move, the pathway comprising a first section having a first transit difficulty and a second section having a second, different, transit difficulty; wherein the method comprises: introducing a sample comprising a motile biological structure to the input region; allowing the motile biological structure to move along the pathway; determining a location of the motile biological structure; incrementing a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and incrementing a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.
2. The method of claim 1 , wherein the sample comprises multiple motile biological structures and the method comprises: allowing multiple motile biological structures to move along the pathway: determining a location of multiple motile biological structures; and for each motile biological structure whose location is determined: incrementing a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and incrementing a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.
3. The method of claim 1 or 2, wherein the transit difficulty of a section is based at least in part on one or more dimensions of the section.
4. The method of claim 3, wherein the transit difficulty of a section is based on a minimum, maximum or average width, height or diameter of the section.
5. The method of any preceding claim, wherein the transit difficulty of a section is based on a shape of the section.
6. The method of any preceding claim, wherein the transit difficulty of a section is based on the presence of one or more obstacles in the section.
7. The method of any preceding claim, wherein the transit difficulty of a section is based on a material property of the assessment structure in the section such as density or stiffness.
8. The method of any preceding claim, wherein the transit difficulty of a section is based on a surface texture of the assessment structure in the section.
9. The method of any preceding claim, wherein one or more sections extend in a plurality of planes.
10. The method of any preceding claim, wherein the transit difficulty of a section is determined based on one or more properties of the motile biological structure being tested.
11. The method of any preceding claim, wherein the first and second sections are arranged serially.
12. The method of any preceding claim, wherein the first and second sections are arranged in parallel.
13. The method of any preceding claim, wherein the pathway includes one or more further sections, and the method comprises incrementing a motility score of a motile biological structure by a value corresponding to a transit difficulty of a furthersection if the motile biological structure has passed through said further section of the pathway.
14. The method of claim 13, wherein the pathway comprises at least five sections.
15. The method of any preceding claim, comprising determining a location of the motile biological structure(s) at least two different times, and updating a motility score for the motile biological structure for the at least two different times.
16. The method of any preceding claim, wherein the movement of the motile biological structure along the pathway is primarily or entirely due to inherent activity of the structure.
17. The method of any preceding claim, wherein the biological assay apparatus induces movement of the motile biological structure(s) along the pathway by haptotaxis and / or durotaxis and / or topotaxis and / or chemotaxis and / or geotaxis and / or rheotaxis and / or galvanotaxis and / or thermotaxis.
18. The method of any preceding claim, wherein the biological assay apparatus comprises a plurality of input regions and corresponding assessment structures, and the method comprises: introducing samples comprising one or more motile biological structures to each input region; allowing the motile biological structures to move along the pathway of each assessment structure at the same time; determining locations of the motile biological structures and, for each motile biological structure in each assessment structure: incrementing a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and incrementing a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.
19. The method of claim 18, wherein the assessment structures are imaged by a single microscope.
20. The method of any preceding claim, comprising obtaining the transit difficulties and / or information about the pathway from which the transit difficulties can be determined from the biological assay apparatus or from a source external to the biological assay apparatus.
21. The method of any preceding claim, comprising generating supplementary motility information of the motile biological structure.
22. The method of claim 21 , comprising generating the supplementary motility information using an artificial intelligence algorithm.
23. The method of claim 22, wherein generating the supplementary motility information comprising inputting one or more images of the biological assay apparatus to the artificial intelligence algorithm and the artificial intelligence algorithm outputting the supplementary motility information.
24. The method of any of claims 21-23, wherein the wherein the supplementary motility information comprises information relating to at least one of: interactions between the motile biological structure and the assessment structure, interactions between the motile biological structure and another motile biological structure, and interactions within the motile biological structure.
25. A system for assessing a motile biological structure comprising: a biological assay apparatus; an imaging apparatus arranged to image the biological assay apparatus; and a processing module; wherein the biological assay apparatus comprises: an input region for receiving a sample comprising a motile biological structure; and an assessment structure defining a pathway extending from the input region and along which the motile biological structure can move, the pathway comprising afirst section having a first transit difficulty and a second section having a second, different, transit difficulty; and the processing module is arranged to: cause the imaging apparatus to produce an image of the biological assay apparatus; use said image to determine a location of a motile biological structure moving along the pathway; and increment a motility score of the motile biological structure by a first value corresponding to the first transit difficulty if the motile biological structure has passed through the first section of the pathway; and increment a motility score of the motile biological structure by a second value corresponding to the second transit difficulty if the motile biological structure has passed through the second section of the pathway.