System and method for generating a three-dimensional structure within a volume of curable material

Abstract

System for generating a three-dimensional structure within a volume of curable material, such as a curable hydrogel, containing at least one feature of interest, such as a labelled cell or organoid, wherein the system comprises: − a holding system for holding a container containing the volume of curable material and the at least one feature of interest; − an imaging system arranged for imaging said volume containing the at least one feature of interest for determining feature positional data representative of the three-dimensional position of said feature of interest in the volume of curable material; − a controller arranged to provide, on the basis of the feature positional data, three- dimensional structure data representative for the three-dimensional structure to be generated; − a printing system comprising an illumination system arranged for directing light in said container for locally curing said curable material, wherein the controller is arranged to control the illuminating system for selectively curing said curable material on the basis of three-dimensional structure data for generating said three-dimensional structure in said material.

Description

[0001] SYSTEM AND METHOD FOR GENERATING A THREE-DIMENSIONAL STRUCTURE

[0002] WITHIN A VOLUME OF CURABLE MATERIAL

[0003] The present invention relates to a system and method for generating a three-dimensional structure within a volume of curable material. The invention further relates to a three-dimensional structure of cured material obtainable by this system and method.

[0004] A typical bioprinting workflow involves ladening a curable material, such as a hydrogel, with cells, selecting a 3d model, and then printing this model so that the cells are encapsulated within the model. Grigoryan et al. (Science 364, 458-464 (2019)) has demonstrated the rudimentary idea for parametrically generated vessel-like 3D models in the context of bioprinting or biofabrication.

[0005] This approach pays no regard to the spatial distribution of the cell within the construct. As an example, features such as channels must be blindly or randomly distributed, thus sacrificing the minute intricacies seen in native tissues and reducing cell viability.

[0006] It is a goal of the present invention, next to other goals, to provide an improved system for generating a three-dimensional structure, wherein preferably the above mentioned drawback is at least partly alleviated.

[0007] This goal, amongst other goals, is met by a system according to appended claim 1. More specifically, this goal, amongst other goals, is met by a system for generating a three-dimensional structure within a volume of curable material containing at least one feature of interest wherein the system comprises: a holding system for holding a container containing the volume of curable biomaterial and the at least one feature of interest; an imaging system arranged for imaging said volume containing the at least one feature of interest for determining feature positional data representative of the three-dimensional position of said feature of interest in the volume of curable biomaterial; a controller arranged to provide, on the basis of the feature positional data, three- dimensional structure data representative for the three-dimensional structure to be generated; a printing system comprising an illumination system arranged for directing light in said container for locally curing said curable biomaterial, wherein the controller is arranged to control the illuminating system for selectively curing said curable material on the basis of three-dimensional structure data for generating said three-dimensional structure in said material. With this system, it is possible to create unique scaffolds that are tuned to the contents (feature of interest) of the container by scanning and subsequent printing of structures around those features. The feature of interest preferably comprises a cell or organoid and preferably the feature of interest is labelled. The features of interest are not limited to cells or organoids, and can comprise any arbitrary feature, such as for example existing printed structures and bulk objects. The container containing the volume of curable biomaterial may additionally or alternatively contain another suitable material known in the art. The container containing the (bio)material and the at least one feature of interest is also referred to as the container containing the print volume, or simply the print volume.

[0008] Preferably, the curable material comprises a biomaterial, such as a hydrogel. Preferably the curable material comprises a photocurable material. As an example, the curable material may comprise a gelatine methacryloyl (GelMA), Polyethylene Glycol Diacrylate) (PEGDA), silk methacrylate (SilkMA), Silk, hyaluronic acid methacrylate (HAMA), hyaluronic acid norbornene (HA-NOR), dextran methacrylate (DexMA), dextran tyramine (Dex-Tyr), alginate methacrylate (AlgMA), gelatin tyramine (Gel-Tyr), gelatin desaminotyrosine (GelDAT), gelatin norbornene (gelNOR) or gelatin allyl glycidyl ether (gel AGE).

[0009] The curable material preferably comprises a visible light photo initiator, such as lithium phenyl 2,4,6 trimethylbenzoyl phosphinate (LAP), or Riboflavin, or Eosin, or Tris(bipyridine)ruthenium(II) chloride / sodium persulfate (Ru / SPS).

[0010] Additionally or alternatively, the system may be arranged for generating a three-dimensional structure within a volume of material containing at least one feature of interest, wherein the material is otherwise modifiable, for instance chemically, not necessarily being curable in the sense of the material hardening under the influence of light. For example, the material may be suitable for spatio-temporal modifications using light, including for example photochemical grafting, stiffness gradients, and optogenetic stimulation. In such a system, the printing system is thus arranged for directing light in said container for locally modifying said material, in particularly chemically modifying said material, wherein the controller is arranged to control the illuminating system for selectively modifying said material on the basis of three-dimensional structure data for generating said three-dimensional structure in said material.

[0011] The imaging system preferably provides image data of at least the feature of interest in the container, which image data can preferably be used for determining feature positional data representative of the three-dimensional position of said feature of interest. The controller is then arranged to provide, on the basis of the feature positional data, three- dimensional structure data representative for the three-dimensional structure to be generated. The structure data represents three-dimensional information of the structure to be printed into the container. Preferably, the three-dimensional structure data is generated on the basis of the feature positional data. The structure to be printed can then be tailored to the feature of interest.

[0012] Determination of the feature positional data does not need to take place in the imaging system. The controller, or another processor, may determine the positional data. Embodiments are envisioned wherein the controller is arranged to process the image data received from the imaging system, thereby determining feature positional data on the basis of the image data. Embodiments are also envisioned wherein the imaging system is arranged to determine the feature positional data on the basis of the image data. In general, it is not critical to the functioning of the system exactly which subcomponents perform which operations on which data.

[0013] Preferably, the controller is further arranged to generate, on the basis of a three-dimensional structure model and the feature positional data, the three-dimensional structure data. The structure data is preferably generated automatically, i.e., without human intervention.

[0014] More preferably, the three-dimensional structure model comprises a parametric model arranged to generate the structure data on the basis of the feature positional data as parameter. This allows efficient tailoring of the structure to be printed to the positional data of the feature of interest in an automated manner. The controller is then preferably arranged to receive, as an input, the feature positional data and the parametric model, and to apply the feature positional data as a parameter in the parametric model for generating the structure data.

[0015] A parametric model in the context of this disclosure is a model that is designed to provide the three-dimensional structure data adapted on the basis of the input of a parameter, in particular the feature positional data in this context. This concept is also referred to as parametric design. Parametric design is a type of design approach that uses parameters or variables to create and modify designs. It involves creating a model or algorithm that defines the relationships between the design variables and how they affect the final design. In parametric design, a designer will create a parametric model, which contains a set of parameters that can be manipulated to create different variations of the design. The design is then changed on the basis of the feature positional data as a parameter. Generative, or iterative, design processes can optionally be applied to the process for generating the structure data, wherein multiple iterations are performed to optimize the structure data. Said parameters can then be varied for each iteration to adjust the structure data, to arrive at a final structure data which is optimized for the desired application.

[0016] Generating the structure data may further include simulating, for instance simulating properties of the structure data. The structure data may for instance be updated based on simulation results, for instance in an iterative manner as described above for optimizing the structure. Simulations may for instance include physical modelling / simulations on the structure data generated by the parametric model. As an example, it is possible to couple a fluid dynamics simulation to the parametric model (in conjunction with any other data streams) in order to guide / optimize vessel diameters to achieve a specific flow rate or pressure in certain regions, for instance on the basis of the identified features of interest.

[0017] The container may contain different types of features of interest, for instance different type of cells or cells having different markers or other identifiable properties, as will be explained further below. Thus preferably, the imaging system is arranged to image said volume for determining feature positional data representative of the three-dimensional position of a first feature of interest and at least one second feature of interest, different from said first feature of interest. The at least first and second features of interest may be features of interest of separate objects, such as cells or organoids, or different features of the same object.

[0018] It is then preferred that the imaging system is arranged to detect and identify the different features of interest and that the structure data (the model to be printed) is generated on the basis of the different types of features of interest. Preferably, the controller is arranged to generate, on the basis of the three-dimensional structure model and the feature positional data of the first and second features of interest, the three-dimensional structure data.

[0019] The at least one feature of interest may for example also include a light-absorbing feature that, when present in a path of light used for selectively curing or otherwise changing the properties of the curable material in the container, or vessel, disturbs the selective curing process. The imaging system may thus additionally be arranged for identifying one or more light-absorbing features on the basis of the feature positional data, and conducting for instance object space optimization of tomographic reconstructions to adapt the three-dimensional structure data representative for the three-dimensional structure to be generated, or having been generated, to account, and at least partially compensate, for said one or more light-absorbing features. It should be noted that the parametric model may also use additional parameters or data streams, alongside or even as an alternative to the positional information, to generate and dictate the structure data. The controller, or another component, may be arranged to determine, for instance on the basis of the image data, further parameters which can be used as an input for the parametric design. Examples of the additional parameters or data streams include:

[0020] Spectral data or color information (including intensity): For example, the parametric model may use a label, for instance a fluorescent label of a scanned feature of interest, to generate a specific geometry around that feature, or differentiate / scrutinize it from other features within the same volume. For example, a parametric model may be designed such that within a single volume, features having a first label may have a different surrounding three- dimensional structure than a feature having a second label. As an example, features labeled with Cy3.5 may be encompassed by a dense network of channels, while features labeled with Cy5 may receive a sparse network. Spectral data could also be derived from various fluorescent assays such as live / dead, DNA staining, pH indication etc. In some cases, pigmentation could also be a quantifiable parameter.

[0021] Feature dimensions: As above but imaged features may be differentiated / scrutinized based on their dimensions. For example the parametric model may select only features of interest, for instance spheroids, of specifics size (radii).

[0022] Morphology: As above, but characteristics such as surface irregularity, ellipticity, internal structure, and branching / outgrowths, may also be quantified and used as input parameter for the parametric model.

[0023] Spatial distribution: Aside from the positional data itself, there are additional parameters that can be inferred from the spatial distribution of the data which can be used as additional input to the parametric model. This can be relative positioning (i.e. the location of features / clusters relative to others), proximity or distance metrics (e.g. location within the volume), density distribution, alignment and orientation, and inter-cluster relationships.

[0024] Preferably, the holding system comprises a rotating member for supporting the container. Preferably, the rotating member is a rotational stage, more preferably an encoded rotational stage. Other devices which are suitable for rotating an object to a precise predetermined angle, such as a stepper motor, may also be used.

[0025] Preferably, the container is a cylindrical container, and preferably the rotating member and container are arranged such that the container rotates around the central axis of the cylindrical shape of the container. The system may include the container. Preferably, the container is arranged in an outer container. Preferably, the outer container contains a second compound with a refractive index corresponding to the refractive index of the volume of curable biomaterial. Preferably, the rotating member is arranged such that the container rotates inside the outer container, while the outer container remains stationary. For this purpose, the rotating member may be arranged in the outer container and may additionally be arranged inside the second compound. The purpose of the container being arranged in the second compound, in the outer container, is to compensate for the refraction resulting from the container being cylindrical. For this purpose, the outer container preferably comprises at least one planar sidewall section.

[0026] Preferably, the imaging system is arranged to generate image data associated with a rotational angle of the container. A plurality of image data points of the one or more features of interest generated for a plurality of rotational angles of the container can then be used by the controller to determine feature positional data representative of the three-dimensional position of said one or more features of interest.

[0027] As the plurality of image data points are associated with rotational angles of the container, the resulting feature positional data may be defined in a cylindrical or spherical, preferably a cylindrical, coordinate system, wherein an angular coordinate corresponds to the rotation angle of the container. The feature positional data may be translated to another coordinate system, such as the three dimensional Cartesian coordinate system.

[0028] The three-dimensional structure data, based on the three-dimensional structure model and the three-dimensional feature positional data may also be defined in a Cartesian coordinate system. After generating the three-dimensional structure data, said data may be translated into cylindrical or spherical, preferably cylindrical, coordinates.

[0029] Preferably, the controller is arranged to control the rotating member and the printing system in accordance with the three-dimensional structure data.

[0030] According to a preferred embodiment, the imaging system comprises a light source arranged for illuminating the feature of interest, wherein the imaging system is arranged to image the illuminated feature of interest. Preferably, the light source is arranged to induce fluorescence in the container. This may be used to identify the features of interest or to distinguish between different types of features of interest in the container. For differentiating between two different features of interest, for instance two differently labelled cells, the imaging system is arranged to selectively illuminating the features of interest. The two features of interest may have different excitation wavelengths. The light source may be arranged illuminate the container with illuminating light having at least two different wavelengths for determining feature positional data representative of the three-dimensional position of the first feature of interest and the second feature of interest. Alternatively, for differentiating different features of interest, label free approaches can be used. These approaches include differentiation based on morphological differences such as shape, geometry, granularity, and / or differentiation based on optical differences such as opacity. These morphological parameters, which may be determined from the image data, may also be used as parameters in the parametric model as mentioned above. The structure data surrounding a particular feature of interest may then be adapted on the basis of the morphological parameter of said, or another, feature of interest.

[0031] Additionally or alternatively, the imaging system may be arranged to capture and differentiate between captured light having different wavelengths for differentiating the first feature of interest from the second feature of interest. The imaging system may hereto be provided with filters for selectively filtering the captured light.

[0032] According to a preferred embodiment, the imaging system comprises a light sheet imaging device, in particular light sheet microscope. Light sheet microscopy is an imaging technique that uses a thin sheet of light to illuminate a sample, allowing for high-resolution, 3D imaging of biological samples. The principles of light sheet microscopy involve illuminating a thin section of the sample with a sheet of light, while imaging the sample from a perpendicular and changing angle, preferably using the rotating holder as mentioned above. This allows for high-resolution 3D imaging of the sample without the need for extensive sample preparation or staining.

[0033] Preferably, the light sheet imaging device comprises a light sheet optical path for generating a light sheet through the axis of the container containing the printing volume. This light sheet optical path may comprise a free space or a fibre-coupled laser source. Both said optical fibre and said laser source may be single mode or multi mode. More than one laser source may be used to provide light at multiple excitation wavelengths. The one or more laser beams may pass through one or more beam reduction optics to reduce the beam diameter. When using more than one laser sources, it is preferred that the imaging device additionally comprises an optical instrument to combine the two laser beams, for example an interference filter, dichroic filter, a thin film filter and / or a semitransparent mirror. A first laser source may be arranged behind the dichroic filter, whereby the first laser beam from the first laser is transmitted through the dichroic filter. A second laser may be arranged such that the emitted second laser beam hits the dichroic filter at an angle, whereafter the second laser beam is reflected in the same direction as the first laser beam. This explanation of how the laser beams combine applies to all listed optical instruments for combining the laser beams.

[0034] Preferably, the light sheet comprises a fan-shaped beam. To form said light sheet, optical instruments such as a Po well-lens (also known as laser line generating lenses), a cylindrical lens or a galvanometric scanning mirror, which comprises one or more mirrors mounted on one or more galvanometric rotary motors, may be used. Combinations of the aforementioned optical instruments, as well as other instruments known in the art which can provide a light sheet - a fanshaped beam - may also be used. Said light sheet may then be focused using focusing optics such as cylindrical lenses, f-theta lenses, or a microscope objective, or other suitable optical instruments, to intersect with the container containing the print volume. Alternatively, a patterned or structured light sheet illumination regime may be employed through the use of spatial light modulation, diffractive optical elements or beam interference.

[0035] Preferably, the imaging system comprises one or more imaging devices arranged to receive the light emitted by the contents of the container, specifically the one or more objects of interest, more specifically fluorescent signals emitted by the one or more objects as a result of the light sheet illumination. The one or more imaging devices are thereby arranged to generate light sheet scans.

[0036] Preferably, the one or more imaging devices are arranged to receive light emitted in a direction perpendicular to the light sheet propagation path. The resulting images then show the one or more objects of interest in a plane parallel to the light sheet propagation path which allows for more efficient processing of the resulting images, and a lower error. The one or more imaging devices may be two imaging devices, each arranged opposite to each other, on opposite sides of the light sheet propagation path. Using two imaging devices improves the accuracy of the light sheet scans as more datapoints are made available.

[0037] Preferably, the one or more imaging devices comprise charge coupled devices (CCD) and / or complementary metal-oxide semiconductor (CMOS) image sensors. The one or more imaging devices preferably additionally comprise optical instruments to focus the received light on the image sensors. These optical instruments may be tube lenses, microscope objectives, camera lenses or telecentric lens assemblies. Preferably, the one or more imaging devices additionally comprise bandpass filters to isolate one or more desired wavelengths, or a range of wavelengths, from the light received by the one or more imaging devices. Additionally or alternatively, the imaging system may employ forms of imaging such as optical tomography, optical coherence tomography, diffraction tomography or ultrasound imaging to generate feature positional data representative of the three-dimensional position of said feature of interest in the printing volume. These forms of imaging use backscatter and / or diffraction on features of interest, instead of fluorescence. The aforementioned forms of imaging do not rely on fluorescence and may therefore be used to generate feature positional data of opaque objects.

[0038] When using the rotating holding member as mentioned above, the imaging device is preferably arranged to provide a light sheet in the plane containing the rotation axis of the rotating member. The controller may then be arranged to rotate the rotating member and to control the imaging device for obtaining images of a plurality of cross-sections of the container for determining the feature positional data.

[0039] With the system, it is possible to provide, preferably automatically generate, structure data, i.e., the structure to be printed in the container, on the basis of determined positions of features of interest in the container. Generally, a printing system arranged for printing a three-dimensional structure in the container is then provided. The printing system is arranged to generate the three-dimensional structure in the container on the basis of the three-dimensional structure data.

[0040] The system can be used with any three-dimensional printing system known in the art, such as extrusion 3D printing or other forms of photopolymerization 3D printing such as stereolithographic and digital light processing 3D printing.

[0041] Other compatible printing techniques include multi-photon lithographic printing and xolography, among other methods (including also FRESH printing, also called Embedded printing, which however uses extrusion heads), as well as Dynamic Interface Printing (DIP, Vidler et al. Nature (2024) l tos:Z / ^.org / 10,l)B8 / s41.586.;024-08077-6), and acoustic printing (Kuang et al. Science. (2023). doi: 10.1126 / science.adil563) (Habibi et al. Nature (2022) https: / / doi.org / 10.1038 / s41467- 022-29395-1).

[0042] According to a preferred embodiment, the printing system is however arranged for volumetric printing, also known as volumetric additive manufacturing. Volumetric printing allows for a relatively rapid printing speed and overcomes geometric and surface quality limitations inherent in for example extrusion 3D printing or other forms of photopolymerization 3D printing such as stereolithographic and digital light processing 3D printing. In addition, volumetric printing does not require support members to be printed as well to support the printed object during printing. This is beneficial as these support members can hinder the functionality of the printed object, and may require additional work to remove them. Volumetric printing allows for 3D printing using a resin without making contact with the resin itself. Furthermore, the nature of the technique allows for unconstrained complexity, with the imaging system, for instance in the form of a light sheet imaging modality, allowing for scanning to be performed at the single-cell level over multiple channels. Furthermore, by understanding the locations of features within the containers, corrections (for occlusion or refraction) can also be applied to the printing process to further improve output quality. Preferably, the printing system is arranged for computed tomographic axial lithography.

[0043] The printing system preferably comprises a printing light beam which is preferably conditioned to be homogenous, spatially modulated, addressable, and collimated. Preferably, the printing system comprises an illumination source for providing a printing light beam to be conditioned. The printing light beam preferably comprises a laser beam generated by a laser, and may therefore be referred to as a printing laser beam.

[0044] According to a preferred embodiment, the printing system comprises a spatial light modulator for conditioning the printing beam for selectively curing said curable biomaterial. The spatial light modular may for instance comprise an array of deflectable micro mirrors, for instance in the form of a Digital Micromirror Device (DMD). The micro mirrors are used to create patterns of light that can be used to selectively illuminate specific regions of the container, in accordance with the structure data.

[0045] Computed tomographic axial lithography comprises projecting, by the printing light beam, a two dimensional image of the rotating three dimensional structure to be generated onto the container containing the print volume which rotates at the same rate. Said projected two dimensional image may be telecentric or non-telecentric. The illumination source may be a single wavelength illumination source, a narrowband spectral illumination source, or a broadband spectral illumination source. To generate the projection, spatial light modulators such a digital micromirror device (DMD), a liquid crystal on silicon modulator, an acousto-optic modulator or deflector, and electro-optic deflector, a micromechanical scanner, a galvanometric scanner, or an electrically and / or optically-addressed spatial light modulator may be used. Preferably, a light beam originating from one of the aforementioned illumination sources is directed onto one of the aforementioned light modulator devices, and subsequently directed towards the container containing the printing volume. Preferably, between the spatial light modulator and the container, additional optical elements to transfer the light beam from the spatial light modulator to the container are arranged. Preferably, said optical elements comprise two lenses, and preferably an aperture, such as a diaphragm or an iris, is arranged between said two lenses. The lenses are preferably placed at a distance of two focal lengths of the lenses relative to each other. In other words, he lenses are preferably placed at a distance of two focal lengths of the lenses relative to each other, forming a 4f relay system. The one lens is placed at one focal length from the spatial light modulator, and preferably the other lens is placed at one focal length from the container. The lenses and diaphragm preferably serve as a Fourier filter, the purpose of which is to block undesired diffraction orders resulting from the spatial light modulator.

[0046] The rotating member is preferably arranged to be controlled, by the controller, to rotate in synchronization with the spatial light modulator.

[0047] When using a light sheet imaging device, the spatial light modulator may be arranged to provide the light sheet. Thus, the printing system and the light sheet imaging system are comprised by the same components. This embodiment beneficially allows for a limited number of components to be used. In this embodiment, the light modulator device is preferably arranged to be able to generate a light sheet.

[0048] In a preferred embodiment, the light sheet imaging device and the printing device are separate, and the spatial light modulator of the printing device is thus not required to also provide a light sheet. The separation of the light sheet imaging device and the printing device allows each to be optimizes for their respective functions. This typically allows the image system to provide image data having higher resolutions.

[0049] Preferably, the light sheet imaging device and the printing device are arranged to project the light sheet and the printing light beam respectively in opposite directions. Preferably, the propagation directions of the light sheet and the printing light beam are axially aligned in opposite direction, and thus aimed at a relative angle of 180° to each other. This axial alignment beneficially does not require an angular translation between the three-dimensional feature positional data generated by the imaging system and the three-dimensional structure data used by the printing device, other than “mirroring” the positional data and the structure data.

[0050] The axial alignment is however not essential, as the three-dimensional feature positional data and / or the three-dimensional structure data can be translated to compensate for the angle between the light sheet and the printing light beam if they are not axially aligned. Having the axial alignment between the light sheet and the printing light beam thus eliminates the need for an angular translation between the positional data and the structure data, thereby eliminating a source of error.

[0051] According to a further aspect, a method is provided for generating a three-dimensional structure within a volume of curable material, such as a curable hydrogel, containing at least one feature of interest, such as a labelled cell or organoid, in particular using a system as mentioned above, wherein the method comprises: providing a container containing the volume of curable material and the at least one feature of interest; determining feature positional data representative of the three-dimensional position of said feature of interest in the volume of curable material; providing, on the basis of the feature positional data, three-dimensional structure data representative for the three-dimensional structure to be generated; generating said three-dimensional structure by selectively curing said curable material on the basis of three-dimensional structure data for generating said three-dimensional structure in said material.

[0052] Preferably and as mentioned above, the step of determining feature positional data comprises imaging said volume containing the at least one feature of interest.

[0053] Also here, it is possible to generate the three-dimensional structure using any known three- dimensional printing technique. The container may then not contain curable material. It is however preferred, as mentioned above, that said three-dimensional structure is generated by selectively curing said curable material. Preferably, the step of selectively curing said curable biomaterial comprises directing light in said container for locally curing said curable biomaterial. Preferably, the step of generating said three-dimensional structure comprises volumetric printing. The benefits of volumetric printing have already been mentioned above.

[0054] The at least one feature of interest may additionally include a light-absorbing feature that, when present in a path of light used for selectively curing or otherwise changing the properties of the curable material in the container, or vessel, disturbs the selective curing process. The method may thus comprise additional steps of identifying one or more light-absorbing features on the basis of the feature positional data, and conducting for instance object space optimization of tomographic reconstructions to adapt the three-dimensional structure data representative for the three- dimensional structure to be generated, or having been generated, to account, and at least partially compensate, for said one or more light-absorbing features. According to a preferred embodiment and as mentioned above, the step of providing the three- dimensional structure data comprises generating, on the basis of a three-dimensional structure model and the feature positional data, the three-dimensional structure data, wherein the three- dimensional structure model comprises a parametric model arranged to generate the structure data on the basis of the feature positional data as parameter.

[0055] A further preferred embodiment further comprises determining feature positional data representative the position of a first feature of interest and at least one second feature of interest, different from said first feature of interest, wherein the step of generating the structure data preferably comprises generating with said parametric model the structure data on the basis of the positional data representative of the position of the first feature of interest and the second feature of interest as parameters.

[0056] Preferably, the parametric model is arranged to generate a three-dimensional tubular network structure, preferably a three-dimensional tubular network structure, wherein the parametric model is arranged to vary at least one geometry parameter of said structure, preferably chosen from the group consisting of a distance of a tubular structure to the feature of interest, a local diameter of the tubular structure and a density of the tubular structure on the basis of an input parameter. The parametric model can also be arranged to generate any other two-dimensional or three-dimensional structure. For example, structures that encapsulate features of interest and / or other structures, positive structures that interconnect several features are also possible. The structure may thus be locally modified based on the input parameters.

[0057] Generating the structure data preferably comprises varying at least of the geometry parameters between the first and the second feature of interest. The geometry parameters may also be varied on the basis of other parameters, such as morphological parameters obtained from the image data as mentioned above. The benefits of this are already explained above.

[0058] The method may thus comprise determining, for instance from the image data, further parameters, wherein step of generating the structure data comprises generating, further on the basis of these further parameters and the parametric model, the structure data. Preferably, the structure data is locally adapted on the basis of the (local) parameters such as the identified first and second (or more) features interest and / or the morphological parameters as defined. The method may further include a second step, and even further steps, of generating a second, or further, three-dimensional structure(s) following a first step of generating the three-dimensional structure, in particular by selectively curing said curable material. This allows for so-called ‘overprinting’, i.e. generating an additional (second) structure around a previously (first) generated structure. The curing, or otherwise changing the properties of the material as mentioned further above, may again take place by directing light in said container for locally curing, or otherwise changing, said biomaterial. It may for instance be possible that in a further step of generating the three-dimensional structure, light with a different wavelength is used, resulting in a different property of the exposed biomaterial.

[0059] Additionally or alternatively, the biomaterial may be exchanged between printing steps, leaving said three-dimensional structure, including any feature of interest. The three-dimensional structure and / or the feature of interest of the first printing step may then be the feature of interest of a second printing step. In particular, the method may comprise repeating at least one, preferably all, of the steps of: providing a container containing a volume of curable material, the at least one feature of interest and preferably a previously generated first structure; determining second feature positional data representative of the three-dimensional position of said feature of interest and / or the first generated structure in the volume of curable material; providing, on the basis of the second feature positional data, three-dimensional structure data representative for a second three-dimensional structure to be generated; generating said second three-dimensional structure by selectively curing said curable material on the basis of three-dimensional structure data for generating said second three- dimensional structure in said material.

[0060] This is especially beneficial in combination with removing a generated structure from the volume of curable material (the first container) after the first printing step, and subsequently placing the generated structure in a new volume of curable material (a second container) for a second printing step, for example with different properties than the previous curable material. It is also possible to leave the generated structure in the first container for a second printing step, for example in case of removing the container with the generated structure from for example the holding system, and subsequently placing the container back in the holding system.

[0061] It is then preferable to generate a transformation matrix to transform the orientation of the at least one feature of interest and / or the generated structure in the first printing step to the orientation of the at least one feature of interest and / or the generated structure for the second printing step. This transformation matrix may be generated by determining second feature positional data representative of the three-dimensional position of the structure and / or the at least one feature of interest in the volume of curable material in the second printing step and aligning said feature positional data to the three-dimensional structure data of the structure generated in the first vessel and / or the feature positional data of the at least one features of interest obtained in the first printing step.

[0062] According to a further aspect, a three-dimensional structure of cured material, such as a cured hydrogel, containing at least one feature of interest, such as a labelled cell, spheroid, cell aggregate, or organoid, is provided.

[0063] More generally, a method for providing three-dimensional structure data is provided. This method may then comprise the additional step of generating said three-dimensional structure, for instance using a three-dimensional printing technique as mentioned. This generic method, which is preferably a computer implemented method, may comprise the steps of: providing feature positional data; generating, on the basis of the feature positional data, three-dimensional structure data representative for the three-dimensional structure to be generated; outputting the three-dimensional structure data.

[0064] The positional data may be received, for instance from an imaging system as described above. More preferably, the step of providing feature positional data comprises: receiving image data containing image data of a volume containing the at least one feature of interest; determining feature positional data representative of the three-dimensional position of said feature of interest in the volume.

[0065] The further details as mentioned above for the method also apply to this generic method, in particular those details about generating the three-dimensional structure data comprises using a parametric model and locally modifying the design on the basis of the identified first and second features of interest and / or local morphological parameters.

[0066] Another aspect related to computer readable instructions, preferably stored on a carrier, that, when executed on a controller or processor, cause the generic method as described above to be performed. The controller refers to a generic computer, and may comprise a single computer or a network of computers or processors.

[0067] The present invention is further illustrated by the following Figures, which show a preferred embodiment of the system according to the invention, and are not intended to limit the scope of the invention in any way, wherein:

[0068] Figure 1 shows an overview of the method;

[0069] Figure 2 shows a first embodiment of the system;

[0070] Figure 3 shows a second embodiment of the system;

[0071] Figure 4 shows a schematic of the angles of incidence of the various light beams;

[0072] Figure 5 shows a close up of the light sheet and the container;

[0073] Figures 6A-D show different examples of the three-dimensional structure model;

[0074] Figure 7 shows another example of a three-dimensional structure model;

[0075] Figure 8 shows a schematic example of a three-dimensional structure model; and Figure 9 shows an example of the three-dimensional structure data and the three- dimensional structure model.

[0076] Figure 1 shows different steps S1-S8 of the method. In SI a container containing a biomaterial and objects of interest, such as cells or organoids is provided. In S2 the contents of the container are scanned with the rotating light sheet scanner, and a series of images is taken of the contents of the container is taken. Three-dimensional feature data is generated based on these images, and after processing and segmentation the feature data is translated from a cylindrical coordinate system to Cartesian coordinates in S3. In S4 three-dimensional structure data is generated based on the feature data and other parameters. In S5 the structure data is translated into a dataset to be used in the printing process, for example a series of two dimensional projections of the three-dimensional structure associated with a corresponding series of rotational angles. In S6 the structure is printed by rotating the container to a rotational angle and projecting the projections associated with the respective angle onto the biomaterial in the container. S7 and S8 are optional. In S7 the container is removed from the rotating member and placed in a linear light sheet scanner to scan the printed structure, and generate three-dimensional data of the printed structure for verification in S8. S7 may also be performed by the rotating light sheet scanner, essentially repeating S2.

[0077] Figure 2 shows an embodiment of the system. The system comprises a container 1 containing objects or features of interest 20, such as cells or organoids 20, and light curable biomaterial 21. The container 1 is placed on a rotating member (not shown) in an outer container 2, which contains a bath of a compound 3 which has a refractive index corresponding to the refractive index of the biomaterial 21. The light sheet scanner device comprises a first scanning laser 111 and a second scanning laser 112, and a semi-transparent mirror 12. The first and second lasers 111, 112 are angled such that the first laser beam Bsi intersects the dichroic filter 12, and passes through said dichroic filter 12. The second laser 112 is arranged at an angle such that the second laser beam Bs2 is projected onto the semi-transparent mirror 12 and reflected in a direction parallel to the first laser beam Bsi- The two laser beams then combine into a single scanning laser beam Bs- The scanning laser beam Bs passes through beam reduction optics 13, 14 to reduce the diameter of the laser beam. The scanning laser beam Bs then passes through a Powell-lens (a laser line generating lens) which changes the shape of the beam Bs into a fan-shape. The beam Bs then passes through a cylindrical lens 16 to focus it into a light sheet. The light sheet then passes through the transparent face, oriented at a perpendicular angle, of the outer container and into the container.

[0078] The light generated and emitted by the fluorescence of objects of interest 20 in the container 1, induced by the scanning light sheet, is then received by the imaging device 18. The light BR first passes through a band pass filter 17 to isolate the desired wavelength(s). The face of the outer container pointing towards the imaging device 18 is oriented perpendicular to the light sheet, and the imaging device 18 is also pointed in a direction perpendicular to the light sheet.

[0079] Printing laser 4 generates a printing light beam Bp, which passes through beam reducing optics 41, 42 and is transmitted by an optical fiber 5 and optic 43 towards a spatial light modulator 6. The now modulated printing light beam Bppasses through optical element 7, diaphragm 8 and optical element 9 towards a mirror 10 which reflects the printing light beam Bp towards a face of the outer container 2, oriented perpendicular to the printing light beam, through the outer container 2 into the container 1 and the biomaterial 21. Optical elements 7 and 9 are lenses, and serve to transfer the projection generated by the spatial light modulator 6 from the printing light beam to the centre of the container 2. Between the lenses 7 and 8 a diaphragm is arranged (not shown).

[0080] Figure 3 shows an alternative embodiment of the system. The system comprises a single scanning laser 11 generating a scanning beam Bs, which is transmitted through beam reduction lenses 13, 14 and through Powell-lens 15, then reflected by flip mirror 151 towards cylindrical lens 16 and into the outer container 2 and container 1. The printing laser is not shown, but the printing laser beam Bp is modulated by the digital micromirror device 6 and transmitted through optics 7 and 9, the printing beam is then reflected by mirror 61 towards the outer container 2 and the inner container 1. The first imaging device 18 is arranged in a non-perpendicular direction relative to the scanning beam Bs, and a flip mirror 184 is used to direct light BR towards the imaging device 18. A second imaging device 181 is arranged on the opposite side of the container, and parabolic mirror 182 is used to direct light BR2 towards the second imaging device 181. Bandpass filters 17 and 171 are used to isolate the desired wavelength(s) from the fluorescent light emitted by the objects of interest 21 in the container 1.

[0081] To perform light sheet scanning, fluorescent light BR is preferably reflected by the flip mirror 184 into the first imaging device 18, passing through the bandpass filter 17.

[0082] The flip mirror 184 can be flipped out of the path of BR. This allows the use of a second, optional, Schlieren imaging setup comprising parabolic mirror 183, parabolic mirror 182 and imaging device 181. A small light source, preferably a pinhole LED is arranged to provide a light source which is focused by the parabolic mirror 183 towards the container 2, through container 2 and onto the parabolic mirror 182, and then into the imaging device 181, through bandpass filter 171.

[0083] An additional third imaging device 30 is provided, in combination with a translating outer container 31. The third imaging device 30 can be used in combination with the translating outer container 31 to perform linear light sheet scanning. The container 1 may be placed into the translating outer container 31 to perform said linear light sheet scanning. When the linear light sheet scanning is performed, flip mirror 151 is flipped out of the path of the scanning beam Bs so that the scanning beam is directed to the second cylindrical lens 34. Hereby the same light source 11 can be used to perform both the rotational light sheet scanning and the linear light sheet scanning. The translating outer container 31 is arranged to translate along direction Ds.

[0084] Figure 4 shows a schematic of the angles of incidence of the various light beams, namely the printing beam Bp, the scanning beam Bs and the fluorescent light BR. Angle a is the angle between the axis along which the printing beam Bp and the scanning beam Bs propagate, and is 90°. Angle B is 180°.

[0085] Figure 5 shows a close up of the scanning beam or the light sheet Bs and the container 1 containing the biomaterial 21 and the objects of interest 20. During scanning and printing the container 1 rotates around its central axis AR.

[0086] Figures 6A-D show different examples of parametric configurations of the three-dimensional structure model 22a-d. In figure 6A the structure model comprises a scaffolding of channels of a certain diameter, and passing by the objects 20 at a certain approach distance. In figure 6B the diameter of the channels 22b is enlarged, and the approach distance is increased. In figure 6C a different structure is provided for the same orientation of objects 20, and in figure 6D a structure model is provided for a different orientation of the objects 20.

[0087] Figure 7 shows another example of a three-dimensional structure model in which two types of objects 20a and 20b have been differentiated. The capability of the scanning system to recognize different types of objects allows, for example, for different modeling parameters to be used. For example, the approach distance di is set at a larger value for object of a first type 20a than the approach distance d for objects of a second type 20b. In the shown example, the different approach distances are not transferred to the three-dimensional structure data to be printed shown on the right.

[0088] Figure 8 shows another example of a three dimensional structure model. In this example, the features of interest comprise organoids 20, and a three dimensional structure 22 has been generated based on the location of said organoids and other parameters.

[0089] Figure 9 shows the translation of the three dimensional structure model 22 and the positional data of the features 20 to the three dimensional structure data to be used to generate the three dimensional structure using the printing system.

[0090] The functions attributed to the controller may be provided through the use of dedicated hardware as well as hardware capable of executing software in association with appropriate software. When provided by a processor being part of the controller, the functions may be provided by a single dedicated processor, by a single shared processor, or by a plurality of individual processors, some of which may be shared. Moreover, explicit use of the term “processor” or “controller” should not be construed to refer exclusively to hardware capable of executing software, and may implicitly include, without limitation, digital signal processor (DSP) hardware, network processor, application specific integrated circuit (ASIC), field programmable gate array (FPGA), read only memory (ROM) for storing software, random access memory (RAM), and non-volatile storage. Other hardware, conventional and / or custom, may also be included.

[0091] It should be appreciated by those skilled in the art that any block diagrams herein represent conceptual views of illustrative circuitry embodying the principles of the present invention. Similarly, it will be appreciated that any flowcharts, flow diagrams, state transition diagrams, pseudo code, and the like represent various processes which may be substantially represented in computer readable medium and so executed by a computer.

[0092] The present invention is not limited to the embodiment shown, but extends also to other embodiments falling within the scope of the appended claims.

Claims

Claims1. System for generating a three-dimensional structure within a volume of curable material, such as a curable hydrogel, containing at least one feature of interest, such as a labelled cell, spheroid or organoid, wherein the system comprises:- a holding system for holding a container containing the volume of curable material and the at least one feature of interest;- an imaging system arranged for imaging said volume containing the at least one feature of interest for determining feature positional data representative of the three- dimensional position of said feature of interest in the volume of curable material;- a controller arranged to provide, on the basis of the feature positional data, three- dimensional structure data representative for the three-dimensional structure to be generated;- a printing system comprising an illumination system arranged for directing light in said container for locally curing said curable material, wherein the controller is arranged to control the illuminating system for selectively curing said curable material on the basis of three-dimensional structure data for generating said three-dimensional structure in said material.

2. System according to claim 1, wherein the controller is further arranged to generate, on the basis of a three-dimensional structure model and the feature positional data, the three- dimensional structure data.

3. System according to claim 2, wherein the three-dimensional structure model comprises a parametric model arranged to generate the structure data on the basis of the feature positional data as parameter.

4. System according to claim 2 or 3, wherein the imaging system is arranged to image said volume for determining feature positional data representative of the three-dimensional position of a first feature of interest and at least one second feature of interest, different from said first feature of interest, wherein the controller is arranged to generate, on the basis of the three-dimensional structure model and the feature positional data of the first and second features of interest, the three-dimensional structure data.

5. System according to any of the preceding claims, wherein the holding system comprises a rotating member for supporting the container.

6. System according to claim 5, wherein the controller is arranged to control the rotating member and the printing system in accordance with the three-dimensional structure data.

7. System according to any of the preceding claims, wherein the imaging system comprises a light source arranged for illuminating the feature of interest, wherein the imaging system is arranged to image the illuminated feature of interest.

8. System according to claim 7, wherein the light source is arranged to induce fluorescence in the container.

9. System according to claim 4 and 7 or 8, wherein the light source is arranged to illuminate the container with illuminating light having at least two different wavelengths for determining feature positional data representative of the three-dimensional position of the first feature of interest and the second feature of interest.

10. System according to claim 4 and 7, 8 or 9, wherein the imaging system is arranged to capture and differentiate between captured light having different wavelengths for differentiating the first feature of interest from the second feature of interest.

11. System according to any of the claims 7 to 10, wherein the imaging system comprises a light sheet imaging device, in particular light sheet microscope.

12. System according to claims 5 and 11, wherein the imaging device is arranged to provide a light sheet in the plane containing the rotation axis of the rotating member, wherein the controller is arranged to rotate the rotation member and to control the imaging device for obtaining images of a plurality of cross-sections of the container for determining the feature positional data.

13. System according to any of the preceding claims, wherein the printing system is arranged for volumetric printing.

14. System according to claim 13, wherein the printing system is arranged for computed tomographic axial lithography.

15. System according to any of the preceding claims, wherein the printing system comprises a spatial light modulator for conditioning the beam for selectively curing said curable biomaterial.

16. System according to claims 11 and 15, wherein the spatial light modulator is arranged to provide the light sheet.

17. Method for generating a three-dimensional structure within a volume of curable material, such as a curable hydrogel, containing at least one feature of interest, such as a labelled cell or organoid, in particular using a system according to any of the preceding claims, wherein the method comprises: providing a container containing the volume of curable material and the at least one feature of interest; determining feature positional data representative of the three-dimensional position of said feature of interest in the volume of curable material; providing, on the basis of the feature positional data, three-dimensional structure data representative for the three-dimensional structure to be generated; generating said three-dimensional structure by selectively curing said curable material on the basis of three-dimensional structure data for generating said three-dimensional structure in said material.

18. Method according to claim 17, wherein the step of determining feature positional data comprises imaging said volume containing the at least one feature of interest.

19. Method according to claim 17 or 18, wherein the step of selectively curing said curable biomaterial comprises directing light in said container for locally curing said curable biomaterial.

20. Method according to claim 19, wherein the step of generating said three-dimensional structure comprises volumetric printing.

21. Method according to any of the preceding claims 17 - 20, wherein the step of providing the three-dimensional structure data comprises generating, on the basis of a three-dimensional structure model and the feature positional data, the three-dimensional structure data, wherein the three-dimensional structure model comprises a parametric model arranged to generate the structure data on the basis of the feature positional data as parameter.

22. Method according to claim 21, further comprising determining feature positional data representative of the position of a first feature of interest and at least one second feature of interest, different from said first feature of interest, wherein the step of generating the structure data comprises generating with said parametric model the structure data on the basis of the positional data representative of the position of the first feature of interest and the second feature of interest as parameters.

23. Method according to claim 21 or 22, wherein the parametric model is arranged to generate a three-dimensional tubular network structure, wherein the parametric model is arranged to vary at least one geometry parameter chosen from the group consisting of a distance of a tubular structure to the feature of interest, a local diameter of the tubular structure and a density of the tubular structure on the basis on an input parameter.

24. Method according to claim 22 or 23, wherein generating the structure data comprises varying at least of the geometry parameters between the first and the second feature of interest.

25. Method according to any of the preceding claims 17 - 24, further including a second step of generating a second three-dimensional structure following a first step of generating the three-dimensional structure by selectively curing said curable material.

26. Three-dimensional structure of cured material, such as a cured hydrogel, containing at least one feature of interest, such as a labelled cell, spheroid or organoid, obtainable by the method according to any of the preceding claims.

27. Computer readable instructions stored on a carrier that, when executed on a controller according to any of the preceding claims 1-16, cause a method to be performed comprising the steps of:- providing feature positional data;- generating, on the basis of the feature positional data, three-dimensional structure data representative for the three-dimensional structure to be generated;- outputting the three-dimensional structure data.

28. Computer readable instructions stored on a carrier, when executed on a controller, cause a method to be performed according to any of the preceding claims 17 - 26.

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