Patterning device for preparation of three-dimensional structures and method for production thereof

Abstract

To provide an improved production of three-dimensional structures with materials comprising particles.SOLUTION: Provided is a patterning device (1) for the preparation of three-dimensional structures of a sample (S) comprising particles (P) free to migrate in a matrix (M). The device comprising a pattern generator (3), a holder (2), where the sample (S) is placed, connected to the pattern generator (3). The patterning device (1) further comprises at least one transformation device (D) adapted for the transformation of the matrix (M) into a modified matrix (M') in which the particles (P) are no longer free to migrate. The invention is also directed to processing for the production of a three-dimensional structure.SELECTED DRAWING: Figure 1a

Description

[Technical Field]

[0001] The present invention relates to an integrated device for fabricating three-dimensional structures, in particular three-dimensional structures of biological materials such as artificial biological tissues. The preparation of such three-dimensional structures is based on Faraday waves (FW) and can be combined with additive manufacturing techniques. In particular, the patterning device enables the simple and useful production of functional biological tissues by inducing self-assembly processes. Such an integrated device therefore comprises one or more pattern generators and may further comprise one or more additive manufacturing elements. [Background technology]

[0002] Faraday wave (FW)-based processes are known for the production of artificial biological tissues. A method using such an approach is described, for example, in WO2019038453. In this case, specific patterns are generated in layers of particles spread in a hydrogel matrix using acoustic vibrations. Multiple layers must be combined to obtain a three-dimensional structure while maintaining the particles within the matrix.

[0003] Other limitations of currently used techniques relate to the reduced variety of materials available for providing artificial biological tissue and the complexity of the biological materials produced.

[0004] The equipment used in applying known processes is not adapted to fabricate three-dimensional structures in a highly reproducible manner, particularly since the various equipment is used and handled differently, which is a source of variability in the samples produced and limits the number and variety of three-dimensional structures that can be fabricated.

[0005] Known methods are time-consuming and expensive, moreover, not adapted for the production of three-dimensional structures at the point of care.

[0006] As such, there appears to be a need for improvements in the currently applied processes and equipment used to fabricate three-dimensional structures. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Application Publication No. 2019 / 038453 Summary of the Invention [Problem to be solved by the invention]

[0008] One of the objectives of the present invention is to propose a patterning device that allows the rapid, clear, and highly reproducible creation of three-dimensional structures in a material. Such materials exhibit particles suspended in a matrix that are movable within the matrix under acoustic or other vibration waves, along with the matrix itself, whose properties can be modified. In particular, the viscosity or rheological properties and other physical and chemical properties of the matrix can be modified from a state in which particles immersed in the matrix are free to move when exposed to vibration waves to a state in which the particles are immobilized within the matrix. The particles thus generate a pattern within the matrix that is adapted to the generation of three-dimensional structures.

[0009] In this disclosure, the term "pattern" refers to a non-uniform arrangement of particles under the influence of an oscillating wave. In particular, a pattern may reflect the shape of a wave in a matrix, defined by the concentration of particles within the matrix. The pattern therefore defines a gradient concentration with local maxima and minima. In other words, the reference to "pattern" in this disclosure excludes a uniform spread of particles. A pattern may relate to a geometric arrangement with periodic or regular variations. Alternatively, a pattern may relate to a non-geometric arrangement.

[0010] The term "particles" as used herein refers to any particles that can be suspended in a matrix and move when exposed to vibration waves. More specifically, particles can be organic, inorganic, or metallic. Such inorganic particles include tricalcium phosphate, hydroxyapatite, other calcium phosphates, calcium sulfate, magnesium carbonate, calcium carbonate, and any inorganic salt or complex. Organic particles include any organic compound or group of compounds such as organic oligomers or organic polymers, fatty acids, liposomes, and encapsulated organic compounds. Organic particles also include potentially active molecules such as drugs or drug combinations. Organic particles also refer to biological particles in the sense of biological materials such as cells, cell aggregates, tissue fragments, organoids, spheroids, bacteria, microsomes, and components of biological materials such as proteins, protein-loaded microparticles, or signaling molecules, as well as any peptides, polypeptides, nucleic acid oligomers, and nutrients for biological cells. The biological cells can be of any type, including osteoblasts, fibroblasts, keratinocytes, human mesenchymal stem cells, chondrocytes, or human umbilical vein endothelial cells, or mixtures thereof. It should be understood that the particles can include mixtures of organic components, inorganic components, metallic components, and biological materials.

[0011] The matrix refers to a material suitable for suspending particles, such as a hydrogel, paste, liquid, or other material. The matrix may comprise gelatin or a gelatin derivative such as gelatin methacrylate, hyaluronic acid or a hyaluronic acid derivative such as hyaluronic acid methacrylate, collagen, fibrin / thrombin, Matrigel, Atrigel, agarose, tyramine hyaluronate, alginate, or a mixture thereof. The matrix may further comprise a component or mixture of components adapted to modify the physical properties of the matrix, at least to increase the viscosity and prevent migration of particles in the matrix after modification.

[0012] It is understood that the particles themselves preferably do not undergo polymerization or reticulation under the treatment of the present disclosure. Thus, the particles are not connected to each other and remain unbound. Conversely, the matrix containing the particles undergoes multiple modifications, such as viscosity modification or partial polymerization, to better fix the particles contained therein and maintain their non-uniform concentration generated by the acoustic waves. It is further understood that once the three-dimensional pattern is produced, the matrix remains bound to the particles. Because the particles remain unbound and unpolymerized, a surrounding matrix is ​​required to maintain the pattern resulting from the wave motion. If the particles are biological materials, the surrounding matrix can also serve as a nutrient environment. Thus, three-dimensional patterning refers to an assembly that includes both the matrix and the particles.

[0013] It is also understood that vibrational waves exclude the frequency range associated with ultrasound, which is particularly true when the particles refer to biological material.

[0014] It is therefore clear that the process of the present disclosure differs from conventional 3D printing, in which multiple particles are cured or polymerized and then the remaining surrounding material is removed.

[0015] It is further understood that in the three-dimensional patterns according to the present disclosure, the particles and matrix provide two distinct compositions, each having different properties under at least heat or ultraviolet (UV) irradiation. The three-dimensional structures described in the present disclosure may thus define multiphase structures.

[0016] In this disclosure, a "sample" refers to a collection of particles in a matrix. Preferably, the matrix has a gel-like consistency in which the particles are embedded. The properties of the matrix may be altered to prevent particle migration.

[0017] Above and below, the expression three-dimensional structure refers to a structure resulting from a pattern produced according to the present process or from the combination of patterns produced according to the present process. A living three-dimensional structure means a structure that arises from the combination of patterns and that contains living organisms, such as living cells.

[0018] It is a further object of the present invention to improve the viability of the manufactured artificial tissue.

[0019] The object of the present invention is also to propose an improved process for the production of three-dimensional structures of biological material.

[0020] It is also an object of the present invention to propose a process adapted to the production of three-dimensional structures with a greater variety and more complex combinations of starting materials.

[0021] It is also an object of the present invention to propose a method for producing three-dimensional structures at the clinical site (point of care). Point of care in this context means any specialized institution dedicated to receiving and treating patients, such as a hospital, private hospital, medical laboratory, analytical laboratory, etc. Point of care also means any research institute, pharmaceutical company, university, public or private training institution. [Means for solving the problem]

[0022] According to the present invention, these objects are achieved by the apparatus and process described in the appended claims.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood with the aid of the description of some embodiments given as examples and illustrated by the following figures, in which: FIG. [Brief explanation of the drawings]

[0024] [Figure 1] 1a, 1b, 1c and 1d are schematic diagrams of devices according to various embodiments. [Figure 2]2a, 2b and 2c show the schematic layout of the directional control device. [Figure 3] 3a and 3b show examples of the holding portion. [Figure 4] 4a, 4b and 4c show examples of lateral damping devices according to some embodiments. [Figure 5] 5a, 5b, 5c and 5d show examples of lateral damping devices according to other embodiments. [Figure 6] Figures 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i and 6j show several examples of lighting systems according to the invention. [Figure 7] Figures 7a, 7b, 7c, 7d, 7e, 7f, 7g and 7h show several examples of container fastening means according to the invention. [Figure 8] 8a, 8b, 8c and 8d show examples of containers according to some embodiments. [Figure 9] 9a and 9b show examples of containers according to other embodiments. [Figure 10] 10a and 10b show examples of holder arrangements according to embodiments. [Figure 11] FIG. 11 is a schematic diagram of the functional elements of a printing device. [Figure 12] 12a and 12b show an example of a process according to the present invention. [Figure 13] FIG. 13 shows an example of a process involving 3D printing of a support part. [Figure 14] FIG. 14 shows an example of a sequential process where each layer builds on the previous layer. [Figure 15] Figure 15 shows an example of parallel processing where each layer is built independently of the others. [Figure 16] FIG. 16 shows an example of a pattern obtained by this process. [Figure 17] Figures 17a, 17b and 17c show examples of containers containing internal functional elements and resulting patterns. DETAILED DESCRIPTION OF THE INVENTION

[0025] 1a, 1b, 1c, and 1d show a general arrangement of a patterning device 1 according to various embodiments of the present invention. The patterning device 1 comprises a pattern generation unit 3 and a holder 2 connected to the pattern generation unit 3. The holder 2 is adapted to hold a sample container 46 containing particles P to be arranged in a three-dimensional structure. The sample container 46 is arranged on the holder 2 using a container fixing means 4. A mixture comprising particles P and a matrix M is hereinafter referred to as sample S. The holder 2 is connected to the pattern generation unit 3 so as to transmit vibration waves W generated by the pattern generation unit 3 to the container fixing means 4 comprising the sample container 46 and the particles P contained therein. For this purpose, the holder 2 comprises a holder plate 21 on which the container fixing means 4 is arranged or which is integrated with the container fixing means 4, and a holder connecting means 22 that allows mechanical connection to the pattern generation unit 3. The pattern generation unit 3 has a generator connecting means 31 adapted to cooperate with the holder connecting means 22 and maintain the holder 2 in a predetermined position. The holder plate 21 of the holder 2 is provided with container positioning means 100 (FIGS. 7a to 7h) adapted to keep the container fastening means 4 firmly located on the holder plate 21.

[0026] The pattern generator 3 is understood here to be equivalent to a sound wave generator in the sense that it allows for the generation of compression waves similar to sound waves. It is, however, not limited to a mere speaker. The waves generated by the pattern generator 3 may or may not be audible to a human user. The quality of the waves emitted by the pattern generator 3 must be very high for the generation of a clear pattern in the sample, which may be difficult or impossible with a speaker.

[0027] The container 46 may be open or closed with a lid.

[0028] The particles P are contained in a matrix M, the properties of which can be modified during the process to generate a three-dimensional structure. For this purpose, the matrix M may contain components that can react under light, in particular ultraviolet (UV) light, heat, or other physical or chemical conditions. The patterning device 1 comprises at least one transformation device D adapted for transforming the matrix M. Such a transformation device D may comprise light, such as UV, UV-visible, or infrared light, a hot plate, an oven, a microwave generator, or any other suitable device. In particular, the patterning device 1 comprises a light-emitting system 6 adapted to initiate the transformation of the matrix M under light irradiation. Alternatively, or additionally, the matrix M may contain chemical components that can react under heat. Such reactions include chemically or temperature-induced polymerization or crosslinking. For this purpose, the patterning device 1 may comprise a heat source, such as infrared light. The infrared light may be included in the light-emitting system 6 or may be part of a separate device.

[0029] The patterning apparatus 1 comprises an image recording unit 7 focused on the sample S. The image recording unit 7 is adapted to monitor the pattern of particles P in the sample container 46 before, during, or after the sample container 46 is exposed to acoustic waves, thereby enabling real-time monitoring of the patterning process. Alternatively, or additionally, the image recording unit 7 can record the shape of the pattern, including the result of exposing the sample S to the waves. The image recording unit 7 may be a video system, such as a camera, an infrared camera, an ultra-high speed camera, or any related device, focused on the sample S during production. The image recording unit 7 may be coupled to an image analysis unit 8 adapted to recognize the pattern (including a predetermined pattern) of particles P in the sample container 46.

[0030] The patterning device 1 preferably comprises a frame 9 in which the light emitting system 6 and the image recording unit 7 are arranged. In particular, at least one of the light emitting system 6 and the image recording unit 7, and preferably both, are adjustable on the frame 9. The light emitting system 6 is advantageously connected to the frame 9 so as to have at least two positions, a first position above the holder 2 and the sample container 46 and a second position remote from the first position. For this purpose, the illumination system 6 can be orientated in the xy plane. It is connected to the frame 9, for example, by a rotatable connection 12a (FIG. 1a) or 12b (FIG. 1b). The rotatable connection 12a, 12b may be connected directly to the frame 9 or to the guide 13 (FIG. 1b). Alternatively or additionally, the illumination system 6 can be adjusted along a vertical axis z to adjust the distance between the receptacle fixing means 4, including the sample receptacle 46, and the emitted light 61 of the illumination system 6. For this purpose, the rotatable connections 12a, 12b may slide along vertical beams of the frame 9 or along guides 13. The position of the lighting system 6 may be adjusted manually or by a motor (not shown). A lock 14 can be used to lock the lighting system 6 at a given height above the holder 2, at an angular position, or at both a height and an angular position.

[0031] The pattern generating unit 3 may also be arranged on the frame 9 by one or more damping devices 10 that reduce noise and vibrations of the patterning device 1. Alternatively, the pattern generating unit 3 is independent from the frame 9. According to one embodiment, the pattern generating unit 3 is arranged on a set of sound-absorbing blocks 10a on the crossbeams of the frame 9 (FIG. 1a). According to another possible embodiment, the pattern generating unit 3 is arranged on a support 15 that includes a counterweight 16, making it possible to reduce the effects of vibrations generated by the pattern generating unit 3. The support 15 may be connected to the frame 9 by a set of sound-absorbing blocks 10b (FIG. 1b).

[0032] The frame 9 may comprise one, two, three or more vertical columns. Additionally, it may comprise at least one ceiling cross beam on which the image recording unit 7 is disposed. Other arrangements of the frame 9 are possible.

[0033] The frame 9 is arranged on a number of feet 11, at least one of which is adjustable, preferably two or more feet 11. The frame of the patterning device 1 advantageously comprises three feet 11, two of which are adjustable for adjusting the level of the patterning device 1, in particular the level of the holder 2 in a certain horizontal orientation. For this purpose, the patterning apparatus 1 comprises level control means 17 adapted to control the orientation of the plate 21 of the holder 2 with respect to the horizontal plane xy. Such orientation may, in some cases, preferably be precisely adjustable in order to avoid defects in the pattern generated by the pattern generation unit 3 on the sample S. The orientation of the holder 2, in which the container fixing means 4 is arranged, is preferably essentially horizontal, lying in the xy plane. Deviations in the orientation of the holder 2 with respect to the xy plane should preferably be about 0.5° or less, not more than 0.3°, not more than 0.1°, even more preferably not more than about 0.05°. The level control means 17 may be a visual orientation control means such as a spirit level (FIGS. 2a, 2b, 2c). The spirit level may be removably arranged on the holder 2, on the pattern generating unit 3 connected to the holder 2, or elsewhere on the patterning device 1. This allows for precise positioning of the orientation of the top surface of the plate 21 of the holder 2. The level control means 17 may, for example, be arranged in a hole 17a adapted to receive it without deviation. For this purpose, the edge of the hole 17a and the edge of the level control means 17 may have complementary conical shapes so that the respective positions of the patterning device 1 and the level control means 17 are always the same. With such a configuration, the level control means 17 can be removed and stored when the wave generator 3 is activated. Other devices, such as an accelerometer or a laser detection device, may be used instead. Alternatively, the level control means 17 or other device used to determine the orientation of the plate 21 may be firmly contained in or integrated into part of the patterning device 1 , such as the holder 2 . This arrangement avoids inaccuracies that may occur with removable devices, mainly over time, after several positions and displacements of the level control means 17. The level control means 17 is directly readable by the user. Alternatively, or additionally, the level control means 17 may be recorded and analyzed by the image recording and analysis unit 7 or another suitable device. Alternatively, or additionally, the level control means 17 may be the subject of an image projected onto a screen or display, visible to the user. Alternatively, or additionally, the level control means 17 may generate electronic signals, such as warning or error signals, and / or signals indicating whether the level complies with the requirements.

[0034] According to one embodiment, the patterning device 1 is arranged according to a vertical axis A, in which the pattern generating unit 3 is at the bottom, the holding unit 2 is above the pattern generating unit 3, and the light emitting system 6 surrounds or is positioned around the holding unit 2. Also here, the image recording unit 7 is located at the top and is adapted to be focused on the sample S being prepared.

[0035] The patterning apparatus 1 is conveniently arranged so that the illumination system 6 can be activated over the sample S being prepared in the sample container 46 in conjunction with activation of the pattern generation unit 3. Additionally, the image recording unit 7 may be activated in conjunction with the illumination system 6 and the pattern generation unit 3. The illumination system 6 may remain over the sample S being prepared in the sample container 46 whether it is activated or not. In this way, illumination of the sample S can be switched on and off without removing or replacing the illumination system 6 over the sample S.

[0036] The patterning device 1 is easily adaptable to a variety of applications. In particular, the light emitting device 6 may be removed from the patterning device 1 and replaced with a different light emitting system 6. Alternatively, the light emitting system 6 may comprise a plurality of different emitting lights 61 or emitting groups of lights 61, each light being independently operable as required. In particular, lights 61 or groups of lights 61 with different wavelengths or different powers may be used. The light emitting system 6 may comprise a light protection screen (not shown) capable of preventing the emitted light from diffusing around the printing device 1. Alternatively or additionally, the user may be provided with separate safety glasses or a face shield.

[0037] The holder 2 is preferably easily removable from the pattern generator 3 and replaceable with another holder 2 according to specific needs. By way of example, the holder 2 may be adapted to accommodate individual dishes 48a (FIG. 3a) or well plates 48b (e.g., single well plates, 6-well, 12-well, 24-well, 48-well, 96-well, or any other number of well plates) (FIG. 3b), or both. The holder connection means 22 may be adapted to be manually connected and disconnected from the generator connection means 31, in particular, without tools. For example, a spring-loaded clamping mechanism may be used to hold the holder 2 in place. Alternatively, the holder 2 may be arranged on a rotating platform with multiple holders 2 of different types. Such a rotating platform may be connectable and disconnectable to the pattern generator 3 by removable connection means.

[0038] In another embodiment, the image recording unit 7 may also be easily removed from the frame 9 and replaced by another image recording unit 7. For example, a camera active in the visible range may be replaced by an infrared camera, a high-speed camera, or another type of image recording unit 7. Replacement of the image recording unit 7 is preferably possible without tools. A manual clamping or locking system is advantageously used.

[0039] Advantageously, patterning device 1 is provided with a set of various accessories, having two or more different emission lights 61 or groups of lights 61, two or more different image recording units 7, and two or more different holders 2. Patterning device 1 can thereby be tailored as needed to suit desired experimental or production requirements. In a convenient arrangement, each element, such as light-emitting system 6, image recording unit 7, or holder 2, arranged on patterning device 1 is automatically recognized by control unit 81. The corresponding setup can be automatically uploaded to control unit 81 for appropriate operation of the corresponding device. Alternatively, the characteristics of the elements may be manually loaded into control unit 81 by human-machine interface (HMI) device 82.

[0040] In a preferred arrangement, the position of the holder 2 along the horizontal axes x and y is not adjustable. In other words, the position of the holder 2 in the xy plane is predetermined. This prevents lateral movement of the holder 2, particularly when the pattern generating unit 3 is operating. In this way, the position of the container fixing means 4 on the top surface of the plate 21 is fixed, and so is the lateral position of the holder 2. The term "predetermined" here should be understood as non-adjustable or non-tunable by the user. If the holder 2 is part of a turntable, the position of the corresponding turntable is predetermined.

[0041] Additionally, displacements in the xy plane due to vibrations are prevented or at least minimized. To this end, one or more lateral damping devices 51 (FIGS. 4a, 4b, 4c, 5a, 5b, 5c, and 5d) are arranged to prevent lateral vibrations of the holder 2 and the sample S during preparation. Such an arrangement further improves the quality of the pattern and, consequently, the resolution and feasibility of three-dimensional structures within the sample container 46. In particular, the holder connection means 22 or the generator connection means 31 may be directly or essentially directly surrounded by the lateral damping devices 51a (FIG. 4a). Alternatively, the holder connection means 22 and the generator connection means 31 are not directly connected to each other but are connected via an intermediate connection 5, which itself is surrounded by the lateral damping devices 51a (FIG. 4b). Alternatively, the holder 2 itself is surrounded by the lateral damping devices 51a (FIG. 4c). For this purpose, the edge of the holding part 2 may, for example, be extended downwards to form a skirt fitting (a portion shaped like a hem) on the lateral damping device 51 a. Such a lateral damping device 51 a may comprise a tube 510 a surrounding either the holding part connecting means 22, or the generator connecting means 31, or the intermediate connecting part, or the edge of the holding part 2. The lateral damping device 51 a further comprises a support part 511 a, independent of the pattern generator 3, that can hold the tube 510 a in place.

[0042] The damping arrangement described herein may be important for providing a good quality pattern. In particular, the damping elements may be arranged and designed to absorb at least some of the reflected waves and resonant frequencies that interfere with pattern generation. Preferably, the damping elements are adapted to absorb all frequencies not emanating directly from the pattern generating unit 3, so that the sample receives the waves generated by the pattern generating unit 3 exclusively or substantially exclusively, without distortion.

[0043] The patterning device 1 may further comprise an illumination system that allows visualization of the sample during and after processing. In particular, multiple photographs of the pattern may be easily taken by an image recording device, either in a continuous manner, at regular time intervals, or after each step of processing. Such an illumination system is therefore independent of the conversion device D for converting the sample and any light system shown therein. The illumination system does not affect the sample itself, but merely visualizes it. The illumination system may therefore provide the necessary light directly to the sample surface. The illumination angle of the sample is variable, providing illumination from the side, thereby improving sample visualization by taking advantage of light reflections from the sample surface at different angles and providing better contrast. Alternatively, or additionally, the illumination system is configured to provide light from below the sample so that the light traverses the sample and can be imaged by the image recording device, in other words, the illumination system is configured to provide transmitted illumination to the sample. The light used to illuminate the sample may be either unpolarized or polarized to better visualize patterns formed below the sample surface. Illumination of different wavelengths may also be used to improve visualization by taking advantage of fluorescent moieties potentially contained in the sample or by other methods related to illumination and absorption of specific wavelengths. It is envisioned that other combinations of polarization and / or color filters may be placed between the illumination system and the image recording unit 7 to improve visualization of the sample before, during, and / or after patterning.

[0044] According to one embodiment, the illumination system is an external module adapted or adaptable to the patterning device to illuminate the sample. Such an illumination system may therefore be easily removed or replaced as needed. Such a removable arrangement allows, for example, to adapt either the wavelength or angle of illumination, or to include or remove a backlight function.

[0045] According to another embodiment, the illumination system is integrated into the patterning device, eg it can be combined, combined or integrated with the conversion device D or other functions of the patterning device.

[0046] 5a, 5b, 5c, and 5d show alternative configurations in which the holder connection means 22 and the generator connection means 31 are connected by an intermediate connection 5, which in turn is connected to the frame 9 of the patterning device 1 by means of one or more flexible arms 51b. Such flexible arms 51b may comprise a rigid portion 511b that prevents lateral movement of the holder 2 and a flexible attachment portion 512b at the end of the arm 51b that allows vertical displacement of the holder 2 (FIGS. 5a, 5b). Alternatively, the flexible arms 51b may take the form of leaf springs 513b (FIG. 5c). One end of each leaf spring is coupled to the frame 9 of the patterning device 1 and the other end is connected to the intermediate connection 5. Alternatively, the flexible arm 51b may comprise multiple rigid portions 511b and multiple flexible portions 512b to form a leaf spring specifically adapted for the retainer 2. Other arrangements or combinations of these arrangements may be used. The flexible arms 51b may be connected to the intermediate connection 5 or to two opposing sides, or if the patterning device 1 comprises three or more vertical pillars 9, one or more flexible arms 51b may be connected to each of these pillars 9.

[0047] 6a to 6i illustrate various alternatives related to the light-emitting system 6. The light-emitting system 6 comprises one or more light-emitting lamps 61 arranged on one or more light frames 62 so as to illuminate the sample S in the sample container 46 when the light-emitting system 6 is arranged on the holder 2. The light-emitting lamps 61 may be, for example, UV or UV-visible light, typically applied for polymerization of reactive chemical components at these wavelengths and present in the sample S. The light-emitting lamps 61 may be any suitable light-emitting device, such as conventional UV or UV-visible light or specific LEDs. Depending on the arrangement of the light-emitting lamps 61, the light-emitting system 6 may comprise one or more reflective surfaces 64 that focus the light toward the sample S arranged on the holder 2. The light frames 62 may be part of the holder 2 and illuminate the sample S from below (FIG. 6h). In such a configuration, the plate 21 of the holder 2 comprises a recess 66 closed by a transparent sheet in which the container fixing means 4 is arranged. Combinations of one or more of the configurations described herein are possible. For example, the sample S may be illuminated from below, above, or from the edge, or from a combination of these directions. The light frame 62 or a group of light frame 62 is arranged to keep the sample S visible to the image recording unit 7. For example, the light frame 62 may be circular with a central hole 63 corresponding to the light path of the image recording unit 7. The orientation of the emitted light 61 or its distance from the sample S, or both, may be adjusted manually or automatically.

[0048] The light frame 62 may be adapted to receive several types of different emitted light 61, for example irradiating in different optical spectral regions or at different wavelengths. In addition, the light frame 62 may comprise an infrared light adapted to heat the sample S. This is particularly convenient if a temperature-induced polymerization of the matrix M is required instead of a polymerization caused by UV irradiation. The matrix M of the sample S may contain different chemical components that can react under different conditions. The light-emitting system 6 is advantageously adapted to alternately activate several lights 61 or groups of lights 61.

[0049] The container fixing means 4 may have any shape. For example, the container fixing means 4 may have a square, rectangular, circular, or oval enclosure. Figures 8a, 8b, 8c, and 8d show several examples of the container fixing means 4. The container fixing means 4 comprises a base 41 for holding a sample container 46 in which matrix M and particles P are disposed. The container fixing means 4 further comprises a removable cap 42 disposed on the base 41 and adapted to fit, preferably in an airtight manner, to the base 41. The removable cap 42 can be adjusted to fit around the edge of the base 41 and maintained by friction alone (Figure 8a). A sealing member 44 can be disposed between the base 41 and the removable cap 42 to increase the friction while maintaining the container fixing means 4 in a closed, preferably sealed, state. Alternatively, the removable cap 42 may be adapted to fit the base 41 by a cap clamp arrangement 43. Such a cap clamp arrangement 43 may, for example, comprise a first member 43a connected to the cap 42 and a second member 43b connected to the base, the first member 43a and the second member 43b adapted to cooperate with each other. The cap clamp arrangement 43 may be adjustable to accommodate multiple closed positions (FIGS. 8b, 8c), including either a gradual closing force (FIG. 7c) or multiple predetermined closed positions (FIG. 8b). Alternatively, only one closed position is possible (FIG. 8d).

[0050] The vessel securing means 4 may itself be modular. A given base 41 may be adaptable to receive various types of caps 42. FIG. 9b shows various configurations in which a given base 41 is combined with different caps 42a, 42b, 42c. Such caps 42a, 42b, 42c may be used to facilitate securing sample vessels 46 of different shapes and sizes on a given base 41. Alternatively, cap fittings 45 may be used to facilitate securing sample vessels 46 of different shapes and sizes on a given base 41. FIG. 9a shows several examples in which a given base 41 is combined with different cap fittings 45a, 45b, 45c. A single removable cap 42 may thereby be usable with the base 41.

[0051] The container securing means 4 may be partially or completely transparent to light, particularly UV-visible light. Either the base 41 or the cap 42 of the container securing means 4, or both the base 41 and the cap 42, may be transparent to such light. The cap 42 may also be completely transparent to such light, or partially transparent to such light. The cap 42 and the base 41 may independently be made of glass, quartz, a hard or soft polymer, or a combination of these materials.

[0052] The container fastening means 4 is preferably fixed to the surface of the plate 21 of the holder 2 by a positioning means 100 that allows the container fastening means 4 to be removably positioned in a predetermined position on the holder 2. The positioning means 100 may be an intermediate element 101 having a first positioning element 100a and a second positioning element 100b, as shown in FIG. 7a. The first positioning element may be a spring adapted to maintain the container fastening means 4, and the second positioning element may have a geometric shape complementary to the geometric shape of the holder 2 so as to secure the intermediate element 101 to the holder 2. Such an intermediate element 101 may comprise a clip (FIG. 7a), a rotating clip (FIG. 7b), a clamping holder (FIGS. 7c, 7d), a spring combined with a press arm (FIG. 7h). Alternatively, the container fastening means 4 may itself comprise the necessary positioning means, such as a rail (FIGS. 7e, 7f) or a flexible clip (FIG. 7g).

[0053] The plate 21 of the holder 2 may be arranged to directly receive the container fastening means 4. For this purpose, it is provided with all the container positioning means 100 necessary to maintain the container fastening means 4 in the appropriate position (FIG. 10a). Alternatively, the plate 21 of the holder 2 is arranged to receive a container adapting portion 24 (FIG. 10b). The container adapting portion 24 allows for easy replacement of the container fastening means 4 on the holder 2, including replacement with a container fastening means 4 adapted to a sample container 46 of a different size or shape. The container adapting portion 24 is maintained on the plate 21 of the holder 2 by adapting portion connecting means 23a, 23b. The adapting portion connecting means 23a, 23b preferably allow manual assembly or disassembly of the container adapting portion 24 from the plate 21. The container adapting portion 24 preferably includes all the container positioning means 100 necessary to maintain the container fastening means 4 in the appropriate position after the container adapting portion 24 has been placed on the plate 21.

[0054] In a preferred embodiment, the holder 2 may be provided with one or more sensors 25a, 25b. At least one of the temperature sensor 25a and the vibration sensor 25b is included in the plate 21 of the holder 2; preferably, both the temperature sensor and the vibration sensor are integrated or combined in the holder 2. The plate 21 advantageously additionally comprises a management device 26, which allows the container 46 containing the particles P and the matrix M to be heated at a desired temperature higher than the ambient temperature. The temperature sensor 25a, together with the temperature management device 26, allows the temperature of the sample S during preparation to be adjusted. The temperature can be defined according to a predetermined value or a set of values ​​adapted to the preparation of the sample S, in particular the polymerization of the chemical components present in the matrix M. The heating duration, the temperature range, and the temperature change rate can be the subject of several programs. The temperature can therefore be adjusted between 20°C and 200°C, preferably between 20°C and 100°C, and most preferably between 20°C and 50°C. The temperature control device 26 may additionally be adapted to cool the sample S to a temperature below ambient temperature, for example, less than 15°, or 10° C., or 5° C. In a highly preferred configuration, the temperature control device 26 is a temperature adjustment device adapted to adjust or vary the temperature of the sample S during preparation at a temperature between about 4° C. and about 40° C.

[0055] The temperature control device 26 may represent a separate conversion device D that allows for the transformation of the matrix M of the sample S. Alternatively, the temperature control device 26 is part of a conversion device D that includes multiple heating devices, such as a heating plate, a set of infrared lights, a microwave generator, and other devices that can be used to heat the sample S and transform the matrix M.

[0056] The plate 21 or the container fitting portion 24 may be provided with one or more level control means 17 .

[0057] The sensors 25a, 25b, temperature management device 26 and any other electrical elements that the holder 2 comprises may be connected to the control unit 81 by electrical connections 27 preferably located on the holder 2. Such electrical connections 27 may be adapted to be independently plugged into or unplugged from the control unit 81. Alternatively, when the holder 2 is placed on the pattern generating unit 3, the sensors 25a, 25b, temperature management device 26 and any other potential electrical elements present on the holder 2 are automatically plugged in.

[0058] The holder 2 may further be connected to or equipped with a cooling device (not shown) adapted to cool or freeze the sample S. The cooling device may be integrated into the patterning device 1. Any known cooling device may be used and adapted for this purpose.

[0059] Although a user can manually add or inject material into the sample S, for example before, during, or after the patterning process under acoustic waves, the device 1 may advantageously be provided with an injection device 200 comprising one or more injectors 201 adapted to inject material into the sample container 46. The matrix and particles are injected into the sample container 46 onto the particles P and matrix M already present in the sample container 46, or into the majority of the matrix M already present in the sample container 46 (FIG. 1c). The injection device 200 may be coupled to the frame 9 of the device 1 so as to be positioned above the sample S or remote from the sample S. A rotary connection means 202 may be used for this purpose. The injection device 200 can be used to add particles P, matrix M, or matrix elements or any other material to the sample S being prepared, as well as to a combination of multiple different elements. The chemical and biological components present in the added material may be different from the chemical and / or biological components already present in the sample container 46 .

[0060] The use of such an injection device 200 integrated into the patterning device 1 facilitates the production of three-dimensional structures. In particular, layers L with different matrices M may be arranged one above the other. Such an injection device 200 is preferably adapted for injecting biomaterials involved in the generation of the three-dimensional structures. The injection device 200 is adapted for any kind of injection procedure, not limited to 3D bioprinting procedures. In one embodiment, such an injection device 200 represents a 3D bioprinting device, preferably adapted for 3D printing of biomaterials.

[0061] Alternatively, or in addition, the injection device 200′ may be used to 3D print materials not directly included in the three-dimensional structure. For example, accessories can be printed on demand using non-biological materials. The term accessory refers to any device or feature used in the preparation of a three-dimensional structure, including the sample container 46 and any functional element integrated or combined with the sample container 46. In particular, the sample container 46 can be manufactured on demand in a predetermined shape. Examples of sample containers 46 and the resulting patterns are shown in FIGS. 17a, 17b, and 17c. In this example, a circular sample container 46 includes a groove V that traverses the interior space of the sample container 46. The groove V can itself have any shape and contributes to the shape of a given sample container 46. Such a groove V provides at least one opening V around the periphery of the sample container 46, allowing for the inclusion of additional material in the biomaterial loaded in the sample container 46. As shown in FIG. 17b, the grooves V may have a circular shape within the interior space of the sample container 46. Of course, the shape of such grooves V is not limited to this particular shape. Other shapes including multiple openings may be provided, or a web of grooves may alternatively be constructed within the sample container 46. The grooves V may be used to circulate a nutrient fluid K, such as artificial or non-artificial blood, physiological fluid, or other relevant substances necessary for growth and survival. FIG. 17C illustrates the resulting three-dimensional structure in which cells are organized according to a specific pattern and perfused by the circulating fluid K. As such, the shape of the sample container 46 should be understood to include the interior shape of any other shape or combination of shapes, as well as its peripheral shape. It should also be understood that while the acoustic wave pattern is determined by the peripheral shape of a given sample container 46, this does not preclude the incorporation of additional three-dimensional shapes within the sample container 46.

[0062] The shape of the sample container 46 can be determined based on the expected pattern of particles P resulting from acoustic exposure. For example, Library B comprises a set of variables (B1...Bm). Each set of variables corresponding to a given experiment can be used to determine the most appropriate shape of the sample container 46 according to the known patterns recorded in Library B. The patterns may be recorded in Library B by an image recording device 7 or other suitable recording device. In such a set of variables, the term "m" refers to an integer comprised between 1 and about 100, preferably between 1 and about 50. Such variables include one or more of acoustic wave properties such as its amplitude A and its frequency F, the shape of the sample container 46, the rheological properties of the matrix M, and other relevant variables such as environmental conditions such as temperature, atmospheric conditions, pressure conditions, gravity conditions, and other monitored variables of the experiment.

[0063] According to an optional embodiment, the shape of the sample container 46 is determined based on multiple sets of variables (B1...Bm) using an artificial intelligence module such as a computing program or artificial intelligence 83 of the patterning device 1 (FIG. 13). Such an artificial module 83 may be integrated into or combined with the command unit 81. Such an artificial module 83 may be integrated into the patterning device 1 or remote from the patterning device 1 and connected to it by conventional communication means such as the Internet or private protocols. Secure connection arrangements may be envisaged to grant access to authorized users. Library B may also be integrated into the patterning device 1 or remote from the patterning device 1. Access to Library B may be via secure arrangements.

[0064] The patterning apparatus 1 may comprise one or two different injection devices 200, a first injection device 200 dedicated to injecting biological material into the sample S, including 3D bioprinting, and a second injection device 200′ for 3D printing of accessories. Alternatively, a single injection device may be used, and elements of the injection device 200, such as the cartridge and nozzle, and any other features adapted for 3D printing of materials, may be interchanged or substituted as needed.

[0065] According to one important aspect, the injection device 200 integrated into or associated with the patterning device 1 is adapted to inject material at predetermined locations on the surface or bulk of the sample S. For this purpose, it may be movable in at least two planar directions X, Y. In addition, it may be movable along the Z axis to a vertical position. In addition, the injection device 200 may be oriented in a position different from the vertical position. It may be provided with conventional articulation means in a manner that allows displacement of the injection device 200 itself or parts thereof over six degrees of freedom. The positioning of the injection device 200 can be precisely controlled (piloted) by the control unit 81. The additional material to be injected into the sample S comprises particles P, one or more matrix components, or a mixture thereof, as defined above. The injected particles P may be the same as or different from the particles P already present in the sample S.

[0066] If an injection device 200' for injection of non-biological material is integrated or combined with the patterning device 1, it may move in at least two planar directions X, Y. The injection device 200' may additionally be movable in a vertical position along the Z axis. In addition, the injection device 200' may be oriented in a position different from the vertical position. It may be provided with conventional articulation means in a way that allows displacement of the injection device 200' itself or parts thereof over six degrees of freedom. The positioning of the injection device 200' can be precisely controlled by the control unit 81.

[0067] Additionally, the injection device 200, 200' is adapted to inject a predetermined volume. The actuation of the injection device and the volume injected can be precisely controlled by the control unit 81.

[0068] The polymerization process of the matrices in each layer may be the same or different. For example, the matrix M1 of the first layer L1 may be polymerized under light irradiation, and the matrix M2 of the second layer L2 may be polymerized under heat depending on the properties of the chemical components of the matrices M1, M2. Heat may be provided by the light-emitting system 6, if infrared light is present, or by the temperature control device 26, or by both the light-emitting system 6 and the temperature control device 26.

[0069] The pattern generating unit 3 comprises one or more vibration generating units 32, or sets of vibration generating units 32, all of which may be identical or different. Each vibration generating unit 32 or set of vibration generating units 32 of the pattern generating unit 3 is adapted to provide wave vibrations that are transmitted to the sample S via the holding unit 2. The vibration generating units 32 can, for example, be mechanically connected to a generator connection means 31 to transmit vibrations to the sample S via mechanical vibrations (FIG. 1a). The vibration generating units 32 can also be speakers that generate sound waves and transmit them to the sample S via air or another suitable medium. Alternatively, or additionally, the pattern generating unit 3 comprises one or more vibration generating units 32 that are arranged near the holding unit 2 and are not mechanically connected to the generator connection means 31 (FIG. 1c). The vibrations generated by such vibration generating units 32 are directed toward the holding unit 2, allowing the sample S to vibrate in a predetermined manner.

[0070] According to one embodiment, the pattern generator 3 includes or is connected to multiple vibration generators 32 arranged in a three-dimensional space (FIG. 1d). In particular, vibration generator 32c may be arranged below the holder 2, vibration generator 32d may be arranged above the holder 2, and vibration generators 32a and 32b may be arranged laterally of the holder 2 to provide multiple sources of vibration waves. Vibration generators 32c and 32d provide vibration sources on different planes. It should be understood that vibration generator 32d may be below the holder 2 on a different plane than vibration generator 32c. The stacked layers L of the sample S may thus be subjected to specific vibrations. In addition, vibrations can be combined to provide a specific pattern in a given layer L. Next, a layer L with an increased thickness compared to known processes can be used to build a pattern while exposed to vibration waves. Alternatively or additionally, lateral vibration generators 32a and 32b are provided. The waves generated by these transverse vibration generators 32a, 32b can be used in succession with or simultaneously with the waves generated by the planar vibration generators 32c, 32d. A patterning device 1 arranged in this way allows for the provision of three-dimensional patterns in a given layer L, instead of the two-dimensional patterns typically obtained under exposure to vibration waves. Such a device allows for an improvement in the speed and variety of three-dimensional structures produced.

[0071] The holder 2, and thus the container fixing means 4 including the sample container 46, may be surrounded by a wall defining a closed space 35. Such a closed space 35 allows the sample S to be placed under special conditions, such as a special atmosphere, a specific pressure condition, a specific temperature condition, a specific gravity condition, or a combination of several specific conditions. Special atmospheres include oxygen-rich atmospheres and oxygen-poor or oxygen-free atmospheres. Specific pressure conditions include high-pressure conditions, such as 2 bar, 3 bar, 4 bar, or more, as well as pressures lower than ambient pressure that can be reached by one or more vacuum pumps. Special gravity conditions primarily include zero gravity conditions, near zero gravity conditions, or at least low gravity conditions.

[0072] Additionally or alternatively, the holder 2 may be connected to the pattern generator 3 by an actuator 36, which allows vertical adjustment of the position of the sample S in the closed space 35. When the vibration generators 32a, 32b are arranged along the vertical axis z, the height of the sample S can be adjusted by the actuator 36 so as to properly receive the waves generated by the vibration generators 32a, 32b. In addition, the holder 2 may be precisely positioned at the convergence point of various wave sources to provide a specific pattern.

[0073] The various elements of the patterning device 1 are described separately for ease of presentation, but can be combined without limitation. The patterning device 1 may then be considered as a modular arrangement. The combination of the above elements, including the pattern generator 3, the conversion device D, and the implantation devices 200, 200′, allows for the generation of three-dimensional structures without or substantially without displacement of the sample S. In other words, all steps required to generate such three-dimensional structures can be performed by the patterning device 1.

[0074] According to one important aspect, the patterning device 1 includes all necessary functional elements to perform the preparation of the three-dimensional structure without, or substantially without, manipulation of the sample S. In other words, all necessary steps are performed with optimized time and reproducibility. Additionally, all or substantially all steps of the process can be performed at the point of care.

[0075] The closed space 35 may be provided by a closable opening, preferably comprising a cap 38 arranged on top. Such a closable opening 37 allows for the injection of additional material by an injection device 200. Alternatively, such an injection device 200, 200' is included in the closed space 35 or is integral therewith. The upper wall of the closed space 35 also comprises a transparent area allowing for light irradiation and image recording of the sample S. The transparent area may be provided on the cap 38 of the closable opening 37.

[0076] The patterning device 1 further comprises a control unit 81 adapted to operate the functional elements of the patterning device 1 (FIG. 11). In particular, the control unit 81 is adapted to switch on or off the image recording part 7, the pattern generation part 3, the light emitting system 6, and / or one or more injection devices 200, 200′. Additionally, the control unit 81 is adapted to receive information from one or more sensors, such as the vibration sensor 25b, the temperature sensor 25a, the image recording part 7, the level control means 17, and any other sensors. A control unit 81 operates (pilots) the above elements of patterning apparatus 1 according to a predetermined program or under instructions from a user. Any predetermined program or instructions from a user can be sent to control unit 81 via a human machine interface (HMI) 82, such as a keyboard, a tactile screen, or any other known human machine interface.

[0077] The control unit 81 is further connected to the image analyzer 8 and can receive from such image analyzer 8 an image of the pattern appearing on the sample S being prepared. Thus, the control unit 81 can automatically recognize whether the prepared pattern matches the expected pattern. The control unit 81 can then automatically steer the elements of the patterning device 1 according to the image of the prepared pattern. As an example, the control unit 81 may operate the pattern generator 3 according to a predetermined program including a predetermined frequency or set of frequencies, a predetermined duration, and a predetermined amplitude of the acoustic waves, while the image recorder 7 allows monitoring of the pattern formation. If the pattern is deemed to correspond to the predetermined target pattern, the control unit 81 can activate a conversion device D, such as the light-emitting system 6 or the temperature management 26, to convert the matrix M of the sample S into a modified matrix M', in which the particles P are no longer mobile. Alternatively, the control unit 81 may be adapted to activate the conversion device D before the pattern is completely reached, so as to limit or avoid diffusion of the particles P within the matrix M once they reach the appropriate position. Alternatively, the control unit 81 may activate both the pattern generator 3 and the conversion device D to allow the matrix M to be progressively converted into a modified matrix M′ during the movement of the particles P.

[0078] According to one embodiment, the image recording unit 7 is connected to the library B and is able to store recorded images, such as the final pattern obtained at the end of a sequence of steps including at least a patterning step and a transforming step. Such an image of the pattern may be associated with several other variables in the library B, including acoustic wave variables such as amplitude A and frequency F, as well as the shape of the container 46 on which the patterning took place.

[0079] With respect to the movement of particles P in sample S, if the control unit 81 determines that the movement of particles P is too slow or inappropriate, it can activate the heating plate to increase the fluidity of matrix M. Such matrix M remains untransformed under the heating of temperature control device 26 and can be polymerized or crosslinked under UV irradiation once particles P reach their expected positions.

[0080] The patterning device 1 may further comprise or be connected to an artificial intelligence module 83 (FIG. 11). Such an artificial intelligence module 83 may be used to automatically determine appropriate conditions for generating a given pattern. In addition, it may be used for modeling patterns based on patterns actually generated and stored in library B.

[0081] According to one embodiment, an artificial intelligence module 83 may be used to determine the shape of the sample vessel 46 according to the shape of the pattern to be obtained.

[0082] The present invention further comprises a process for generating a three-dimensional structure based on particles P spread in a matrix M, as shown in Figures 12a and 12b. This process comprises a dispersion step Q1 in which the particles P are uniformly dispersed in a first layer L1 of matrix M, which is fluid enough to allow easy movement of the particles P. The dispersion step Q1 may comprise a homogenizer or a movable table or any other mixing device adapted to homogenize the particles P in the matrix M. Alternatively, the dispersion step Q1 may comprise a pattern generator 3, e.g., generating vibrations that allow homogenizing the particles P in the matrix M. The dispersion step Q1 may be optional if the particles P are already uniformly mixed with the matrix M.

[0083] The process may comprise a pre-loading step Qa in which the sample container 46 is loaded with a matrix M. The loading step Qa allows the sample container 46 to be filled with a predetermined amount of a predetermined matrix M. If the matrix M already contains the required particles P, the dispersion step Q1 may be optional. Even if particles P are already present in the matrix M, the loading step Qa may be followed by the injection of additional material. Such injection of additional material may be performed manually or by one or more integrated injection devices 200.

[0084] According to a preferred embodiment, the particles P are uniformly or regularly dispersed in the matrix M on the plane defined by the x-axis and the y-axis. In addition, the particles P are uniformly or regularly dispersed in the matrix M across the thickness of the matrix M, i.e., across the vertical axis z. In other words, no particle concentration gradient is expected within the matrix M before the acoustic wave patterning step is applied. More specifically, the viscosity of the matrix M is adapted to maintain the particles P uniformly suspended in the matrix M while allowing their movement under the application of the acoustic waves. Thus, the particles P are not allowed to fall to the bottom of the sample container 46 before the vibration waves are applied.

[0085] Next, a patterning step Q2 is applied in a way that moves the particles P in the matrix M according to a predetermined pattern. This process comprises a transformation step Q3 in which the matrix M is changed into a matrix M', e.g. to prevent the movement of the particles P. Such a transformation step Q3 may be performed by a transformation device D of the patterning device 1, such as the light emitting system 6 or the temperature management device 26 or any other transformation device D that the patterning device 1 comprises.

[0086] The dispersion step Q1, the patterning step Q2 and the conversion step Q3 may be performed sequentially, meaning that each of the required devices, such as the pattern generator 3 and the conversion device D, is activated and deactivated one after the other. Alternatively, these steps may overlap or partially overlap each other, meaning that two or more pattern generators 3 and conversion devices D may be activated simultaneously.

[0087] This process may further comprise a second dispersion step Q1', in which particles P2 are dispersed in a second layer L2 of a second matrix M2. The second dispersion step Q1' can be performed in the same way as the first dispersion step Q1, or in a different way. The second dispersion step Q1' may be optional if particles P2 are already uniformly dispersed in the corresponding matrix M2 when the matrix M2 is placed with the sample S. Particles P2 may be identical to or different from particles P. The second matrix M2 may be identical, similar, or different from matrix M. In particular, the second matrix M2 may be transformed during the transformation step Q3' under different conditions than the transformation step Q3 of matrix M, such as using a different transformation device. The transformation device D must be activated. For example, if the transformation step Q3 of the matrix is ​​performed under light irradiation, the transformation step Q3' of the second matrix M2 may be performed under heating. Inverse transformation steps Q3 and Q3' may be applied.

[0088] According to one important aspect, multiple of the dispersion step Q1, patterning step Q2, and conversion step Q3 are performed without manipulating the sample container 46, and the sample S is in place during all or a substantial portion of the processing. This is true even when multiple layers L are constructed. This is true even when multiple layers L are constructed in sequence one on top of the other.

[0089] The process may additionally comprise at least one step Q4 of injecting additional material onto or into the bulk of the layer without manipulating the sample container 46. Such injection may be performed manually. Preferably, the injection device 200 is used for this purpose. Such injection of additional material may be performed before or after the application of acoustic waves. Alternatively, injection of additional material may be performed by the injection device 200 during patterning of the sample S under the application of acoustic waves. It should be understood that the injection device 200 may be used on demand at any time during the patterning process so that an accompanying operation can be applied to the sample S without the need to manipulate the sample S. For example, additional material may be injected into a given matrix M successively at one or more precise locations by the injection device 200 while a wave motion is being applied to the sample S.

[0090] The process according to the present disclosure leads to a three-dimensional structure of particles within a matrix. In particular, the process does not involve the removal of material such as the matrix surrounding the patterned particles.

[0091] Combining patterning techniques with material injection, particularly in combination with 3D printing or additive manufacturing processes, can produce highly regular patterns containing irregular or random particle distributions, which are particularly advantageous for, for example, research testing or investigations.

[0092] The process may further comprise steps of determining the shape of the sample container 46 and printing on demand the sample container 46 having the determined shape. Such 3D printing of non-biological materials can be performed by an injection device 200' integrated into the patterning device 1 or separate from the patterning device 1. More specifically, the process may comprise a selection step a) in which the shape of a given sample container 46 is predetermined according to the target shape of the pattern to be generated in a given layer L. The selection of the shape of the sample container 46 takes into account several variables, such as the amplitude A and frequency F of the acoustic waves applied to the layer L in which the particles P are dispersed in the matrix M. In addition, the nature of the matrix M, its composition, or at least the rheological properties, may be taken into account, as these variables also affect the behavior of the particles P under acoustic wave exposure. The selection step a) can be performed based on the previous set of variables B1...Bm stored in the library B. The selection step a) may additionally comprise an artificial intelligence program loaded into the artificial intelligence module 83 or into a separate artificial intelligence module and trained for this purpose.

[0093] According to one aspect of the present disclosure, the artificial intelligence unit 83 is capable of instantly analyzing images of the patterned particles during the patterning process and comparing such images with a predetermined target pattern. The artificial intelligence unit 83 is therefore capable of adjusting or providing instructions to the control unit 81 to adapt the pattern generator accordingly until the desired pattern is obtained exactly or with minimal acceptable deviation.

[0094] Once the shape of the sample container 46 is determined in selection step a), the corresponding sample container 46 is generated. It is preferably manufactured on demand in container manufacturing step b). Container manufacturing step b) advantageously uses additive manufacturing techniques, more specifically, the injection device 200′ described above. While the sample container 46 can be manufactured from any material compatible with 3D printing techniques, such as polystyrene or related polymers, the sample container 46 is preferably made from a biocompatible polymer, such as polyvinyl alcohol or any other biocompatible polymer and its mixture. If a crosslinking step is required, the chemical components and polymerization conditions are selected so as not to damage the biomaterial being prepared. The 3D printing process for the sample container 46 is performed, for example, at ambient temperature or a temperature below 40°C, preferably between about 20°C and about 35°C. If UV crosslinking is performed, the light irradiation is preferably determined so as not to damage the biomaterial being prepared.

[0095] The loading step Qa) defined above makes it possible to fill the sample vessel 46 with matrix M and particles P.

[0096] Then the patterning step Q2) and the conversion step Q3) described above are applied.

[0097] Figures 16a, 16b, and 16c provide an example in which a first sample container 4a having a hexagonal shape is filled with a first matrix M1 containing a first type of particles P1 to form a first layer L1 having a first pattern. Figure 16b shows a second layer L2 having a second pattern, which is generated based on a second sample container 4b having a round shape. The second sample container 4b is placed on top of the first layer L1. Figure 16c shows an alternative second pattern obtained based on the circular second sample container 4b.

[0098] As an example, the patterns shown in Figures 16b and 16c were obtained as follows. Matrix M was made from a hydrogel of gelatin 70% methacryloyl (GelMA70%) and 5% w / v photoresist (Irgacure). In the first layer L1, 800 μL was loaded into the square frame. In the second layer, 400 μL was loaded into the circular frame. ·The particles P were tricalcium phosphate (TCP) with dimensions between 32 μm and 75 μm. ·The vibration frequencies were selected as follows: The first layer L1 has 78Hz, The second layer, L2 (Fig. 16b), contains 60 Hz; in the second layer L2 (Fig. 16c), 65 Hz; The vibration duration was chosen as follows: After reaching liquid motility by adjusting the frequency and amplitude, it takes less than 10 seconds for the first layer L1 and less than 10 seconds for the second layer L2. The temperatures were selected as follows: GelMA temperature: 37°C The experimental temperature was room temperature (25°C). The transformation of matrix M was performed as follows: Each layer of the sample was cross-linked in two steps. First, once the pattern was formed, a mild UV light was placed on top to stabilize the pattern by partial cross-linking. The patterning chamber was then transferred to a UV light oven to complete the cross-linking of the hydrogel. The cross-linking process was carried out at room temperature (25°C) with UV light at a wavelength of 365 nm.

[0099] The above process allows for the production of multiple layers (Ln), where "n" denotes an integer between 1 and 50, preferably between 2 and 10. According to one embodiment, different layers L1 to Ln are produced in a separate manner, either in parallel or sequentially. This means that each layer L1 to Ln is produced independently of the other layers, as shown, for example, in FIG. 15. In the case of parallel production, multiple sample vessels 4a to 4e are exposed to acoustic waves W1 to Wn generated by multiple independent pattern generators S1 to Sn. The shape of each of the sample vessels 4a to 4n, as well as the characteristics of the acoustic waves W1 to Wn, are determined individually and may vary for each sample vessel 4n. Once the layers Ln are produced, they may be combined to produce the desired three-dimensional structure.

[0100] According to another embodiment, layers L1 through Ln are created sequentially and combined, meaning that once a given layer Ln is created, the next layer Ln+1 is created on top of the previous layer Ln. In other words, after a pattern is created in a sample container 4n, a container 4n+1 of the next rank n+1 is placed on top of the previous layer Ln, with a geometry appropriate for this anticipated new pattern. This new sample container 4n+1 is then filled with the corresponding particles Pn+1 dispersed in a suitable matrix Mn+1, and experimental conditions, including the amplitude An+1 and frequency Fn+1 of the acoustic waves, are applied to create the desired pattern. Polymerization of the matrix Mn+1 is then carried out under appropriate conversion conditions. According to this approach, acoustic waves are applied to a combination of sample containers 4a to 4n associated with already prepared layers L1 to Ln, in addition to the layer Ln+1 being prepared. Particles Pn must remain immobilized in the previously modified matrix Mn of these prepared layers Ln.

[0101] The recording step f) may occur at the end of each manufacturing cycle, which manufacturing cycle comprises the above-mentioned selection step a), frame manufacturing step b), filling step c), patterning step d) and converting step e).If layers L1 to Ln are made layer by layer in succession to one another, a monitoring step may be used to monitor the already constructed layers to verify whether they are damaged under the repeated conditions of the process.

[0102] When all layers L1 to Ln have been produced and stacked, the process may include an incubation step g) during which the three-dimensional structure of the biomaterial is allowed to grow and produce the expected biological tissue. [Explanation of symbols]

[0103] 1. Patterning device 10 Damping device 10a, 10b Silencer blocks 11 feet 12a, 12b Rotatable connection 13 Information Department 14 Fixator 15 Support part 16 Counterweight 17 Level control section 2 Holding part 21 Holding plate 22 Retaining portion connecting means 23a, 23b Adaptation part connection means 24 Container fitting part 25a, 25b sensors 26 Temperature control device 3 Pattern generation section 31 Generation unit connection means 32 Vibration generating unit 4 Container fixing means 41 Base 42 Removable Cap 43 Cap Clamp Configuration 44 Sealed part 45, 45a, 45b, 45c Cap fitting part 46, 4a, 4b, 4c, 4d sample container 48a Dish 48b well plate 5 Intermediate joint 51, 51a, 51b Horizontal damping device (and part thereof) 6 Lighting System 61 Emitted light 62 Light frame part 63 holes 7 Image recording unit 8. Image Analysis Department 9 Frame 81 Control Unit 82 Human-machine interface device 83 Artificial Intelligence Unit 100 Container positioning means 100a First positioning element 100b Second positioning element 101 Intermediate Elements 200 Injection device 200' injection device D. Apparatus for chemical transformation of samples L, L1, L2 layers P particles M Matrix S sample

Claims

1. A patterning device (1) for preparing a three-dimensional structure of a sample (S) comprising particles (P) freely moving within a matrix (M), the patterning device comprising: a pattern generating unit (3); and a holding unit (2) connected to the pattern generating unit (3) and adapted to hold a sample container (46) comprising the sample (S); A patterning device (1), at least one transformation device (D) adapted to transform the matrix (M) into a modified matrix (M'), in which the particles (P) remain disconnected from one another and are arranged according to a pattern, while the particles (P) no longer move freely; Image recording unit (7) A patterning device (1), further comprising:

2. 2. The device according to claim 1, characterized in that the at least one conversion device (D) is a light-emitting system (6), or a temperature-control device (26), or a combination of a light-emitting system (6) and a temperature-control device (26).

3. 3. Apparatus according to claim 2, characterized in that the illumination system (6) is adjustable in at least one of its height and angular position with respect to the sample (S).

4. 4. Device according to claim 2 or 3, characterized in that at least one conversion device (D) can be used concomitantly or in series with the pattern generator (3).

5. 5. Device according to any one of claims 1 to 4, characterized in that the holding part (2) comprises at least one temperature sensor (25a) and a vibration sensor (26b).

6. Apparatus according to any one of the preceding claims, characterised in that the patterning device comprises one or more attenuation means (51).

7. 7. Apparatus according to any one of claims 1 to 6, characterized in that it further comprises a control unit (81) adapted to control one or more pattern generators (3) and at least one conversion device (D).

8. 8. Device according to claim 7, characterized in that the control unit comprises or is connected to an artificial intelligence unit (83).

9. 9. Apparatus according to any one of claims 1 to 8, characterized in that it further comprises an illumination system allowing the sample to be visualized and recorded.

10. 10. The device according to claim 1, wherein the pattern generating unit (3) comprises at least one vibration generating unit (3) or a set of vibration generating units (32) for applying sound waves or other vibrational waves to the sample (S).

11. 11. The device according to claim 10, characterized in that at least one vibration generator (32) or set of vibration generators (32) emits waves from a position lateral to the sample (S).

12. 12. Device according to any one of the preceding claims, characterized in that it further comprises level control means (17).

13. 13. Apparatus according to any one of the preceding claims, characterized in that it further comprises at least one injection device (200, 200').

14. 14. Apparatus according to claim 13, characterized in that at least one injection device is adapted for 3D bioinjection of biomaterials.

15. A set comprising a plurality of accessories including two or more emitting lights or groups of lights 61, two or more different image recording devices 7 and two or more different holders 2.

16. 1. A process for producing a three-dimensional structure of particles in a sample (S) comprising particles (P) moving freely within a layer (L1) of a matrix (M), comprising: a patterning step (Q2) of structuring particles (P) in said layer (L1) to achieve a predetermined pattern while applying acoustic waves to the sample (S); a transformation step (Q3) of said matrix (M) into a modified matrix (M') in which the particles (P) no longer move freely; In the process comprising: A process for producing three-dimensional structures of particles, characterized in that the patterning step (Q2) and the transformation step (Q3) are carried out without handling the sample (S).

17. a second patterning step (Q2') of arranging second particles (P2) in a second layer (L2) of a second matrix (M2) to create a second predetermined pattern while applying acoustic waves to the sample (S); a second transformation step (Q3') of transforming the second matrix (M2) into a modified second matrix (M2') in which the second particles (P2) are no longer free to move; Furthermore, 17. Process according to claim 16, characterized in that a second layer (L2) is on top of said layer (L1).

18. further comprising one or more injection steps (Q4) of injecting additional material into one or the other of the layers (L1, L2) or onto one or the other of the layers (L1, L2), Process according to claim 16 or 17, characterized in that one or more implantation steps (Q4) are performed using an implantation device (200) integrated or combined with the patterning device (1).

19. 19. Process according to claim 16 or 18, characterized in that it further comprises one or more container manufacturing steps b), wherein one or more sample containers (46) are printed on demand by means of an injection device (200').

20. 20. Process according to claim 19, characterized in that the sample container (46) has a predetermined geometry according to the geometry of the target pattern.

21. 21. A sample (S) characterized in that the sample (S) is printed under acoustic waves according to a process according to claim 16 or 20 using a patterning device (1) according to claims 1 to 14.

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