EMBEDDING BATH

Abstract

The invention provides a construct (1) comprising a number N of material types (100, 110, . . . ), wherein N is at least 2, wherein at least two of the material types (100, 110, . . . ) comprise granular material (101) comprising particles (10), wherein the granular material (101) at least defines an exterior surface (6) of the construct (1), wherein the construct (1) is self-supporting, and wherein the construct (1) is (i) self-healing or is (ii) configured for being self-healing by changing a liquid (15) content of the construct (1); wherein the different material types (100, 110, . . . ) mutually differ in at least one characteristic (19) selected from the group consisting of a physical characteristic and a chemical characteristic.

Claims

1. A construct (1) comprising a number N of material types (100, 110, . . . ), wherein N is at least 2, wherein at least two of the material types (100, 110, . . . ) comprise granular material (101) comprising particles (10), wherein the granular material (101) at least defines an exterior surface (6) of the construct (1), wherein the construct (1) is self-supporting, and wherein the construct (1) is (i) self-healing or is (ii) configured for being self-healing by changing a liquid (15) content of the construct (1); wherein the different material types (100, 110, . . . ) mutually differ in at least one characteristic (19) selected from the group consisting of a physical characteristic and a chemical characteristic.

2. The construct (1) according to claim 1, wherein the construct (1) is self-healing and wherein the granular material (101) defining the exterior surface (6) is self-supporting.

3. The construct (1) according to claim 1, wherein the construct (1) comprises compartments (2000), wherein each compartment (2000) comprises a subset of the material types (100, 110, . . . ).

4. The construct (1) according to claim 1, wherein the construct (1) has embedding bath properties, wherein the construct (1) is configured for locally supporting a further material (300) being provided into the construct (1).

5. The construct (1) according to claim 1, wherein one or more of the material types (100, 110, . . . ) comprising granular material (101) further comprises an interstitial material (109) arranged between the particles (10), wherein the interstitial material (109) comprises one or more materials selected from the group consisting of a gas, a liquid (15), and a powder.

6. The construct (1) according to claim 1, wherein the material types (100, 110, . . . ) comprising granular material (101) mutually differ in at least one characteristic (19) selected from the group of particle characteristics consisting of a characteristic number averaged average size (d) of the particle (10), a shape of the particle (10), a stiffness of the particle (10), and a surface property of the particle (10).

7. The construct (1) according to claim 1, wherein the material types (100, 110, . . . ) comprising granular material (101) mutually differ in at least one characteristic (19) selected from the group of payload (320) characteristics consisting of a bioactive compound (330) contained within the particle (10), a subset of smaller particles contained within the particle (10), a charge of the particle (10), a biological cell (350) contained within the particle (10), a catalyst contained within the particle (10) or configured at a surface of the particle (10), a photo-initiator contained within the particle (10) or configured at the surface of the particle (10), a magnetic load of the particle (10), an interpenetrating polymer network contained within the particle (10), and a vesicle contained within the particle (10).

8. The construct according to claim 7, wherein the bioactive compound (330) comprises one or more growth factors selected from the group consisting of an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a platelet-derived growth factor (PDGF), an angiopoietin (Ang), a transforming growth factor beta (TGFβ), a cytokine, a hormone, a bone morphogenic protein (B1V11.sup.3), a cytokine, and a hormone.

9. The construct (1) according to claim 1, wherein the particles (10) of the granular material (101) have a number average particle size (d) selected in the range of 25-250 μm and wherein the particles (10) of the granular material (101) of at least one of the material types (100, 110, . . . ) have a size distribution characterized by a coefficient of variation equal to or smaller than 10%, and wherein particles (10) of at least one of the material types (100, 110, . . . ) comprising granular material (101) comprise a hydrogel.

10. A cartridge frame (4) comprising a construct (1) according to claim 3, wherein the cartridge frame (4) comprises one or more terminals (5) for providing one or more types of stimuli to at least one of the compartments (2000), wherein the types of stimuli are selected from the group consisting of a fluid flow through the compartment (2000), a provision of a chemical component to the compartment (2000), a provision of an electrical signal to the compartment (2000), a provision of a magnetic signal to the compartment (2000), and a provision of a drug to the compartment (2000).

11. A method for producing a construct (1), wherein the method comprises providing a number N of material types (100, 110, . . . ) at a substrate (2), wherein N is at least 2, wherein at least two of the material types (100, 110, . . . ) comprise granular material (101) comprising particles (10), wherein the granular material (101) is self-supporting, and wherein at least one of the material types (100, 110, . . . ) comprising granular material (101) is provided at the substrate (2) using an additive manufacturing technique to provide at least part of an exterior surface (6) of the construct (1).

12. The method according to claim 11, wherein the material types (100, 110, . . . ) comprising granular material (101) are in a jammed state.

13. The method according to claim 11, wherein the method further comprises depositing at least one of the material types (100, 110, . . . ) comprising granular material (101) at a pre-assemble substrate (2a) to provide one or more building elements (2001) comprising the respective material type (100, 110, . . . ) with a determined shape, and successively providing the one or more building elements (2001) at the substrate (2).

14. The method according to claim 11, further comprising a remodeling stage, wherein the remodeling stage comprises forcing particles (10) of at least part of the material types (100, 110, . . . ) comprising granular material (101) provided at the substrate (2) to move relative to each other, wherein a spatial arrangement of the material types (100, 110, . . . ) at the substrate (2) is adjusted.

15. A method for the manufacture of an engineered tissue (1000) from one or more biological cells (350), the method comprising: providing a construct (1) comprising a number N of material types, wherein N is at least 2, wherein at least two of the material types comprise granular material comprising particles, wherein the granular material at least defines an exterior surface of the construct, wherein the construct is self-supporting, and wherein the construct is (i) self-healing or is (ii) configured for being self-healing by changing a liquid content of the construct, wherein the different material types mutually differ in at least one characteristic selected from the group consisting of a physical characteristic and a chemical characteristic, wherein the construct (1) is self-healing, locally dispensing a further material (300) in the self-healing construct (1), wherein the further material (300) comprises one or more elements selected from the group consisting of (i) one or more biological cells (350), (ii) a protein and/or peptide and/or a growth factor, and (iii) a liquid and/or a solid, and growing the tissue (1000) from the one or more biological cells (350) arranged in the construct (1); wherein the one or more biological cells (350) originate from the provided construct (1) and/or from the further material (300); wherein the one or more biological cells (350) are selected from the group consisting of a cell, a cell aggregate, a spheroid, and an organoid.

16. The method according to claim 15, wherein the further material at least comprises the one or more biological cells (350).

17. The method according to claim 15, wherein the one or more cells (350) are selected from a mammalian cell, a fish cell, an insect cell, a plant cell, a yeast cell, and bacteria, and wherein the one or more cells (350) comprise one or more cells (350) selected from the group consisting of a stem cell, an induced pluripotent stem cell, an omnipotent stem cell, an adult stem cell, a progenitor cell, a somatic cell, a genetically modified organism.

18. The method according to claim 15, wherein the further material (300) comprises further particles (310) comprising biological cells (350), wherein a number average size of the further particles (310) is larger than a number average size (d) of the particles (10) of the construct (1).

19. An engineered tissue (1000) obtainable by the method according to claim 15, wherein the engineered tissue (1000) comprises a tissue selected from the group consisting of an organ, a subsystem of an organ or a combination of organs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: FIGS. 1 and 2 schematically depict some aspects of the construct and the method for producing the construct; FIGS. 3A and 3B schematically depict some further aspects of the method for producing the construct; FIGS. 4 and 5 schematically depict some further aspects of the invention; FIG. 6 depicts some embodiments of particles of the invention; and FIG. 7 depicts some further aspects of the invention. The schematic drawings are not necessarily to scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0110] In FIG. 1 schematically an embodiment of the construct 1 of the invention is depicted. The construct 1 is depicted in 3D as is very schematically indicated by the X, Y, and Z axis, and indicated by the in the Y direction overlapping particles 10. For simplicity reasons, the depicted embodiment comprises only two material types 100, 110. Both of the material types 100, 110 comprise granular material 101. The granular material 101 of the material types 100, 110 comprises a plurality of (different) particles 10. The granular material 101 at least defines an exterior surface 6 of the construct 1. The construct 1 may further be self-healing. The construct 1 is especially self-supporting, especially indicating that the construct substantially will not lose its shape under its own weight. Hence, the (3D) shape depicted in the figure will substantially last for an extended period of time. The extended period of time may be in the range of minutes, hours, days, weeks or even more. In such extended period of time the dimensions, especially the size/dimensions of the external surface 6 may only change a few percent, especially less than 10%, such as less than 5%, even more especially less than 2%, or no more than 1% (relative to its initial dimensions, especially when being produced). This extended period of time is especially achieved in ambient conditions where the volume fraction (v.sub.f) of the particles may not change, for instance by preventing evaporation of the interstitial liquid.

[0111] The construct is further especially (also) self-healing. The self-healing capacity may allow 3D printing a further material 300, e.g. comprising a biological cell 350, in the construct 1, see e.g. FIG. 4 and FIG. 5A. The volume fraction (v.sub.f) of the particles 10 in the granular material 101 may especially be selected high enough to allow printing the granular material 101 and provide a self-supporting construct 1. Further, the volume fraction of the particles 10 in the granular material 101 may be selected to be low enough to provide the self-healing properties of the construct 1. The range in which the volume fraction is selected may depend on further physical parameters of the granular material 101 and/or the particles 10, such as the stiffness/elasticity of the particles 10 and e.g. the size distribution of the particles 10 in the granular material 101. Especially, the selected volume fraction (v.sub.f) is larger than the random close packing of identical non-deformable spheres (64% v/v), especially larger than the densest packing of identical non-deformable spheres (74%), such as at least 80% (v/v), or at least 85% v/v. In embodiments the volume fraction of the particles in the granular material is selected in the range of 80-90% v/v to provide a self-healing and especially self-standing construct 1. In further specific embodiments, the construct 1 is configured for being self-healing by changing a liquid 15 content of the construct 1, especially by changing the v.sub.f of the particles 10 in the granular material 101.

[0112] The different material types 100, 110 of the construct mutually differ in at least one characteristic 19, especially a physical characteristic and/or a chemical characteristic. In embodiment comprising more than two material types 100, 110, . . . , the types 100, 110, . . . may also differ in at least one such characteristic 19. For instance, the material types 100, 110, 120, 130, 140 depicted in FIG. 2 also mutually differ in at least one characteristic 19 selected from the group consisting of a physical characteristic and a chemical characteristic. The material types 100, 110, 120, 130, 140 may e.g. differ in an average size d of the particle 10 of the material type 100, 110, 120, 130, 140. They may further e.g. differ in a material of the particle 10, a stiffness of the particle 10, or 15 a liquid in the interstitial cavities 25 between the particles 10. They may further e.g. differ in a size distribution over the material type 100, 110, . . . .

[0113] Herein, the . . . . such as in phrases like “more than two material types 100, 110, . . . . ”

[0114] etc. refer to the (optional) further material types (e.g. 120, 130); and that the material types at least comprise material type 100 and 110.

[0115] The construct 1 is compartmentalized. The construct 1 especially comprises (or is made up of different) compartments 2000. A subset of the material types 100, 110, . . . may define or be comprised by such compartment 2000. The embodiment of FIG. 1 shows four (discrete) compartments 2000, wherein two compartments 2000 comprise the first material type 100, and two other compartments 2000 comprise the second (granular) material type 110.

[0116] It is noted that the granular material 101 type 100 may (exclusively) consists of particles 10 (see e.g. FIG. 2). Yet, in further embodiments, the granular material 101 consists of particles 10 and one or more other materials, e.g. a liquid 15, especially arranged in the interstitial cavities 25 between the particles 10 (see e.g. FIG. 1). Furthermore, in embodiments, granular material 101 of a first compartment 2000 may further comprise such liquid 15, whereas granular material of a further compartment 2000 of the same construct 1 substantially only comprises particles 10. Hence, in embodiments one or more of the material types 100, 110, . . . comprising granular material 101 further comprises an interstitial material 109 arranged between the particles 10, especially in the interstitial cavities 25. Such interstitial material 109 may especially comprise one or more materials selected from the group consisting of a gas, a liquid 15, and a powder.

[0117] Although not depicted in FIG. 1 or FIG. 2, not all material types 100, 110, . . . of the 30 construct 1 necessarily comprises granular material 101. For instance, one or more of the material types 100, 110, 120, . . . of the construct may comprise non-granular material 102. The embodiment schematically depicted in FIG. 3A at the bottom, e.g. comprises a space defined by two granular material types 130, 140 that comprises a material type 150 comprising a fluid such as a gaseous fluid, a liquid 15 or an emulsion as an example of a non-granular material 102. In other examples, e.g. the non-granular material 102 comprises a solid. Also these non-granular materials 102 may define /be comprised by a compartment 2000 of the construct 1. A solid compartment 2000 (a compartment 2000 comprising a solid material) may e.g. provide an additional stiffness to the construct 1. Especially, the further material 300 is provided in the granular material 101.

[0118] It is further noted that the numbering of the material types 100, 110, 120, 130, etc. are merely used to indicate different material types 100, 110, . . . The reference numbers of these material types 100, 110, 120, 130, etc. do not refer to the type, i.e. granular 101 or non-granular 102, of the material type 100, 110, . . . . Hence, in a first embodiment, material types 100, 110, 120, 130, and 140 may comprise granular material 101 and material type 150 may comprise non-granular material 102 (like FIG. 3A at the bottom). In a further embodiment, material types 100, 120, and 140, for instance comprise non-granular material 102, and material types 110, 130, and 150 comprise granular material 101. In FIG. 4, e.g., material type 110 configured in a space between particles of (granular) material type 100 is a non-granular material 102.

[0119] The embodiment of the construct 1 in FIG. 2 is especially (being) produced using an embodiment of the method of the invention as is schematically illustrated at the top side of the figure wherein (a part of) one of the material types 110 (at this stage) is provided, especially printed in 3D, at the substrate 2. It is noted that because already a number of material types (at least party) 100, 110, 120, 130, 140 are deposited at the substrate 2, the presently provided (part of the) material type 110 (only) indirectly contacts the substrate 2. Herein, providing a material type 100, 110, . . . at the substrate 2 refers to providing it such that it may directly or indirectly contact the substrate after providing it 100, 110 to the substrate 2. It is further noted that the figure depicts an embodiment, wherein the material type 110 is provided in two periods of time to the substrate 2. As such, at least two of the compartments 2000 of the final construct 1 may comprise the material type 110.

[0120] In the method, a number (especially at least 2) of material types 100, 110, . . . is provided at the substrate 2, especially wherein at least two of the material types 100, 110, . . . . comprise granular material 101, see e.g. FIG. 2 wherein five material types 100, 110, 120, 130, 140 comprise granular material 101. At least one of the material types 100, 110, 120, 130, 140 is provided at the substrate 2 using an additive manufacturing technique. As such at least part of the exterior surface 6 of the construct 1 is provided. The additive manufacturing may comprise 3D printing of the material type. In specific embodiments all material types 100, 110, . . . comprising granular material 101 are 3D printed. The substrates 2 of the embodiments in FIGS. 1 and 2 (as well as the pre-assemble substrate 2a depicted in FIG. 3A) are flat substrates, especially on top of which the granular materials 101 are deposited. The granular material 101 may keep its shape (while only being supported at the bottom by the support 2) because of its self-standing capacity.

[0121] The material types 100, 110, 120, may all be provided or deposited in one run at the substrate 2. Yet in embodiments at least one of the material types 100, 110, . . . comprising granular material 101 is initially provided at a pre-assemble substrate 2a, thereby providing one or more building elements 2001 comprising the respective material type 100, 110, . . . with a determined shape. Successively, the one or more building elements 2001 may be provided (as a building elements 2001/a compartment 2000) at the substrate 2, as is schematically depicted in FIG. 3A. One or more of the (at the substrate 2) provided material types 100, 110, . . . may further provide space, especially wherein successively a further material type 100, 110, . . . is provided.

[0122] Furthermore, in embodiments, the method may further comprise a remodeling stage as is depicted in FIG. 3B. In the remodeling stage particles 10 of at least part of the material types 100, 110, . . . provided at the substrate 2 may be forced to move relative to other particles 10 (of the same material type 100, 110, . . . and/or another material type 100, 110, . . . (especially relative to each other). As such, a spatial arrangement of the material types 100, 110 at the substrate 2, and e.g. also of the compartments 2000 is adjusted.

[0123] For providing a construct 1 to be free standing while also being able to function as an embedding bath (potentially after changing the volume fraction), especially a combination of characteristics of the particle 10 and/or of the granular material 101 seem relevant. The volume fraction of should be high enough to provide the self-standing capacity. The volume fraction of is especially selected above that of a random close packing (approx. 64% v/v) and even more especially above the maximum packing of non-deformable spheres (approx. 74% v/v). The volume fraction of may especially be in the range of 75-95% v/v, such as in the range of 75-90% v/v. Further, it is hypothesized that the particles 10 should be elastically deformable. In order to achieve a volume fraction of of over 74%, the particles 10 may especially be selected for being deformable. Especially, the deformation is elastic and not plastic. Plastic deformation may facilitate particle aggregation and may in embodiments prevent functioning as an embedding bath.

[0124] Further, having granular material 101 comprising particles 10 that may be free-sliding in along other particles 10 and especially are non-sticky may further facilitate the printing of further material 300 in the granular material 101 and the self-healing of the granular material 101. A motion of the injection device 500 or of a first particle 10 in the preferably does not drag another particle 10 with it, for example by particle-particle adhesion forces. Further, smooth sliding between particles 10 may be facilitated by minimizing of friction between the particles 10.

[0125] A further relevant parameter may be an extend of attraction between the particles 10. Minimizing the attraction between the particles 10 may facilitate free movement of the particles 10 relative to each other. Attraction of particles 10 may result in aggregation and/or in particles 10 that are not able to move freely with respect to each other when being sheared (e.g. when injecting a further material 300 in the embedding bath). In embodiments, particles 10 may be selected that show no interaction between particles 10 or even show repulsion between particles 10.

[0126] Furthermore, the granular material is especially shear thinning. The granular material further may show (especially complete) recovery after being sheared. These last effects may especially facilitate printability of the granular material. The factors may further facilitate the free standing and in embodiments also the shelf-healing of the construct

[0127] In embodiments (e.g. depicted in FIG. 2) the material types 100, 110, 120, 130, 140, . . . comprising granular material 101 may mutually differ in at least one characteristic 19, such as selected from the group of particle characteristics and/or payload 320 of the particle 10. The group of particle 10 characteristics may e.g. comprise of a characteristic number averaged sized of the particle 10, a shape of the particle 10, a stiffness of the particle 10, a composition of the particle 10, and a surface property of the particle 10. The particle 10 may e.g. comprise a coating 210. The particles 10 of the granular material 101 may in specific embodiments e.g. have a number average particle size d selected in the range of 10-250 μm. The particles 10 of different material types 100, 110, 120, 130, 140 especially differ in average particles size d. For clarity reasons all particles 10 of any of the material types 100, 110, 120, 130, 140 are spherical (globular) and have the same size d. In further embodiment, this may be different. In specific embodiments, at least one of the material types 100, 110, . . . comprises monodispersed particles 10, and especially the particles 10 of said material type 100, 110, . . . have a size distribution characterized by a coefficient of variation CV equal to or smaller than 10%. Further, particles 10 of at least one of the material types 100, 110, 120, 130, 140 may e.g. comprise a hydrogel (particle). Some further of examples of the properties 19 of the particle 10 are further depicted in FIG. 6 (see below).

[0128] The material types 100, 110, 120, 130, 140 may additionally or alternatively (also) mutually differ in a characteristic 19 from the group of payload 320 characteristics. Examples of payload 320 characteristics are e.g. a bioactive compound 330 contained within the particle 10, a subset of smaller particles 10 contained within the particle 10, a charge of the particle 10, a biological cell 350 contained within the particle 10, a catalyst contained within the particle 10 or configured at a surface of the particle 10, a photo-initiator contained within the particle 10 or configured at the surface of the particle 10, a magnetic load of the particle 10, an interpenetrating polymer network contained within the particle 10, and a vesicle 201 such as a liposome or capsosome comprised by the particle 10.

[0129] The bioactive compound 330 may herein especially refer to a biological element such as a biological cell 350, an organoid, an embryoid (body) or a protein or a peptide, especially a growth factor, or an oligonucleotide; or to a chemical factor such as catalyst, photo-initiator. Optionally the protein and/or peptide comprises a synthetic proteins and/or peptide that may function as a growth factor.

[0130] In specific embodiments, one or more of (especially the particles 10 of) the material types100, 110, 120, 130, 140, is be loaded with one or more growth factors such as an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a platelet-derived growth factor (PDGF), an angiopoietin (Ang), a transforming growth factor beta (TGFβ), a cytokine, a hormone, a bone morphogenic protein (BMP), a cytokine, and a hormone.

[0131] In FIG. 5B, the construct 1 is (at least partly) enclosed in a cartridge frame 4. In the given embodiment, the cartridge frame 4 comprises two terminals 5 for providing one or more types of stimuli to at least one of the compartments 2000 (defined in the construct 1). Examples of types of stimuli that may be provided to at least part of the construct 1, or to one or more specific compartments 2000 are e.g. a fluid flow through the compartment 2000, a provision of a chemical component to the compartment 2000, a provision of an electrical signal to the compartment 2000, a provision of a magnetic signal to the compartment 2000, and a provision of a drug to the compartment 2000.

[0132] FIG. 6 schematically depicts some characteristics 19 of the material types 100, 110, . . . , especially of the particles 10 of the material types 100, 110, .. (comprising granular material 101). Particles 10A, 10B, 10C e.g. depicts hydrogel particles. In the depicted embodiments, these hydrogel particles are all loaded. i.e. comprise a payload 320. In particle 10A indicated by some dots, e.g., schematically depicting a bioactive compound 330. Particles 10B and 10C both comprise a cell 350. The particles 10 do not need to be loaded. Particle 10F e.g. depicts a hydrogel particle without a payload 320. The particle 10 may further comprise a coating 210, see particle 10C, being an embodiment of a core-shell particle. The particle 10 may comprise or be a vesicle 201. Especially, a core-shell particle 10 may comprise one or more vesicle 201. A vesicle 201 may especially comprise or be a core-shell particle 10 especially being hollow with an enveloping layer, see particle 10D. Further, the core-shell particle 10H comprises a (loaded) capsosome, schematically indicated by the inner circle with the small circles arranged in it. The vesicle 201 may herein also be called capsule. The particle 10 may comprise a Janus particle 10G having a different composition at a first part of the particle 10 compared to the other part of the particle 10. Further, the particle 10 may comprise a complex particle 10E comprising a combination of aforementioned characteristics.

[0133] The particle 10 may be a microprinted cage as depicted by particle 10J or it may comprise a complete microprinted architecture depicted by particle 101. The particles 10 depicted by particles 10A to 10J show examples of what is also referred to herein as particle classes. Particles 10K to 10P further depict some particle 10 shapes, like spherical (or globular), ellipsoid, sphenoid, fiber (or fibrous), ribbon, and complex, for respectively particle 10K, 10L, 10M, 10N, 10O, 10P.

[0134] The construct 1 described herein and/or obtained using the method to produce the construct may especially have embedding bath properties. The construct 1 may thus be configured for locally supporting a further material 300 being provided into the construct 1. To allow provision of a further material 300 into the construct 1, the construct preferably is self-healing. The method to produce the construct may in embodiment further comprises changing the liquid content of the construct 1, especially to obtain or provide a self-healing construct 1.

[0135] In FIGS. 4, 5A and 5B schematically some aspects of the method for the manufacture of an engineered tissue 1000 from one or more biological cells 350 is depicted. In said method a construct 1 described herein is provided.

[0136] The construct 1 is especially (already) self-healing (and self-supporting). In embodiments, the liquid content of the construct 1 may be changed to provide the construct 1, wherein the construct 1 is self-healing. In embodiments, the volume fraction of the particles in the granular material of may be reduced, such as a few % to provide the self-healing capacity. In the self-healing construct 1 locally a further material 300 is provided/dispensed (especially via a dispensing path 501 very schematically indicated in FIG. 4). The further material 300 is especially selected to comprises one or more elements of (i) one or more biological cells 350, (ii) a protein and/or peptide and/or a growth factor, and (iii) a liquid and/or a solid. In embodiments, the construct before providing the further material, (already) comprises one or more biological cells 350. In other embodiments, the construct 1 does not comprise a biological cell 350 to grow the tissue 1000 from. Hence, in embodiments the further material 300 at least comprises the one or more biological cells 350. After dispensing the further material 300, the tissue 1000 may be grown or cultured from the one or more biological cells 350 arranged in the construct 1.

[0137] The further material 300 may be locally provided in the construct 1, such as via an injection device 500. The further material 300 may be provided in the construct 1 along a spatial path 501 and/or at a discrete location in the construct 1. As such the further material 300 may be provided at determined spatial positions in the construct 1, e.g. in or relative to one or more determined compartments 2000. The method may comprise providing/dispensing (the same type of or different types of) the further material 300 in the construct, especially at the same or at another location in the construct 1. For instance during a first period further material 300 comprising a biological cell 350 is dispensed in the construct 1 (see e.g. FIG. 5A). During another period, e.g. a cell growth medium may be dispensed in the construct 1. Hence the spatial path 501 not necessarily is a continuous path but may refer to multiple different spatial path 501. In embodiments, the further material 300 is dispensed continuously or interruptedly in time in the construct 1.

[0138] The one or more biological cells 350 is (are) especially selected from the group consisting of a cell, a cell aggregate, a spheroid, and an organoid. Moreover, the one or more cells 350 may in embodiments comprise a mammalian cell, or a fish cell, or an insect cell. In further embodiments, the cell 350 may comprise a plant cell, a yeast cell, or a bacterium. Furthermore, the one or more cells 350 may comprise a stem cell, an induced pluripotent stem cell, an omnipotent stem cell, an adult stem cell, a progenitor cell, a somatic cell, and/or a genetically modified organism.

[0139] In specific embodiments, the further material 300 comprises further particles 310 comprising the biological cells 350. In specific embodiments, a number averaged size of the further particles 310 is configured larger than a number average size d of the particles 10 of the construct 1, especially to prevent spontaneous movement of the biological cell 350 between particles 10 of the construct. In FIG. 5A, the further particles 310 have about the same dimension as the particles 10 of material type 110. It is further, very schematically and simplified depicted in FIG. 5A that the construct 1 comprises a plurality of compartments 2000 of different material types 100, 110, 120, 130 providing the cues to grow the tissue 1000.

[0140] In the construct different particles 10 are configured that may especially during the method for the manufacture of the engineering tissue be consumed by growing biological cells 350 and/or that may define a path for the biological cells 350 to grow. Moreover, the particles 10 may shrink, degrade or disintegrate under controlled stimuli e.g. provided via the terminals 5 or via the further material 300. The cells 350, tissue 1000, spheroids or other biological entity present in the construct may (further) as a result differentiate, expand, or e.g. go quiescent or proliferate. This again may result in grows of the cells 350 and especially in the extending of the cells 350 between the particles 10 thereby forming an artificial tissue 1000 architecture as is very schematically depicted in FIG. 5B showing an expansion of the cells 350 in the interstitial cavities 25 between particles 10 as one of the examples of phenomena taking place during cell culturing.

[0141] Hence, using the method, an engineered tissue 1000 may be obtained, schematically depicted in FIGS. 5A and 5B. Examples of the engineered tissue 1000 are e.g. an organ, a subsystem of an organ or a combination of organs.

[0142] In FIG. 7, some pictures of experimental results are schematically depicted, showing the addition of biological cells 350 to the embedding bath (construct 1) and the differential behavior of the cells 350 deposited in constructs 1 of different compositions. FIG. 7A depicts a further material 300 comprising a spheroid of human smooth muscle cells (SMC) 350 together with human umbilical vein endothelial cells (HUVEC) 350 embedded in a bath or construct 1 (after depositing) of monodisperse alginate spheroidal particles 10 with culture medium as the interstitial fluid 109 in the interstitial cavities 25 at day 0.

[0143] FIG. 7B schematically depicts the same formulation (at a lower magnification) after four days of culture. It can be seen that the cells 350 migrate through the interstitial cavities 25 in between the alginate particles 10 to some extent, but that there is limited attachment of the cells 350 to the particles 10.

[0144] FIG. 7C shows the result after depositing a further material 300 comprising a spheroid of SMC together with HUVEC as the biological cell 350 in an embedded bath 1 of monodisperse alginate spheroidal particles 10 that were functionalized with collagen in the interstitial fluid 109 in the interstitial cavities 25, after three days of culture. Due to the collagen coating 210 of the particles 10, the cells 350 can attach to and interact with the particles 10. The embedded (and deposited further material 300) cell spheroid 350 is visible as the dark structure at the right side of the figure. It was experimentally found that that there is extensive migration and elongation of the cells 350 over the particles 10, indicated by the small open spots. The cells 350 migrated and elongated along the particles 10 of which the outlines are depicted by the dotted lines. Some of the larger extensions are depicted with small open spots of which some are indicated with the arrows. It is noted that the image represents a two-dimensional image, and extensions in the third dimension over the surface of the particles 10 are not shown. Based on these experiments, it may be concluded that the migration of the cells 350 among others may be controlled/supported by the type of material types 100, 110, . . . . of the construct 1.

[0145] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of” and “a number of” may be used interchangeably. The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms “about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90%-110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0146] The term “comprise” includes also embodiments wherein the term “comprises”means “consists of”.

[0147] The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term “comprising” may in an embodiment refer to “consisting of”) but may in another embodiment also refer to “containing at least the defined species and optionally one or more other species”.

[0148] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0149] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0150] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0151] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0152] Use of the verb “to comprise” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. The article “a” or “an” preceding an element does not exclude the presence of a plurality of such elements.

[0153] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0154] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0155] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method respectively. The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

EXPERIMENTAL

Initial Demonstration of 3D Printed Patterned Granular Construct

[0156] Patterned constructs have been prepared using 3D printing of granular material. In one example a computer file defining a 3D checker-board profile with squares (fields) of 5×5 mm in square and 5 mm in height. The “closed” squares representing one color of the fields were printed, thereby defining open (i.e. not printed) squares representing the other color of the fields. The (printed) squares are connected (only) at the edges (during printing) as such forming a continuous system. The printing was done using a printing nozzle of 400 μm. To complete the design (construct), 12 layers are deposited on top of each other. The construct was printed using monodisperse circular spheroidal particles with a diameter of 110 μm±7 μm, consisting of 0.8% alginate-di-aldehyde-gelatin (ADA-GEL) +0.25% alginate. The interstitial fluid was phosphate buffered saline (PBS).

[0157] The final construct resembled the design and showed the same checker-board profile with the open squares and closed (printed) squares having substantially the same size. The packing density of the particles in the resulting construct, defined as the fraction of the volume that is occupied by particles instead of interstitial fluid (wherein the total volume is occupied with particles and the interstitial volume) is >74% v/v. This density is above the maximum packing fraction of balls, due to the deformable nature of this formulation of particles i.e. the particles deform to allow for more dense packing. The results show that the construct is self-standing and self-supporting, even when tilted at a 90 degree angle. A 24-hour test wherein the construct was immersed in additional fluid showed that the construct remained stable for at least 24 hours (longer periods were not tested) under immersed conditions.

Demonstration of the Use of a 3D Printed Patterned Granular Construct as an Embedding Bath

[0158] Further experiments were performed to demonstrate the use and capability of the construct as embedding bath. A patterned construct was made by 3D printing granular material to study the effect of deposition of a further material. The printed construct consists of 4 distinct squares with a height of 5 mm being connected (during printing) to each other along the edges thereby forming a larger square of 2×2 of the distinct smaller squares. Given that a printing nozzle of 400 μm is used, 12 layers are deposited on top of each other. The construct was printed using monodisperse slightly elongated spheroidal particles with a feret diameter of 211 μm±7 μm for the long direction and 140 μm±7 μm for the short direction, consisting of 0.8% alginate-di-aldehyde-gelatin (ADA-GEL) +0.2% alginate, coated with poly-L-lysine (PLL). The interstitial fluid used was phosphate buffered saline (PBS). The packing density of the particles in the resulting construct, defined as the fraction of the volume of the particles relative to the sum of the volume of the particles and the volume of the interstitial fluid, was >74%. This is above the maximum packing fraction of balls, due to the deformable nature of this formulation of particles i.e. the particles deform to allow for more dense packing. Experimentally it was shown that a circle of further material (4% Xanthan gum complemented with blue food coloring) could be printed and maintained (embedded0 within the granular construct.

[0159] Before the further material was printed within the granular construct, a small amount of liquid (water containing 0.2 M CaCl.sub.2) was added to the construct to lower the packing density of particles to <74%. With this specific formulation of particles, a lower packing density seemed to be needed to give the construct embedding bath properties. The (3D) location of the embedded circle was stable (in time) within the construct, and the construct itself has not been damaged by printing within it, showing that this granular construct portrays favorable embedding bath properties. Microscopic images of the granular construct after deposition of the further material showed the close packing of the granules and the presence of granules (construct particles) on top of the further deposited material. The region where further material has been deposited, remained stationary after deposition.

Further Experiments General

3D Printing of the Granular Material

[0160] Granular devices/constructs of 100 μm, 150 μm, 200 μm, etc. 0.8% ADAGEL (1:1 ratio; alginate di-aldehyde/gelatin crosslinked hydrogel) containing a PLL coating (incubated in 0.1% PLL (Poly-L-Lysine) solution overnight to attain a thin coating of max 1.5 μm), or 0.3-0.8% Alginate PLL coated (incubated in 0.1% PLL (Poly-L-Lysine) solution overnight to attain a thin coating of max 1.5 μm) microparticles, that on high-volume fraction of can be 3D printed (behave as an ink, shear thinning) and exhibit self-standing behavior upon deposition on a substrate, have been prepared. The particles are able to deform and recover their initial structure after shear (elastic recovery of particles, system is self-healing). Indicative values of storage modulus while at rest are 10,000 Pa-100 Pa (material dependent). The printing of the ADAGEL-PLL particles has been achieved with nozzles that are 2.5× times the particle size. 3D printing is consistent at 4x Nozzle size compared to particle size (i.e. 400 μm nozzle, with 100 micron particles, 600 μm nozzle, with 150 micron particles, etc.).

Embedding Bath Properties (Self-Standing)

[0161] Some printed self-standing high-volume fraction granular compositions can act as embedding baths directly after printing. For other compositions, adding a small liquid volume (water, cell medium, . . . ) on the ink deposit readily reduces the volume fraction of the device, to reach a state of self-standing embedding bath with self-healing properties. Based on the first experiments, a feasible window of operation for 1-2% ADAGEL PLL and 0.5%-2% ADAGEL 0.25% Alginate PLL granular compositions may be between 70%-80% volume fraction (v.sub.f) .

Embedding Bath Properties (Not Self-Standing)

[0162] It appeared that adding more liquid volume may completely unjam the device returning into an embedding bath which is not free standing, or upon further dilution to a liquid solution (resuspension of particles without agglomeration).

Other Particle Compositions

[0163] Similar behavior has been observed by particles consisting of 0.5-2% ADAGEL, 0.8% ADAGEL 0.3-0.5% Alginate, Gelatin and Ag/Col formulations. Similar behavior is expected of gelatin methacryloyl (GelMA) particles. It has been possible to create devices with different compositions, concentrations, particle shapes, with and without coating. Interstitial space composition

[0164] If fibrinogen is added in the liquid phase of the suspension, upon the addition of liquid volume that contains fibrin, the whole granular device can be annealed.

[0165] If cells are added into the liquid volume, such as smooth muscle cells (SMCs), they can proliferate and populate the interstitial space thus annealing the macro volume and deforming it. This has been observed with a concentration of lmillion cells/ml after 1 week of culture in ADAGEL-PLL particles. This effect became even more pronounced when combined with thrombin/fibrin annealing.

Further Experiments—Some Specific Results

[0166] Constructs/bioinks were prepared from 0.8% ADAGEL-PLL, particles with a particle size of about 45 μm.

[0167] First type of constructs/bioinks were obtained by straining the particle solution with a cell strainer to remove the liquid by gravity. The first constructs had a of of 65-70% and were not self-standing.

[0168] Second type of constructs/bioinks were obtained by straining the particle solution with the cell strainer wherein a pressure drop of 0.02 MPa for 30 seconds is applied to remove an extra amount of the liquid compared to the first constructs/bioinks. The second constructs had a of of about 75-80% and exhibited self-standing and self-healing properties. It is noted that the self-standing aspect is observed but sharp features/corners may not always be retained.

[0169] Third type of constructs/bioinks were obtained by straining the particle solution with the cell strainer wherein a pressure drop of 0.04 MPa for 30 seconds is applied to remove an extra amount of the liquid compared to the second constructs/bioinks. The third constructs had a of of about 80-85% and showed self-standing properties and sharp features of corners may retain in the construct. These constructs may further be sculpted with a spatula, which demonstrates the retention of the ability of the particles to move in relation to each other.

[0170] Rheological characterization of the three types ofjammed 0.8% ADAGEL-PLL bio-ink (particle size ˜145 μm) showed for all types shear thinning behavior: Changes of viscosity with shear rate ramping up for all three types of constructs/bioinks. A continuous decrease in viscosity with increasing shear rate indicated that the systems were shear thinning.

[0171] Yielding of the three types of ink was tested under oscillatory shear: The three types of Jammed bioink were sheared at frequency f=1 Hz and 0.01 to 200% strain amplitude. For all types G′<G″ at the lowest strain and G″>G′ at highest strain demonstrating that the tested bioinks became liquid like with increasing strain.

[0172] Next to the three types of constructs, a granular assembly of ADAGEL-PLL 100 μm particles with 7-14 μm 1%w/v microparticles in the interstitial space was prepared and experiments were repeated. The particles were colored to show their presence. The experiments demonstrated that aforementioned behavior/properties, are also possible when 1%w/v (color) particles are added in the liquid phase. The added solid, rigid, neutral charge, color particles that are significantly smaller by an order of magnitude from the hydrogel particles that comprise the granular assembly, do not seem to participate or affect in a significant manner the jamming/unjamming properties of the device but they only seemed to change the color (the color particles were red).

[0173] In yet a further experiment, high vacuum volume fraction samples (based on straining at a pressure drop of 0.04 MPa) were scooped onto a glass slide. Then shaped with a spatula, then cut in half with a spatula, and then one piece was stacked on top of the other and merges. Overhangs of the top piece over the bottom piece can be observed, demonstrating that the construct is free-standing, that the particles in the construct may move freely with respect to each other and that the construct may be remodeled and/or may be assembled from smaller constructs. This specific sample is 2% ADAGEL 150 μm particles. The particles could be resuspended in a liquid solution without the presence of agglomerates.